WO2023111286A1

WO2023111286A1 - Nucleic acid delivery methods and compositions

Info

Publication number: WO2023111286A1
Application number: PCT/EP2022/086392
Authority: WO
Inventors: Adam TAKOS; Virginia MARTINEZ; Andreas PORSE; Camilla KRISTENSEN; Alex SALAZAR
Original assignee: Snipr Biome Aps
Priority date: 2021-12-17
Filing date: 2022-12-16
Publication date: 2023-06-22
Also published as: FR3136481A1

Abstract

The present invention relates to improved methods for the delivery of DNA to target bacteria and archaea.

Description

Nucleic acid delivery methods and compositions

Bacteria and archaea, such as those in human and animal microbiota or in environments, encode methyltransferase enzymes which protect host bacterial DNA against DNA cleavage by host encoded restriction modification (RM) systems. These RM systems target DNA which is not methylated in the same way as the host DNA. The RM system enzymes cleave DNA at specific motifs within the DNA, and the site specific cleavage is prevented by the methylation. In laboratory bacteria the RM systems can be modified to allow for more efficient transformation of foreign DNA, but this is not possible for delivery to bacteria in natural environments such as the microbiome.

WO 2016/205276 discloses methods to methylate bacteriophage or phagemid DNA in production strains before delivery.

Statements of invention

The invention relates to:

A carrier bacteria comprising a conjugative plasmid, the plasmid encoding a methyltransferase enzyme.

A method for delivery of DNA from a carrier bacteria to a target bacteria by conjugation, the method comprising contacting the target bacterial species with the carrier bacteria as disclosed herein, wherein the target bacteria comprises a restriction modification system that cleaves DNA which is unmethylated at a target site of methylation of the methyltransferase enzyme.

A bacteriophage encoding a methyltransferase enzyme, optionally wherein the methyltransferase enzyme protects against a restriction modification system found in a target bacterial which is pathogenic in humans or animals, and/or in a target bacteria which is associated with biofouling or microbial corrosion.

A method for delivery of bacteriophage DNA to a target bacteria, the method comprising contacting the target bacterial species with a bacteriophage encoding a methyltransferase enzyme, wherein the target bacteria comprises a restriction modification system that cleaves DNA which is unmethylated at a target site of methylation of the methyltransferase enzyme. A method for methylation of a bacteriophage DNA or plasmid or phagemid DNA, the method comprising: introducing into a production bacterium (i) a polynucleotide encoding a plurality of methyltransferases, or (ii) polynucleotides which together encode a plurality of methyltransferases, thereby producing a modified production bacterium expressing at least 2 introduced methyltransferases; infecting the modified production bacterium with a bacteriophage particle comprising bacteriophage DNA or phagemid DNA or plasmid DNA, thereby methylating said bacteriophage DNA or phagemid or plasmid DNA; and producing a bacteriophage particle comprising bacteriophage DNA or phagemid DNA or plasmid DNA having a modified methylation pattern, optionally purifying the bacteriophage particle comprising bacteriophage DNA or phagemid DNA or plasmid DNA.

A pharmaceutical composition comprising a carrier bacteria or bacteriophage or phagemid or plasmid as disclosed herein in combination with a pharmaceutically acceptable carrier or excipient.

A method of prevention or treatment of disease caused by a pathogenic target bacteria in a human or animal, the method comprising delivery of a bacteriophage, phagemid or carrier bacteria or plasmid or a pharmaceutical composition as disclosed herein to a human or animal in need thereof.

A bacteriophage, phagemid or carrier bacteria or plasmid or a pharmaceutical composition as disclosed herein for use in a method of prevention or treatment of disease caused by a pathogenic target bacteria in a human or animal, the method comprising delivery of the bacteriophage, phagemid or carrier bacteria or pharmaceutical composition to the human or animal in need thereof.

A method of killing of a target bacteria or the targeted modification of the genome of a target bacteria, the method comprising delivery of a bacteriophage, phagemid or carrier bacteria or plasmid as disclosed herein to the target bacteria, wherein the target bacteria is comprised within in a naturally occurring population and associated with microbial corrosion or biofouling of a substrate or fluid in an industrial or domestic system.

A method for targeted modification of a target bacterial species or strain, the method comprising contacting a target bacteria with a carrier bacteria, bacteriophage or phagemid or plasmid as disclosed herein, such that the target sequence of the target bacterial species is modified, where the conjugatable plasmid, phage or phagemid further comprises a target cell modifying CRISPR array.

Figures

Fig. 1. Conjugation efficiency of small conjugative plasmids methylated in cis (n=3)

Fig. 2. Conjugation efficiency of large conjugative plasmid methylated in cis (n=3)

Fig. 3. Comparison of conjugation efficiency of small conjugative plasmids methylated in cis and in trans (n=3)

Fig. 4. Efficiency of plaquing of phage lysates, methylated either in trans or in cis (n=3) Fig. 5. Efficiency of phage transduction after methylation in trans by a production strain expressing an MTase operon (n=3)

Detailed description

The present invention demonstrates that it is not necessary to pre-methylate DNA using methyltransferases (MTases) in a production bacterial strain before delivery to target bacteria comprising a RM system that would otherwise cleave the introduced DNA and reduce transfer efficiency. Instead, we surprisingly show that methyltransferases may be directly encoded on nucleic acid that is delivered to a target cell, such as on a phage or a conjugative plasmid, and then can be expressed in that target cell to provide protection against host restriction modification. This avoids the need to pre-methylate at the production stage of the phage or plasmid.

In addition we have demonstrated that the methylation of DNA using multiple MTases within a production strain also provides significant protection to the DNA upon subsequent transfer to a target bacteria. This approach can be used in combination with the encoding of MTases in the phage or conjugative plasmids directly if desired.

In addition we compare the methylation of DNA such as plasmid DNA in trans within a cell by a co-resident non-conjugative plasmid, versus cis methylation. Both approaches are effective in preventing restriction of introduced DNA with cis methylation highly effective.

In any method or other aspect herein, where there is disclosed the contacting of a carrier cell with a target cell, in the alternative embodiment there is provided contacting a population of carrier cells with a population of target cells.

In a first aspect, the present invention relates to a carrier bacterial cell comprising a conjugative plasmid, the plasmid encoding a methyltransferase enzyme. Data provided herein show that inclusion of a methyltransferase enzyme on a conjugative plasmid which is delivered to a target bacteria increases the efficiency of conjugative transfer when compared to a plasmid lacking the methyltransferase, in the context of a target bacteria in which that methyltransferase can protect against a host RM system. The invention also provides a population of said carrier cells, e.g., wherein the population is comprised by a pharmaceutical composition for administration to a human or animal subject to prevent or treat a medical condition.

The invention also therefore relates to a method for delivery of plasmid DNA from a carrier bacterial cells to a target bacterial cell by conjugation, the method comprising contacting the target bacterial cell with the carrier cell as disclosed herein, wherein the target cell comprises a restriction modification (RM) system that cleaves DNA which is unmethylated at a target site of methylation of the methyltransferase enzyme.

In this way the plasmid DNA transferred by conjugation is protected from cleavage by the RM system in the target cell by expression of the encoded MTase directly within the target cell.

The contacting of the carrier bacterial cell and target bacteria may be by administration of the former to by a pharmaceutical composition for administration to a human or animal subject to prevent or treat a medical condition, e.g., to a gut, lung, skin or kidney microbiota.

Administration may be orally, by inhalation, topically or parenterally (e.g., IV).

Preferably the plasmid encodes more than one MTase, preferably 2, 3, 4, 5 or even more MTases. Preferably the plasmid encodes more than one Type I MTase. Preferably the plasmid encodes more than one Type II MTase. Preferably the plasmid encodes more than one Type III MTase. Preferably the plasmid encodes a Type I MTase and a Type II and/or III MTase.

The plasmid may encode multiple MTases as a part of an operon, or the expression of different MTases may be controlled by more than one promoter, for example as part of 2 or more operons. Each nucleotide sequence encoding a MTase may be under the control of a respective promoter.

The plasmid may encode phage DNA or encode an entire phage genome.

Where a bacterial cell or bacteria are mentioned herein in relation to the invention, the invention may instead apply mutatis mutandis to an archaeal cell or archaea.

The plasmid to be transferred by conjugation may itself comprise methylated DNA to protect further against target cell restriction enzyme digestion. Methylation of the plasmid may be for example by expressing methylase enzymes in the carrier bacterial cell from a different plasmid to the conjugative plasmid, or resulting from the enzymes of the conjugative plasmid itself, or may result from pre-methylation in a production strain. The invention therefore relates to a carrier bacterial cell having a plasmid and/or genome encoding one or more MTases which provide for methylation of a said conjugative plasmid DNA in the carrier cell.

The carrier bacterial cell may be provided as a population of cells such as 10^3> 10⁴, 10⁵, 10^6> 10⁷, 10⁸, IO^9, IO¹⁰, 10¹¹, IO^12, 10¹³, 10¹⁴ or more cells.

In a second aspect the invention relates to a bacteriophage encoding a methyltransferase enzyme, optionally wherein the methyltransferase enzyme is an enzyme that protects against a RM system found in a bacterial species or strain that is pathogenic in humans and animals.

The invention also provides a nucleic acid (e.g., a DNA) that encodes such a bacteriophage.

The bacteriophage genome may encode more than one MTase.

The invention also relates to a mixture (also called a cocktail herein) of at least 2 bacteriophages, each bacteriophage encoding a different methyltransferase enzyme, such as 3, 4, 5 or even more bacteriophage, each encoding a different MTase. The invention also relates to a mixture (also called a cocktail herein) of first and second bacteriophages, wherein each bacteriophage encodes at least one methyltransferase and wherein the first bacteriophage encodes a methyltransferase that is not encoded by the second bacteriophage. For example, the first bacteriophage and second bacteriophage do not encode any MTase in common.

Data provided herein show that inclusion of a methyltransferase enzyme on a bacteriophage which is delivered to a target bacterial cell increases the efficiency of bacteriophage infection when compared to a bacteriophage lacking that same methyltransferase, in the context of a target cell in which that methyltransferase can protect against a host RM system.

The invention also relates to a method for delivery of a bacteriophage to a target bacterial cell, the method comprising contacting the target cell with a bacteriophage encoding a methyltransferase enzyme as disclosed herein, wherein the target bacteria comprises a restriction modification system that cleaves DNA which is unmethylated at a target site of methylation of the methyltransferase enzyme.

Data provided herein also show that that the expression of multiple MTases in a production strain can improve bacteriophage mediated infection of target bacteria with respect to an unmethylated control.

Advantageously, multiple MTases may be used to protect the plasmid, phagemid or phage nucleic acid from the action of different RM systems within a target bacterial cell. Thus, in an embodiment, the plasmid or phage nucleic acid encodes first and second MTases, wherein the MTases are MTases of different RM systems, e.g., RM systems found together in a single bacterial or archaeal species or strain.

Therefore, in a yet further aspect the invention relates to a method for methylation of a bacteriophage DNA or phagemid DNA or plasmid DNA, the method comprising: introducing into a production host bacterium, such as into a production host bacterium genome (i) a nucleotide sequence encoding a plurality of methyltransferases, or (ii) two or more nucleotide sequences together encoding a plurality of methyltransferases, thereby producing a modified production host bacterium expressing at least 2 introduced methyltransferases, suitably which are not found naturally occurring in the host bacterium cell; infecting the production host bacterium having an altered methylating activity with a bacteriophage particle comprising bacteriophage DNA or phagemid DNA, or transforming the bacterium with a plasmid comprising plasmid DNA, thereby methylating said bacteriophage DNA or phagemid DNA or plasmid DNA; and producing a bacteriophage particle comprising bacteriophage DNA or phagemid DNA, or producing plasmid DNA, having a methylation pattern produced by methyltransferases in the host bacterium, optionally further isolating the bacteriophage particle comprising bacteriophage DNA or phagemid DNA, or isolating the plasmid DNA or plasmid.

Optionally, the method further comprises formulating the isolated bacteriophage, plasmid or DNA with a pharmaceutically-acceptable diluent, carrier or excipient to produce a pharmaceutical composition.

The method for methylation of a bacteriophage DNA or phagemid DNA or plasmid DNA can also employ bacteriophage or phagemid nucleic acid or plasmid DNA as described above which itself encode an MTase, and this method therefore combines production strain methylation (delivered in trans) and methylation by an MTase or MTases encoded by the DNA that is introduced into the target bacteria (delivered in cis).

The invention also relates to a bacteriophage or phagemid or plasmid produced by the above method in the production strain.

Each MTase disclosed herein may be a Type 1, Type II or Type III MTase. In an example, at least one Type I MTase is used.

An MTase may be provided in cis and/or trans.

Where multiple MTases are provided, these may be provided in cis (e.g., on the conjugative plasmid or the phage or phagemid that is delivered to a target cell), or in trans (e.g., by a coresident non conjugative plasmid in a production strain cell or carrier cell), or both. In one aspect the MTase is an Eco0015 MTase, (a Type I MTase) which methylates within the recognition sequence CCANNNNNNNCTTC.

The invention also relates to any MTase that methylates within the recognition sequence CCANNNNNNNCTTC.

In one aspect the MTase is an EcoKl MTase, (type I) which methylates within the recognition AACNNNNNNGTGC.

The invention also relates to any MTase that methylates within the sequence AACNNNNNNGTGC.

Other methylases that may be encoded on the phage, conjugative plasmid or production strain include M. EcoAl, M. EcoAO831, M.EcoMII, M.EcoMIII, Ml M2 Eco31I, M.Ecol l67.

The invention also relates to any MTase that methylates within a sequence as follows. These sequences are, for example, associated with the MTase as listed below:

In particular, we have identified the prevalence of these sites as follows:

* recognition sequence with methylated A in bold and T corresponding to methylated A on complementary strand underlined (where not indicated the methylated bases have not been determined) ** prevalence in a collection of 932 E. coli clinical isolates in total

The most prevalent RM systems found in the genome sequence of the E. coli clinical isolates were assigned an identifier based on a minimum of 99.5% amino acid identity to sequences described in REBASE (http://rebase.neb.com/rebase/rebase.html)

In any aspect the methyltransferase may be a methyltransferase from a pathogenic bacterial strain or species.

In any aspect the methyltransferase may be a methyltransferase from a bacteria associated with bacterial corrosion or biofouling.

The methyltransferase may be a methyltransferase that protects against Type I, Type II or Type III RM systems, and optionally multiple systems, such as Type I and type II, type I and type III or type II and III, or all three.

The methyltransferase may be a methyltransferase that protects against a RM system found in any one of a Staphylococcus, Streptococcus, Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio or Clostridium bacteria. For example, the methyltransferase may be a methyltransferase that protects against a RM system found in E coli. For example, the methyltransferase may be a methyltransferase that protects against a RM system found in C difficile. For example, the methyltransferase may be a methyltransferase that protects against a RM system found in K pneumoniae. For example, the methyltransferase may be a methyltransferase that protects against a RM system found in P aeruginosa. For example, the methyltransferase may be a methyltransferase that protects against a RM system found in S typhimurium. For example, the methyltransferase may be a methyltransferase that protects against a RM system found in H pylori.

The MTase may be any MTAse that protects against an RM system found in a bacterial species disclosed in Table 1 of WO 2017/211753 Al, which list is specifically herein incorporated by reference.

The MTase may be an MTase from Enterobacteriaceae such as E. coli, Salmonella, Yersinia pestis, Klebsiella, Shigella, Proteus, Enterobacter, Serratia, and Citrobacter.

An MTase may be an orphan or promiscuous MTase.

Where more than one MTase is encoded it may be a combination of a promiscuous MTase and a non-promiscuous, specific, MTase. Bacteriophage referred to herein may be any suitable bacteriophage for targeting of a target cell that carries a RM system that cleaves unmethylated DNA, preferably DNA viruses.

Bacteriophage referred to herein may be any phage from a genus selected from Dhakavirus, Gaprivervirus, Gelderlandvirus, Jiaodavirus, Karamvirus, Krischvirus, Moonvirus, Mosigvirus, Schizotequatrovirus, Slopekvirus and Tequatrovirus. Each phage herein may be an enterobacteria phage, E coli phage, Myoviridae phage, Tevenvirinae phage, Tequatrovirus phage, Caudovirales phage, adeno-associated viruses (AAV), herpes simplex viruses, retroviruses or lentiviruses. For example, each virus or phage herein may be from a genus selected from Dhakavirus, Gaprivervirus, Gelderlandvirus, Jiaodavirus, Karamvirus, Krischvirus, Moonvirus, Mosigvirus, Schizotequatrovirus, Slopekvirus and Tequatrovirus. Each phage herein may be a Klebsiella phage (e.g., Klebsiella phage PMBT1, Klebsiella phage PKO111, Klebsiella phage phi KpNIH-6, Klebsiella phage Miro, Klebsiella phage vB_KpnM_KpV477, Klebsiella phage KPV15, Klebsiella phage vB_Kpn_F48, Klebsiella phage KPN5, Klebsiella phage KP27, Klebsiella phage KPI 5, Klebsiella phage KPI or Klebsiella phage JD18), Acinetobacter phage (e.g., Acinetobacter virus 133), Aeromonas phage (e.g., Aeromonas virus 65 or Aeromonas virus Aehl), Escherichia phage (e.g., Escherichia virus RB16, Escherichia virus RB32 or Escherichia virus RB43) or Pseudomonas phage (e.g., Pseudomonas virus 42). Each phage herein may be a Tevenvirinae phage, e.g., a phage selected from Table D. A phagemid herein may be a modified version of the genome of any phage disclosed in this paragraph.

Cocktails (mixtures) of 2 or more bacteriophage may be used in which each of the same type of bacteriophage encodes a different MTase enzyme needed for protection within a target host cell.

A cocktail or mixture herein may a phage population, wherein the population consists of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (or more than 10) different types of bacteriophage or phagemids, wherein each bacteriophage/phagemid is according to the invention.

Alternatively a bacteriophage cocktail may also comprise phage having a different bacteriophage backbone as well as a different MTase - so the cocktail for example could be bacteriophage Pl and M13, wherein the Pl encodes a MTase that is not encoded by the M13, wherein the M13 encodes a MTase that is (or is not) encoded by the Pl; or wherein the Ml 3 encodes a MTase that is not encoded by the Pl, wherein the Pl encodes a MTase that is (or is not) encoded by the M13.

In one embodiment bacteriophage are specific for a single strain or single species of bacteria.

Here, the skilled person will understand that “specific for” refers to the host infectivity profile of each phage. In another embodiment bacteriophage may be used which are capable of infecting two or more bacterial strains or two or more species of bacteria.

In one embodiment each bacteriophage, plasmid, or phagemid is for use in a human or animal in vivo, e.g., for use as a medicament.

In one embodiment each bacteriophage, plasmid, or phagemid is for use in an environment, e.g., for use in remediation of biofouling or microbial corrosion of a surface (e.g., a metal surface).

In one embodiment each bacteriophage, plasmid, or phagemid is used ex vivo or in vitro.

The phage as disclosed herein may be a Pl phage, a M13 phage, a A phage, a T4 phage, a PhiC2 phage, a PhiCD27 phage, a PhiNMl phage, a Bc4 31 v3 phage, a Phi 10 phage, a Phi25 phage, a Phi 151 phage, an A5 1 1 -like phage, a B054 phage, an 01761 -like phage, or a Campylobacter phage, optionally NCTC 12676 or NCTC 12677. The phage may be a lambda phage. The phage may be any phage from a genus selected from Dhakavirus, Gaprivervirus, Gelderlandvirus, Jiaodavirus, Karamvirus, Krischvirus, Moonvirus, Mosigvirus, Schizotequatrovirus, Slopekvirus and Tequatrovirus, such as those disclosed herein.

The phagemid as disclosed herein may be a Pl phagemid, a Ml 3 phagemid, a A phagemid, a T4 phagemid, a PhiC2 phagemid, a PhiCD27 phagemid, a PhiNMl phagemid, a Bc43 1 v3 phagemid, a PhilO phagemid, a Phi25 phagemid, a Phil51 phagemid, an A511 -like phagemid, a B054 phagemid, an 01761 -like phagemid, a Campylobacter phagemid, optionally NCTC12676 or NCTC12677. In an example, the phagemid is a lambda phagemid. The phagemid may be derived from any phage from a genus selected from Dhakavirus, Gaprivervirus, Gelderlandvirus, Jiaodavirus, Karamvirus, Krischvirus, Moonvirus, Mosigvirus, Schizotequatrovirus, Slopekvirus and Tequatrovirus, such as those disclosed herein.

A phagemid may encode a non-self-replicative transduction particle, as is familiar to the skilled person.

A conjugative plasmid as disclosed herein is a plasmid that is transferred by bacterial conjugation. Conjugative plasmids comprise at least an Origin of Transfer, oriT, and may comprise one or more or all of a set of transfer (tra) genes necessary for conjugation. These tra gene functions may be alternatively be provided in whole or in part by the carrier cell comprising the plasmid.

In a preferred embodiment all of the necessary tra genes needed for conjugation are comprised on the plasmid. The carrier bacterial cell comprising the conjugative plasmid may be any suitable bacteria, but is preferably a bacteria that is not pathogenic in humans or animals. The bacterial species may be any species disclosed herein, e.g., E coli or a Bacteriodies species. The carrier bacterial cell may be, for example, a Bacteriodes, Escherichia, Salmonella, Clostridium, Bifidobacterium, Lactobacillus, Pseudomonas, Caulobacter, Listeria, Proteus or Streptococcus cell.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), in combination with CRISPR-associated genes (cas) constitute the CRISPR-Cas system, which confers adaptive immunity in many bacteria and most archaea. CRISPR-mediated immunization occurs through the integration of DNA from invasive genetic elements such as plasmids and bacteriophages that can be used to prevent future infections by invaders containing the same sequence. CRISPR-Cas systems consist of CRISPR arrays of short DNA "repeats" interspaced by hypervariable "spacer" sequences and a set of flanking cas genes. In any aspect of the invention the nucleic acid that is delivered to the target cell can comprise a CRISPR array, suitably an array that comprises at least one spacer substantially complementary to a target DNA sequence in the target bacteria. In this way, the target DNA sequence of the target bacteria can be modified by the CRISPR-cas system using the spacer to specifically target the target sequence. The methylation of the DNA encoding the array - as well as any other components of the CRISPR-cas system that are also delivered to the target cell - are protected from restriction cleavage and therefore will function more efficiently within the target cell. The CRISPR array is also referred to herein as a target cell modifying CRISPR array.

The CRISPR array may be part of the bacteriophage nucleic acid, the phagemid nucleic acid or the conjugative plasmid.

The array suitably comprises a spacer sequence and repeats encoding a crRNA, the crRNA comprising a sequence that hybridises to a target cell DNA target to guide a Cas enzyme to the target.

The modification of the target gene sequence that results from the delivery of a CRISPR array into the target bacteria can include cleavage of one or both strands of the target cell DNA at the target sequence, causing inactivation of gene expression, directly or indirectly, or can result in killing of the cell.

In one aspect the CRISPR array is a nucleic acid molecule that comprises at least two repeat sequences, and at least one spacer sequence, wherein one of the two repeat sequences is linked to the 5' end of the spacer sequence and the other of the two repeat sequences is linked to the 3' end of the spacer sequence. The CRISPR array may comprise 2 or more repeat-spacer sequences, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more n repeat-spacer sequences, suitably wherein all spacers in said array are flanked on both the 5' end and the 3' end by a repeat sequence.

Other components of the CRISPR-cas system may be comprised within (encoded by) the bacteriophage nucleic acid, the phagemid nucleic acid or the conjugative plasmid, and these include one or more of

(i) a cas endonuclease,

(ii) a tracrRNA sequence or a DNA sequence expressing a tracrRNA sequence.

The cas endonuclease functionality may alternatively be provided naturally from within the target bacteria.

When delivered as a part of the bacteriophage, phagemid or plasmid, the cas endonuclease may be a type I, type II, type III, type IV, type V or type VI cas nuclease, such as cas3 or cas9 or cpfl.

The spacer sequence is complementary to a target DNA sequence in the target cell genome, and may be fully (100%) complementary or substantially complementary (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the target DNA. The spacer sequence can have one, two, three, four, or five mismatches as compared to the target DNA. The mismatches can be contiguous or noncontiguous. The spacer sequence may have complete complementarity or substantial complementarity over a region of a target nucleotide sequence that is at least about 20 nucleotides to about 100 nucleotides in length.

The carrier bacterial cell, bacteriophage, phagemid or plasmid disclosed herein preferably encodes an MTase and a payload, e.g., a CRISPR array. For example, the payload and the MTase are encoded on the same DNA.

The delivered nucleic acid may encode a guided nuclease (optionally a Cas) and/or a guide RNA and/or the delivered nucleic acid comprises a CRISPR array for producing a crRNA in the target cell. The guided nuclease may be a Cas nuclease (e.g., a Type I, II, III, IV, V or VI Cas nuclease, e.g., a Cas9, a Cas3, a Casl2, or a Casl3). The guided nuclease may be a TALEN, zinc finger nuclease or meganuclease.

The delivered nucleic acid may comprise or consist of from 1 to lOkb, e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2kb, of DNA. For example, the delivered nucleic acid comprises a CRISPR array (and/or a nucleotide sequence encoding a guide RNA, such as a single guide RNA) and optionally one or more nucleotide sequences which each encodes a respective Cas.

The delivered nucleic acid may encode a CRISPR Cascade protein (e.g., Cas A, B, C, D or E).

The delivered nucleic acid may encode a crRNA. The heterologous DNA may encode a single guide RNA (sgRNA). The heterologous DNA may encode a tracrRNA.

The following are preferred aspects of the components of the CRISPR-cas system:

The Cas may be any Cas (e.g., a Cas2, 3, 4, 5, or 6) of a Type I system. In an embodiment, the Cas may be fused or conjugated to a moiety that is operable to increase or reduce transcription of a gene comprising the target protospacer sequence. For example the nucleic acid encoding the Cas that is introduced into a cell may comprise a nucleotide sequence encoding the moiety, wherein the Cas and moiety are expressed in the host cell as a fusion protein. In one embodiment, the Cas is N-terminal of the moiety; in another embodiment it is C -terminal to the moiety.

Optionally, one or more Cascade proteins may be provided by or to a target cell. For example, a first Cas (Cl) and/or a second Cas (C2) may be provided and the Cascade protein(s) are cognate with the Cl or C2, which is a Cas3. For example, a first Cas (Cl) and/or a second Cas (C2) are provided and Casl or Cas2 is a Cas3 that is cognate with Cascade proteins encoded by the cell.

Optionally, the Cascade proteins comprise or consist of cas5 (casD, csy2), cas6 (cas6f, cse3, casE), cas7 (csc2, csy3, cse4, casC) and cas8 (casA, cas8al, cas8bl, cas8c, caslOd, cas8e, csel, cas8f, csyl).

Optionally delivered nucleic acid is provided which comprises a promoter and a Cas3-encoding or crRNA-encoding sequence that are spaced no more than 150, 100, 50, 40, 30, 20 or lObp apart, e.g., from 30-45, or 30-40, or 39 or around 39bp apart. Optionally herein a ribosome binding site and the Cas3 -encoding or crRNA-encoding sequence are spaced no more than 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 4 or 3bp apart, e.g., from 10-5, 6 or around 6bp apart.

In an example, a promoter herein is in combination with a Shine-Dalgamo sequence comprising the sequence 5’- aaagaggagaaa-3 ’ (SEQ ID NO: 1) or a ribosome binding site homologue thereof. Optionally the promoter has an Anderson Score (AS) of AS >0.5; or an Anderson Score (AS) of 0.5>AS >0.1; or an Anderson Score (AS) of <0.1.

Optionally, delivered nucleic acid is devoid of nucleotide sequence encoding one, more or all of a Casl, Cas2, Cas4, Cas6 (optionally Cas6f), Cas7 and Cas 8 (optionally Cas8f). Optionally, the DNA is devoid of a sequence encoding a Cas6 (optionally a Cas6f). Optionally, the DNA comprises (optionally in 5’ to 3’ direction) nucleotide sequence encoding one, more or all of Cast 1, Cas7 and Cas8al. Optionally, the DNA comprises nucleotide sequence encoding Cas3’ and/or Cas3”. In one embodiment, the DNA comprises nucleotide sequences (in 5’ to 3’ direction) that encode a Cas3 (e.g., Cas3’ and/or Cas3”), Casl l, Cas7 and Cas8al.

Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cast 1 sequence.

Optionally, the delivered nucleic acid comprises a Type IA CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with a Cas3. Thus, the array is operable in a host cell when the hybrid DNA has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the host cell, optionally thereby killing the cell. Similarly, single guide RNAs encoded by the delivered nucleic acid in one embodiment are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the host cell, optionally thereby killing the cell.

Optionally, a target cell comprises a Type IA CRISPR array that is cognate with the Cas3 (Cl or C2). Optionally, a target cell comprises an endogenous Type IB, C, U, D, E or F CRISPR/Cas system. Optionally, the delivered nucleic acid comprises (optionally in 5’ to 3’ direction) nucleotide sequence encoding one, more or all of Cas8bl, Cas7 and Cas5. In one embodiment, the delivered nucleic acid comprises nucleotide sequences (in 5’ to 3’ direction) that encode a Cas3, Cas8bl, Cas7 and Cas5. Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cas8bl sequence. Optionally, the delivered nucleic acid comprises a Type IB CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the delivered nucleic acid has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell. Similarly, single guide RNAs encoded by the delivered nucleic acid in one embodiment are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell.

Optionally, the target cell comprises a Type IB CRISPR array that is cognate with the Cas3.

Optionally, the target cell comprises an endogenous Type IA, C, U, D, E or F CRISPR/Cas system. Optionally, the delivered nucleic acid comprises (optionally in 5’ to 3’ direction) nucleotide sequence encoding one, more or all of Cas5, Cas8c and Cas7. In one embodiment, the delivered nucleic acid comprises nucleotide sequences (in 5’ to 3’ direction) that encode a Cas3, Cas5, Cas8c and Cas7. Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cas5 sequence. Optionally, the delivered nucleic acid comprises a Type IC CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the delivered nucleic acid has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell. Similarly, the single guide RNAs encoded by the delivered nucleic acid in one embodiment are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell.

Optionally, the target cell comprises a Type IC CRISPR array that is cognate with the Cas3. Optionally, the target cell comprises an endogenous Type IA, B, U, D, E or F CRISPR/Cas system. Optionally, the delivered nucleic acid comprises (optionally in 5’ to 3’ direction) nucleotide sequence encoding one, more or all of Cas8U2, Cas7, Cas5 and Cas6. In one embodiment, the delivered nucleic acid comprises nucleotide sequences (in 5’ to 3’ direction) that encode a Cas3, Cas8U2, Cas7, Cas5 and Cas6. Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cas8U2 sequence.

Optionally, the delivered nucleic acid comprises a Type IU CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the delivered nucleic acid has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell. Similarly, the single guide RNAs encoded by the vector in one embodiment are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell.

Optionally, the target cell comprises a Type IU CRISPR array that is cognate with the Cas3. Optionally, the host cell comprises an endogenous Type IA, B, C, D, E or F CRISPR/Cas system. Optionally, the vector comprises (optionally in 5’ to 3’ direction) nucleotide sequence encoding one, more or all of CaslOd, Cas7 and Cas5. Optionally, the delivered nucleic acid comprises a nucleotide sequence encoding Cas3’ and/or Cas3”. In one embodiment, the delivered nucleic acid comprises nucleotide sequences (in 5’ to 3’ direction) that encode a Cas3, CaslOd, Cas7 and Cas5. Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the CaslOd sequence. Optionally, the delivered nucleic acid comprises a Type ID CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a cell when the vector has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell. Similarly, the single guide RNAs encoded by the delivered nucleic acid in one embodiment are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell.

Optionally, the target cell comprises a Type ID CRISPR array that is cognate with the Cas3.

Optionally, the target cell comprises an endogenous Type IA, B, C, U, E or F CRISPR/Cas system.

Optionally, the delivered nucleic acid comprises (optionally in 5’ to 3’ direction) nucleotide sequence encoding one, more or all of Cas8e, Cast 1, Cas7, Cas5 and Cas6. In one embodiment, the delivered nucleic acid comprises nucleotide sequences (in 5’ to 3’ direction) that encode a Cas3, Cas8e, Cast 1, Cas7, Cas5 and Cas6. Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cast 1 sequence. Optionally, the delivered nucleic acid comprises a Type IE CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the vector has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell. Similarly, the single guide RNAs encoded by the delivered nucleic acid in one embodiment are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell.

Optionally, the target cell comprises a Type IE CRISPR array that is cognate with the Cas3.

Optionally, the target cell comprises an endogenous Type IA, B, C, D, U or F CRISPR/Cas system.

Optionally, the delivered nucleic acid comprises (optionally in 5’ to 3’ direction) nucleotide sequence encoding one, more or all of Cas8f, Cas5, Cas7 and Cas6f. In one embodiment, the delivered nucleic acid comprises nucleotide sequences (in 5’ to 3’ direction) that encode a Cas3, Cas8f, Cas5, Cas7 and Cas6f. Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cas8f sequence. Optionally, the delivered nucleic acid comprises a Type IF CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a cell when the vector has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell. Similarly, the single guide RNAs encoded by the delivered nucleic acid in one embodiment are operable with the Cas and Cascade proteins to target and modify (e.g., cut) a target nucleotide sequence in the cell, optionally thereby killing the cell.

Optionally, the cell comprises a Type IF CRISPR array that is cognate with the Cas3

Optionally, the cell comprises an endogenous Type IA, B, C, D, U or E CRISPR/Cas system.

Optionally, the Cas and Cascade are Type IA Cas and Cascade proteins.

Optionally, the Cas and Cascade are Type IB Cas and Cascade proteins.

Optionally, the Cas and Cascade are Type IC Cas and Cascade proteins.

Optionally, the Cas and Cascade are Type ID Cas and Cascade proteins.

Optionally, the Cas and Cascade are Type IE Cas and Cascade proteins.

Optionally, the Cas and Cascade are Type IF Cas and Cascade proteins.

Optionally, the Cas and Cascade are Type IU Cas and Cascade proteins.

Optionally, the Cas and Cascade are E coli (optionally Type IE or IF) Cas and Cascade proteins, optionally wherein the E coli is ESBL-producing E. coli or E. coli ST 13 l-O25b:H4.

Optionally, the Cas and Cascade are Clostridium (e.g., C difficile) Cas and Cascade proteins, optionally C difficile resistant to one or more antibiotics selected from aminoglycosides, lincomycin, tetracyclines, erythromycin, clindamycin, penicillins, cephalosporins and fluoroquinolones.

Optionally, the Cas and Cascade are Pseudomonas aeruginosa Cas and Cascade proteins, optionally P aeruginosa resistant to one or more antibiotics selected from carbapenems, aminoglycosides, cefepime, ceftazidime, fluoroquinolones, piperacillin and tazobactam.

Optionally, the Cas and Cascade are Klebsiella pneumoniae (e.g., carbapenem-resistant Klebsiella pneumoniae or Extended-Spectrum Beta-Lactamase (ESBL)-producing K pneumoniae) Cas and Cascade proteins. Optionally, the Cas and Cascade are E coli, C difficile, P aeruginosa, K pneumoniae, P furiosus or B halodurans Cas and Cascade proteins.

Optionally, each crRNAs or gRNAs comprises a spacer sequence that is capable of hybridising to a protospacer nucleotide sequence of the cell, wherein the protospacer sequence is adjacent a PAM, the PAM being cognate to the Cl or C2, wherein Cl or C2 is a Cas nuclease, e.g., a Cas3. Thus, the spacer hybridises to the protospacer to guide the Cas3 to the protospacer. Optionally, the Cas3 cuts the protospacer, e.g., using exo- and/or endonuclease activity of the Cas3.

Optionally, the Cas3 removes a plurality (e.g., at least 2, 3,4, 5, 6, 7, 8, 9 or 10) nucleotides from the protospacer.

The invention also relates to pharmaceutical compositions comprising the bacteriophage, phagemid, or carrier cells as disclosed herein in combination with a pharmaceutically acceptable diluent, carrier or excipient. Suitable carriers and excipients are well known and include suitably sterile water, saline and buffered saline. In certain aspects, where there is delivery to the GI tract, the delivered carrier cell or phage is suitably protected from acid by an enteric coating, or other coating that can prevents adverse effect on the cell or phage in the gastric environment such as the stomach.

Any composition (e.g., a pharmaceutical composition) herein may be comprised by a sterile container or medical container, e.g., a syringe, IV bag, autoinjector pen or a vial.

Bacteriophage where used may be lyophilised prior to delivery and then reconstituted before use with a suitable diluent.

The delivery of the bacteriophage, phagemid or carrier cell comprising a conjugative plasmid as described herein may form a part of a method of prevention or treatment of disease of a human or animal, optionally in combination with CRISPR-CAS targeted modification of a target sequence within the target bacteria.

In one embodiment the modification at the target DNA of the target bacteria results in killing or attenuation of the target bacteria. Where the target bacteria is pathogenic then the modification provides prevention or treatment of pathogenic bacterial disease directly by said killing or attenuation of the pathogenic bacteria.

Therefore the invention relates to a method of prevention or treatment of disease caused by pathogenic target bacteria in a human or animal, the method comprising delivery of a bacteriophage, phagemid or carrier bacterial cell comprising a conjugative plasmid as disclosed herein, or pharmaceutical composition as disclosed herein, to a human or animal in need thereof, and a bacteriophage, phagemid or carrier bacterial cell according as disclosed herein, or a pharmaceutical composition as disclosed herein, for use in a method of prevention or treatment of disease caused by a pathogenic target bacteria in a human or animal, the method comprising delivery of the bacteriophage, phagemid or carrier bacterial cell or pharmaceutical composition to the human or animal in need thereof.

The modification may also provide indirect treatment or prevention of disease, in the case where bacteria present in a host are negatively affecting the host response to diseases other than bacterial disease. For example, patient responses to cancer treatments have been shown to be impacted by their microbiome make up, see Vancheswaran Gopalakrishnan et al, Cancer Cell. 2018 Apr 9; 33(4): 570-580.

Therefore, the present invention also relates to a method of prevention or treatment of disease, the method comprising delivery of a bacteriophage, phagemid or carrier bacterial cell comprising a conjugative plasmid as disclosed herein, or pharmaceutical composition disclosed herein, to a human or animal in need thereof, wherein the human or animal comprises target bacteria having a target sequence the modification of which results in improved responses to cancer or cancer immunotherapy.

For example, the target bacteria may be Salmonella, Clostridium, Bifidobacterium, Lactobacillus, Escherichia, Pseudomonas, Caulobacter, Listeria, Proteus, and Streptococcus.

The target bacteria may be any bacteria listed in Table 1 of WO 2017/211753 Al, which list is specifically herein incorporated by reference.

The bacteriophage, phagemid and carrier cells and pharmaceutical compositions disclosed herein may also be used in methods in combination with other medical treatments, including, but not limited to

(i) anti cancer agents,

(ii) antibiotics and

(iii) bacterial cells that are used to repopulate the human or animal microbiota.

Examples of suitable medicaments are provided in Table E.

Delivery of bacteriophage, phagemid and carrier cells and pharmaceutical compositions as disclosed may be by any suitable delivery route, such as intravenous, intradermal, subcutaneous, intramuscular, topical, by inhalation or intranasal delivery, or oral delivery.

The present invention may be used for treatment of diseases or conditions such as the following:

(a) A neurodegenerative disease or condition;

(b) A brain disease or condition;

(c) A CNS disease or condition; (d) Memory loss or impairment;

(e) A heart or cardiovascular disease or condition, e.g., heart attack, stroke or atrial fibrillation;

(f) A liver disease or condition;

(g) A kidney disease or condition, e.g., chronic kidney disease (CKD);

(h) A pancreas disease or condition;

(i) A lung disease or condition, e.g., cystic fibrosis or COPD;

(j) A gastrointestinal disease or condition;

(k) A throat or oral cavity disease or condition;

(l) An ocular disease or condition;

(m) A genital disease or condition, e.g., a vaginal, labial, penile or scrotal disease or condition;

(n) A sexually-transmissible disease or condition, e.g., gonorrhea, HIV infection, syphilis or Chlamydia infection;

(o) An ear disease or condition;

(p) A skin disease or condition;

(q) A heart disease or condition;

(r) A nasal disease or condition

(s) A haematological disease or condition, e.g., anaemia, e.g., anaemia of chronic disease or cancer;

(t) A viral infection;

(u) A pathogenic bacterial infection;

(v) A cancer;

(w) An autoimmune disease or condition, e.g., SLE;

(x) An inflammatory disease or condition, e.g., rheumatoid arthritis, psoriasis, eczema, asthma, ulcerative colitis, colitis, Crohn’s disease or IBD;

(y) Autism;

(z) ADHD;

(aa) Bipolar disorder;

(bb) ALS [Amyotrophic Lateral Sclerosis];

(cc) Osteoarthritis;

(dd) A congenital or development defect or condition;

(ee) Miscarriage;

(ff) A blood clotting condition;

(gg) Bronchitis; (hh) Dry or wet AMD;

(ii) Neovascularisation (e.g., of a tumour or in the eye);

(jj) Common cold;

(kk) Epilepsy;

(ii) Fibrosis, e.g., liver or lung fibrosis;

(mm) A fungal disease or condition, e.g., thrush;

(nn) A metabolic disease or condition, e.g., obesity, anorexia, diabetes, Type I or Type II diabetes.

(oo) Ulcer(s), e.g., gastric ulceration or skin ulceration;

(pp) Dry skin;

(qq) Sjogren’s syndrome;

(rr) Cytokine storm;

(ss) Deafness, hearing loss or impairment;

(tt) Slow or fast metabolism (ie, slower or faster than average for the weight, sex and age of the subject);

(uu) Conception disorder, e.g., infertility or low fertility;

(vv) Jaundice;

(ww) Skin rash;

(xx) Kawasaki Disease;

(yy) Lyme Disease;

(zz) An allergy, e.g., a nut, grass, pollen, dust mite, cat or dog fur or dander allergy;

(aaa) Malaria, typhoid fever, tuberculosis or cholera;

(bbb) Depression;

(ccc) Mental retardation;

(ddd) Microcephaly;

(eee) Malnutrition;

(fff) Conjunctivitis;

(ggg) Pneumonia;

(hhh) Pulmonary embolism;

(iii) Pulmonary hypertension;

(jjj) A bone disorder;

(kkk) Sepsis or septic shock;

(111) Sinusitus;

(mmm)Stress (e.g., occupational stress);

(nnn) Thalassaemia, anaemia, von Willebrand Disease, or haemophilia; (poo) Shingles or cold sore;

(ppp) Menstruation;

(qqq) Low sperm count.

NEURODEGENERATIVE OR CNS DISEASES OR CONDITIONS FOR TREATMENT OR PREVENTION

In an example, a neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer disease, geriopsychosis, Down syndrome, Parkinson's disease, Creutzfeldt-jakob disease, diabetic neuropathy, Parkinson syndrome, Huntington's disease, Machado- Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt Creutzfeldt- Jakob disease. For example, the disease is Alzheimer disease. For example, the disease is Parkinson syndrome.

In an example, wherein the method of the invention is practised on a human or animal subject for treating a CNS or neurodegenerative disease or condition, the method causes downregulation of Treg cells in the subject, thereby promoting entry of systemic monocyte-derived macrophages and/or Treg cells across the choroid plexus into the brain of the subject, whereby the disease or condition (e.g., Alzheimer’s disease) is treated, prevented or progression thereof is reduced. In an embodiment the method causes an increase of IFN-gamma in the CNS system (e.g., in the brain and/or CSF) of the subject. In an example, the method restores nerve fibre and//or reduces the progression of nerve fibre damage. In an example, the method restores nerve myelin and//or reduces the progression of nerve myelin damage. In an example, the method of the invention treats or prevents a disease or condition disclosed in WO2015136541 and/or the method can be used with any method disclosed in WO2015136541 (the disclosure of this document is incorporated by reference herein in its entirety, e.g., for providing disclosure of such methods, diseases, conditions and potential therapeutic agents that can be administered to the subject for effecting treatment and/or prevention of CNS and neurodegenerative diseases and conditions, e.g., agents such as immune checkpoint inhibitors, e.g., anti-PD-1, anti-PD-Ll, anti-TIM3 or other antibodies disclosed therein).

CANCERS FOR TREATMENT OR PREVENTION

Cancers that may be treated include tumours that are not vascularized, or not substantially vascularized, as well as vascularized tumours. The cancers may comprise non-solid tumours (such as haematological tumours, for example, leukaemias and lymphomas) or may comprise solid tumours. Types of cancers to be treated with the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukaemia or lymphoid malignancies, benign and malignant tumours, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumours/cancers and paediatric tumours/cancers are also included.

Haematol ogic cancers are cancers of the blood or bone marrow. Examples of haematological (or haematogenous) cancers include leukaemias, including acute leukaemias (such as acute lymphocytic leukaemia, acute myelocytic leukaemia, acute myelogenous leukaemia and myeloblasts, promyeiocytic, myelomonocytic, monocytic and erythroleukaemia), chronic leukaemias (such as chronic myelocytic (granulocytic) leukaemia, chronic myelogenous leukaemia, and chronic lymphocytic leukaemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukaemia and myelodysplasia.

Solid tumours are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumours can be benign or malignant. Different types of solid tumours are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumours, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous eel! carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumour, cervical cancer, testicular tumour, seminoma, bladder carcinoma, melanoma, and CNS tumours (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

AUTOIMMUNE DISEASES FOR TREATMENT OR PREVENTION

Acute Disseminated Encephalomyelitis (ADEM) Acute necrotizing hemorrhagic leukoencephalitis Addison’s disease Agammaglobulinemia

Alopecia areata

Amyloidosis

Ankylosing spondylitis

Anti-GBM/Anti-TBM nephritis

Antiphospholipid syndrome (APS)

Autoimmune angioedema

Autoimmune aplastic anemia

Autoimmune dysautonomia

Autoimmune hepatitis

Autoimmune hyperlipidemia

Autoimmune immunodeficiency

Autoimmune inner ear disease (AIED)

Autoimmune myocarditis

Autoimmune oophoritis

Autoimmune pancreatitis

Autoimmune retinopathy

Autoimmune thrombocytopenic purpura (ATP)

Autoimmune thyroid disease

Autoimmune urticaria

Axonal & neuronal neuropathies

Balo disease

Behcet’s disease

Bullous pemphigoid

Cardiomyopathy

Castleman disease

Celiac disease

Chagas disease

Chronic fatigue syndrome

Chronic inflammatory demyelinating polyneuropathy (CIDP)

Chronic recurrent multifocal ostomyelitis (CRMO)

Churg-Strauss syndrome

Cicatricial pemphigoid/benign mucosal pemphigoid

Crohn’s disease

Cogans syndrome • Cold agglutinin disease

• Congenital heart block

• Coxsackie myocarditis

• CREST disease

• Essential mixed cryoglobulinemia

• Demyelinating neuropathies

• Dermatitis herpetiformis

• Dermatomyositis

• Devic’s disease (neuromyelitis optica)

• Discoid lupus

• Dressier’s syndrome

• Endometriosis

• Eosinophilic esophagitis

• Eosinophilic fasciitis

• Erythema nodosum

• Experimental allergic encephalomyelitis

• Evans syndrome

• Fibromyalgia

• Fibrosing alveolitis

• Giant cell arteritis (temporal arteritis)

• Giant cell myocarditis

• Glomerulonephritis

• Goodpasture’s syndrome

• Granulomatosis with Polyangiitis (GPA) (formerly called Wegener’s

Granulomatosis)

• Graves’ disease

• Guillain-Barre syndrome

• Hashimoto’s encephalitis

• Hashimoto’s thyroiditis

• Hemolytic anemia

• Henoch-Schonlein purpura

• Herpes gestationis

• Hypogammaglobulinemia

• Idiopathic thrombocytopenic purpura (ITP)

• IgA nephropathy • IgG4-related sclerosing disease

• Immunoregulatory lipoproteins

• Inclusion body myositis

• Interstitial cystitis

• Juvenile arthritis

• Juvenile diabetes (Type 1 diabetes)

• Juvenile myositis

• Kawasaki syndrome

• Lambert-Eaton syndrome

• Leukocytoclastic vasculitis

• Lichen planus

• Lichen sclerosus

• Ligneous conjunctivitis

• Linear IgA disease (LAD)

• Lupus (SLE)

• Lyme disease, chronic

• Meniere’s disease

• Microscopic polyangiitis

• Mixed connective tissue disease (MCTD)

• Mooren’s ulcer

• Mucha-Habermann disease

• Multiple sclerosis

• Myasthenia gravis

• Myositis

• Narcolepsy

• Neuromyelitis optica (Devic’s)

• Neutropenia

• Ocular cicatricial pemphigoid

• Optic neuritis

• Palindromic rheumatism

• PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus)

• Paraneoplastic cerebellar degeneration

• Paroxysmal nocturnal hemoglobinuria (PNH)

• Parry Romberg syndrome Parsonnage-Tumer syndrome

Pars planitis (peripheral uveitis)

Pemphigus

Peripheral neuropathy

Perivenous encephalomyelitis

Pernicious anemia

POEMS syndrome

Polyarteritis nodosa

Type E IE & III autoimmune polyglandular syndromes

Polymyalgia rheumatica

Polymyositis

Postmyocardial infarction syndrome

Postpericardiotomy syndrome

Progesterone dermatitis

Primary biliary cirrhosis

Primary sclerosing cholangitis

Psoriasis

Psoriatic arthritis

Idiopathic pulmonary fibrosis

Pyoderma gangrenosum

Pure red cell aplasia

Raynauds phenomenon

Reactive Arthritis

Reflex sympathetic dystrophy

Reiter’ s syndrome

Relapsing polychondritis

Restless legs syndrome

Retroperitoneal fibrosis

Rheumatic fever

Rheumatoid arthritis

Sarcoidosis

Schmidt syndrome

Scleritis

Scleroderma

Sjogren’s syndrome • Sperm & testicular autoimmunity

• Stiff person syndrome

• Subacute bacterial endocarditis (SBE)

• Susac’s syndrome

• Sympathetic ophthalmia

• Takayasu’s arteritis

• Temporal arteritis/Giant cell arteritis

• Thrombocytopenic purpura (TTP)

• Tolosa-Hunt syndrome

• Transverse myelitis

• Type 1 diabetes

• Ulcerative colitis

• Undifferentiated connective tissue disease (UCTD)

• Uveitis

• Vasculitis

• Vesiculobullous dermatosis

• Vitiligo

• Wegener’s granulomatosis (now termed Granulomatosis with Polyangiitis (GPA).

INFLAMMATORY DISEASES FOR TREATMENT OR PREVENTION

• Alzheimer's

• ankylosing spondylitis

• arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis)

• asthma

• atherosclerosis

• Crohn's disease

• colitis

• dermatitis

• diverticulitis

• fibromyalgia

• hepatitis

• irritable bowel syndrome (IBS)

• systemic lupus erythematous (SLE)

• nephritis Parkinson's disease ulcerative colitis.

The improved delivery possible using bacteria carrier bacterial cell, bacteriophage and phagemid as disclosed herein also has a wide range of non-medical applications, such as the treatment of bacteria associated with microbial corrosion or biofouling of a substrate or fluid in an industrial or a domestic system.

Therefore the bacteriophage, phagemid, and carrier cells of the invention as disclosed herein may be in the form of a composition which is a medical, opthalmic, dental or pharmaceutical composition. In an example, the composition is a an antimicrobial composition, e.g., an antibiotic or antiviral, e.g., a medicine, disinfectant or mouthwash. In an example, the composition is a cosmetic composition (e.g., face or body make-up composition). In an example, the composition is a herbicide. In an example, the composition is a pesticide (e.g., when the target bacteria is a Bacillus (e.g., thuringiensis)). In an example, the composition is a beverage (e.g., beer, wine or alcoholic beverage) additive. In an example, the composition is a food additive (e.g., where the target bacteria is an E coli, Salmonella, Listeria or Clostridium (e.g., botulinum)). In an example, the composition is a water additive. In an example, the composition is a additive for aquatic animal environments (e.g., in a fish tank). In an example, the composition is an oil or petrochemical industry composition or comprised in such a composition (e.g., when the target bacteria is a sulphate-reducing bacterium, e.g., a Desulfovibrio). In an example, the composition is a oil or petrochemical additive. In an example, the composition is a chemical additive. In an example, the composition is a disinfectant (e.g., for sterilizing equipment for human or animal use, e.g., for surgical or medical use, or for baby feeding). In an example, the composition is a personal hygiene composition for human or animal use. In an example, the composition is a composition for environmental use, e.g., for soil treatment or environmental decontamination (e.g., from sewage, or from oil, a petrochemical or a chemical, e.g., when the target bacteria is a sulphate-reducing bacterium, e.g., a Desulfovibrio). In an example, the composition is a plant growth stimulator. In an example, the composition is a composition for use in oil, petrochemical, metal or mineral extraction. In an example, the composition is a fabric treatment or additive. In an example, the composition is an animal hide, leather or suede treatment or additive. In an example, the composition is a dye additive. In an example, the composition is a beverage (e.g., beer or wine) brewing or fermentation additive (e.g., when the target bacteria is a Lactobacillus). In an example, the composition is a paper additive. In an example, the composition is an ink additive. In an example, the composition is a glue additive. In an example, the composition is an anti-human or animal or plant parasitic composition. In an example, the composition is an air additive (e.g., for air in or produced by air conditioning equipment, e.g., where the target bacteria is a Legionella). In an example, the composition is an anti-freeze additive (e.g., where the target bacteria is a Legionella). In an example, the composition is an eyewash or opthalmic composition (e.g., a contact lens fluid). In an example, the composition is comprised by a dairy food (e.g., the composition is in or is a milk or milk product; e.g., wherein the target bacteria is a Lactobacillus, Streptococcus, Lactococcus or Listeria). In an example, the composition is or is comprised by a domestic or industrial cleaning product (e.g., where the target bacteria is an E coli, Salmonella, Listeria or Clostridium (e.g., botulinum)). In an example, the composition is comprised by a fuel. In an example, the composition is comprised by a solvent (e.g., other than water). In an example, the composition is a baking additive (e.g., a food baking additive). In an example, the composition is a laboratory reagent (e.g., for use in biotechnology or recombinant DNA or RNA technology). In an example, the composition is comprised by a fibre retting agent. In an example, the composition is for use in a vitamin synthesis process. In an example, the composition is an anti-crop or plant spoiling composition (e.g., when the target bacteria is a saprotrophic bacterium). In an example, the composition is an anticorrosion compound, e.g., for preventing or reducing metal corrosion (e.g., when the target bacteria is a sulphate reducing bacterium, e.g., a Desulfovibrio, e.g. for use in reducing or preventing corrosion of oil extraction, treatment or containment equipment; metal extraction, treatment or containment equipment; or mineral extraction, treatment or containment equipment). In an example, the composition is an agricultural or farming composition or comprised in such a composition. In an example, the composition is a silage additive.

Any aspect of the present invention is for an industrial or domestic use, or is used in a method for such use. For example, it is for or used in agriculture, oil or petroleum industry, food or drink industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotechnology industry, medical industry, healthcare industry, dentistry industry, energy industry, consumer products industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishing industry, leisure industry, recycling industry, cosmetics industry, plastics industry, pulp or paper industry, textile industry, clothing industry, leather or suede or animal hide industry, tobacco industry or steel industry.

These different applications imply that the carrier bacterial cell comprising conjugative plasmids and or bacteriophage used are able to deliver the MTase to bacteria associated with biofouling or microbial corrosion in these industries. Indeed, it will be seen that the invention finds an application in any method which achieves an effect through the specific targeted modification (e.g. killing) of one target bacterial cell in the context of another, such that the relative population of the targeted bacteria and another bacteria is altered. This may have medical and non-medical applications.

All such applications all equally benefit from the combination of the use of MTases as disclosed herein in combination with components of the CRISPR system as described above, to modify the DNA of the target bacteria, for example resulting in target cell killing.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or."

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The term "or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

Any part of this disclosure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Examples

The present invention is described in more detail in the following non limiting Examples.

Methods and materials

Plasmids

All plasmids referred to in this document are described in Table A.

DNA fragments containing ORFs encoding the MTase activities of type I RM systems (M and S subunits) were amplified by PCR from appropriate E. coll strains. For conjugation experiments, the DNA fragments encoding the type I MTases M.Eco0015 and M.EcoKI were cloned into the small conjugative plasmid pl 114 which contains the mobilization and origin of replication elements from pBBRl (Antoine and Locht. Mol. Microbiol. 6(13): 1785-1799 (1992), or into the large conjugative plasmid pCollB9^SL1344 of the Incll incompatibility group (Kroger et al. PNAS. 109(20):E1277-E1286 (2012). In both cases the MTases are encoded on the DNA transferred to recipient cells and so the methylation of this DNA is referred to as “in cis”. For the pl 114 based constructs, a pair of each of the type I RM recognition sites separated by about 850 to 1350 bp was cloned in to provide a substrate for the RM systems being tested.

The MTase ORFs were also cloned into the non-conjugative plasmid pl45 which has a compatible marker (tetracycline resistance (Tet)) and origin of replication (pSClOl) to co-reside in the same cells as the small conjugative plasmid pl 114 (lacking any encoded MTAse). The subsequent methylation of pl 114 by the methylase activity encoded on a co-residing plasmid is referred to as “in trans”. For each of the MTase encoding plasmids, corresponding negative control plasmids were created in which stop codons were introduced into the orfs to abolish MTase activity without altering the rest of the DNA sequence. Chloramphenicol resistance (Cm) was encoded on the transferred DNA for selection of transconjugants while the expression from the MTase ORFs was under the control of the arabinose promoter.

To methylate DNA packaged into a P4 transducing phage particle, seven type I MTases (ORFs of the M and S subunits, which together provide the methylation activity ) were cloned into pl 114 to create the operon construct pl 160. The order of MTases in the operon was EcoMII, EcoAI, EcoKI, EcoAO83I, EcoMIII, Eco0015 and Ecol 167, with an arabinose promoter cloned 5’ of the EcoMII sequence and 5’ of the Eco0015 sequence. For packaging into P4 particles, the plasmid pl 065 was constructed to contain a P4 packaging signal, spectinomycin resistance marker (Spec), the sGFP ORF under control of the BBa_J23100 promoter (Andersen promoter collection) and a pair of each of the type I RM recognition sites (as above) separated by about 850 to 1350 bp. The backbone of this plasmid is based on pCasens3 (Uribe et al. Cell Host & Microbe. 25:233-241 (2019)).

For expression of the Eco0015 RM system to be used in lambda phage spot assays, the plasmid pl 331 was constructed by cloning a PCR fragment containing the hsdR-hsdM-hsdS ORFs of Eco0015 into the plasmid pl45. For weak constitutive expression, the promoter pMO is located 5’ of the M subunit ORF.

Bacterial strains and their cultivation

All strains were grown in lysogeny broth (LB) at 37°C with addition of antibiotics, supplements or with culture conditions as indicated for experiments.

All strains used for molecular biology procedures, including conjugation and preparation of phage lysates are listed in Table B.

All strains used for testing conjugation and phage lysis or transduction efficiency are listed in Table C.

For conjugation experiments the E. coli strain JKE201 (Harms et al. PLos Genet. 13(10): el007077(2017)) was transformed with the conjugation plasmids and used as a donor. This strain contains the RP4 conjugation system on its chromosome and is engineered auxotroph for diaminopimelic acid (DAP), allowing counter selection of transconjugants from donor cells.

For testing benefits of methylation on conjugation and P4 phage transduction, a panel of E. coli clinical isolates harboring type I RM systems, particularly EcoKI or Eco0015 RM systems, were selected as recipient strains. An A. coli C-l wildtype strain (CGSCG Strain #: 3121), referred to as b52, was selected as a comparison strain and has no active type I or type II restriction system.

The efficiency of plaquing (eop, efficiency of plating) of lambda phages were tested by spot assays on several strains. To test for eop against the EcoKI restriction system the strain MG1655 was used. To test for eop against the Eco0015 system, the strain NEB 10-beta was transformed with p!331 (harboring the hsdR-hsdM-hsdS system of Eco0015 under constitutive expression); and to test eop against both EcoKI and Eco0015 in the same strain, MG1655 was transformed with pl331.

The strain b587 was the production strain for P4 packaged DNA.

Conjugation conditions For conjugation experiments, cultures of donor strains were grown in LB supplemented with chloramphenicol (Cm 20 ug ml'¹), DAP (100 uM) and arabinose (0.2% w/v). Overnight cultures of the donor strains were washed in PBS and resuspended in a volume of LB media equal to that of the overnight culture while recipient strains were grown overnight in LB without any supplements and cultures used directly. In a 96-well microtiter plate, 50 ul of donor strain and 10 ul of recipient strain were mixed in 140 ul of LB media, supplemented with DAP and arabinose. The conjugation reactions were left to incubate stationary at 37°C for 18 h. Transconjugant cultures were plated as 10-fold dilutions in PBS on arabinose and Cm supplemented LB agar plates and incubated for 18h at 37 degrees C. Donor cells do not grow as they require DAP, while transconjugant recipients, harboring the Cm gene on donor plasmid, are able to grow and were enumerated as cfu ml'¹ of the transconjugant mix.

Phage lambda engineering

Synthetic phage lambda genomes were designed to be assembled from PCR fragments using commercially available lambda DNA (NEB Cat# N3011 S) as a template for PCR. The genes c/, rexA, and rexB were excluded from the assembly designs and thus subsequent rebooted phage lambda is virulent. The amber mutation Sam7 (45,352 G->A), a mutation in a gene involved in cell lysis that is present in the commercial DNA template, was reverted to wildtype sequence (A- >G). Three genome designs were made, that as described above without any additional sequence is referred to as Design I (see Appendix I for genome sequence). Design II includes the EcoKI hsdM and hsdS ORFs under control of the Anderson promoter pJ23100, which was amplified by PCR from pl 172 and assembled in place of the deleted lambda genes (see Appendix II for genome sequence). Likewise Design III includes the Eco0015 hsdM and hsdS ORFs, which were amplified by PCR from pl 174, but unlike Design II expression is under control of the native lambda sequence and assembled in place of the deleted lambda genes (see Appendix III for genome sequence).

The high-fidelity DNA polymerase Platinum™ SuperFi™ (Invitrogen) was use in all PCR reactions. PCR primers were designed to incorporate Bsal sites to facilitate seamless Golden Gate assembly of PCR fragments. Assembled phage genomes were used to transform E. coli NEB 10-beta cells by electroporation. The transformation was mixed with 3ml of LB top agar (0.4 % w/v agar) containing lOOul of NEB 10-beta overnight culture and supplemented with 5 mM CaCh and 10 mM MgCh. After overnight incubation of plates at 37°C, single plaques were isolated and propagated on relevant production strains to create lysates. Lambda phage propagation and titer determination

For unmethylated lambda phage DNA the phage was grown on NEB 10-beta, for methylation in trans by the EcoKI MTase phage were grown on MG1655, for methylation in trans by Eco0015 MTase phage were grown on NEB-10 beta harboring the plasmid p 1331, for methylation in trans by both EcoKI and Eco0015 MTases phage was grown on MG1655 harboring p 1331, and for production of lambda phage with the genome designs II and III (for cis methylation) the phage was propagated on NEB 10-beta. Strains were grown to early exponential phase at 37°C in LB media supplemented with 5 mM CaCh and 10 mM MgCh before phage addition. After overnight incubation, cultures were spun at 10,000 g for 5 min and the supernatant filtered through a 0.2 pm filter. The titers of these resulting lysates were determined by titration on E. coll NEB 10-beta bacterial overlays. Overlays were created using 3 ml of LB top agar (0.4 % agar) containing lOOul of NEB 10-beta overnight culture and supplemented with 5 mM CaCh and 10 mM MgCh. Lysates were serially diluted 10-fold in SM buffer (50 mM Tris-HCl (pH 7.5), 8 mM MgSO4, 100 mM NaCl), spotted on the bacterial overlay, and after incubation overnight the resulting plaques were enumerated to calculate an eop in pfu ml'¹ of lysate.

Lambda phage spot assay

All phage lysates were diluted in SM buffer (50 mM Tris-HCl (pH 7.5), 8 mM MgSO4, 100 mM NaCl) to a similar eop as determined on E. coli NEB 10-beta. These were further serially diluted 10-fold in SM buffer and spotted on bacterial overlays (made as described above) of the relevant strains. For spot assay of cocktail lysates, equal volumes of the two lysates at identical titers were mixed before further dilution and spotting. To test for eop against the EcoKI restriction system the E. coli strain MG1655 was used. To test eop against the Eco0015 system the strain NEB 10-beta was transformed with pl 331 (harboring the hsdR-hsdM-hsdS system of Eco0015 under constitutive expression). To test against both the EcoKI and Eco0015 systems in the same strain, MG1655 was transformed with pl 331.

P4 transducing phage lysate preparation and titer

The pl 065 plasmid (containing sGFP, spectinomycin marker, type I restriction recognition sites and P4 COS packaging site as described above) was used to transform the E. coli strain b587 which contains all the genetic machinery necessary to catalyze packaging of pl 065 into phage heads. For methylation of the packaged DNA, the plasmid pl 160 (containing an operon of MTases under control of the arabinose promoter) was co-transformed into b587. Overnight cultures of the transformants were grown in LB media (supplemented with Tet + Spec + 5mM CaCh and lOmM MgCh) and incubated at 37°C, shaking. The next day fresh 10 ml cultures were inoculated to ODeoo=0.1 and then grown to ODeoo=0.2 at which point the packaging and lyses genes, and in the case of pl 160 the expression of MTases, were induced by the addition of arabinose (0.2% w/v). Cultures were left to lyse for 3-4h at 37°C with shaking. Lysed cultures were spun down (5min at 10,000xg) and the supernatant filter sterilized using a 2uM filter. To remove residual bacterial DNA the lysate was incubated with DNasel overnight at 37°C.

To determine the lysate titer, lOOul of lysate was mixed with lOOul of overnight culture of the strain b52 and incubated at RT for 30 minutes. This mixture was serially diluted 1 : 10 in SM buffer, spotted on to LB + Spec plates and the GFP positive colonies enumerated to calculate a titer in cfu ml'¹ of lysate. The titer on clinical strains was determined by the same method.

Results of the experiments are shown in Figures 1-5

A. Small conjugative plasmids encoding wildtype (pl 172 and pl 174) or mutated (pl243 and pl244) versions of M.EcoKI and M.Eco0015 respectively are directly compared for conjugation efficiency into A. coll clinical isolates. The b52 strain is used as a negative control as it does not harbour any active restriction system. Bars marked with X are for strains harbouring the EcoKI restriction system while all other strains harbour the Eco0015 restriction system. Dashed line represents limit of detection at 2.3 logs.

B. Unpaired T-test comparison of pl 174 (encoding wildtype M.Eco0015) and pl244 (encoding mutated M.Eco0015) conjugation efficiency into strains harbouring the Eco0015 restriction system (p < 0.05).

A. Large conjugative plasmids based on pCollB9SL1344 and encoding wildtype (p 1284) or mutated (p 1286) versions of M.Eco0015 respectively are directly compared for conjugation efficiency into A. coll clinical collection strains. The b52 strain is used as a negative control as it does not harbour any active restriction system. Bars marked with X are for strains harbouring the EcoKI restriction system while all other strains harbour the Eco0015 restriction system. Dashed line represents limit of detection at 2.3 logs. B. Unpaired T-test comparison of pl284 (encoding wildtype M.Eco0015) and pl286 (encoding mutated M.Eco0015) conjugation efficiency into strains harbouring the Eco0015 restriction system (p < 0.05).

A. The small conjugative plasmid (pl 114) is methylated in trans by a co-resident non- conjugative plasmid encoding either wildtype (pl082 and p819) or mutated (pl369 and p 1368) versions of M.EcoKI and M.Eco0015 respectively (n=3). The conjugation efficiency of the transmethylated plasmids is plotted against the cis-methylation conjugation efficiency data from figure 1 (A), with the plasmids encoding wildtype (pl 172 and pl 174) and mutated (pl 243 and pl244) versions of M.EcoKI and M.Eco0015 respectively. The b52 strain is used as a negative control as it does not harbour any active restriction system. Bars marked with X are for strains harbouring the EcoKI restriction system while all other strains harbour the Eco0015 restriction system. Dashed line represents limit of detection of the assay at 2.3 logs.

B. Unpaired T-test comparison of pl 082 (encoding wildtype M.Eco0015 in trans) and pl 174 (encoding wildtype M.Eco0015 in cis) conjugation efficiency into strains harbouring the Eco0015 restriction system (p < 0.05).

Fig. 4. Efficiency of plaquing of phage lysates, methylated either in trans or in cis (n=3)

On horizontal axis is description of strain background in which phage lysates were made in regards to methylation properties. Categories being: unmethylated phages, phages methylated in trans e.g.. from MTases encoded by the strain genome or on a co-resident plasmid, and methylation in cis e.g.. from MTases encoded on the phage genome. In some instances two lysates were produced separately and then mixed together to make a cocktail. Each graph shows the eop of spotting these lysates on an E. coli strain with particular restriction properties:

A. eop on a strain harbouring no restriction enzymes (NEB 10-beta)

B. eop on strain harbouring the EcoKI system (MG1655)

C. eop on strain harbouring the Eco0015 system (NEB 10-beta containing p!331)

D. eop on strain harbouring both EcoKI and Eco0015 systems (MG1655 containing p!331) Fig. 5. Efficiency of phage transduction after methylation in trans by a production strain expressing an MTase operon (n=3)

Methylated and unmethylated plasmid pl 065 was packaged into a P4 transducing phage particle and the transduction efficiency compared in a panel of E. coli clinical isolates. The methylation of pl 065 DNA was achieved in trans by producing a lysate from a strain harbouring a coresident plasmid (pl 160) encoding an operon of seven MTases. The b52 strain is used as a negative control as it does not harbour any active restriction system. Strains marked with an X encode a restriction system cognate to the MTases encoded by pl 160 and show improved transduction upon methylation of pl 065, strains marked with a Y also encode a restriction system cognate to the MTases encoded by pl 160 but do not show any improvement in transduction efficiency. All other strains do not encode a restriction system cognate to the MTases encoded by pl 160. Dashed line represents limit of detection of the assay at 2.3 logs.

Details of various Mtases used in these experiments is provided in Appendix IV.

Appendix V is the sequence of pl 065

Conclusions

It can be seen that:

Conjugative plasmids encoding MTase enzymes are significantly protected from host RM systems after conjugation;

Protection may be provided by methylation in cis or trans (e.g. by a co-resident non-conjugative plasmid in the conjugation strain);

Cocktails of MTases can provide high levels of protection from RM systems; and

Operons encoding multiple MTases can used to effectively methylate plasmid DNA for delivery by phage transduction. Table A: Plasmids

Table B: E. coli strains general molecular biology

Table C: E. coli strains for testing conjugation and phage transduction efficiency

*RM system designations taken from REBASE known recognition sequences

Table D: Example Tevenvirinae Phage

Acinetobacter virus 133

Aeromonas virus 65

Aeromonas virus Aehl

Dhakavirus

Escherichia virus Bp7

Escherichia virus IME08

Escherichia virus JS10

Escherichia virus JS98

Escherichia virus MX01

Escherichia virus QL01

Escherichia virus VR5

Escherichia virus WG01

Escherichia phage RBI 6

Escherichia phage RB32

Escherichia virus RB43

Enterobacteria phage RB43-GVA

Gaprivervirus

Escherichia virus VR20

Escherichia virus VR25

Escherichia virus VR26

Escherichia virus VR7 Shigella virus SP18

Gelderlandvirus

Salmonella virus Melville

Salmonella virus S16

Salmonella virus STML198

Salmonella virus STP4a

Jiaodavirus

Klebsiella virus JD18

Klebsiella virus PKO111

Karamvirus

Enterobacter virus PG7

Escherichia virus CC31

Krischvirus

Escherichia virus ECD7

Escherichia virus GEC3S

Escherichia virus JSE

Escherichia virus phil

Escherichia virus RB49

Moonvirus

Citrobacter virus CF1

Citrobacter virus Merlin

Citrobacter virus Moon

Mosigvirus

Escherichia virus APCEcOl

Escherichia virus HP3

Escherichia virus HX01

Escherichia virus JS09

Escherichia virus O157tp3

Escherichia virus O157tp6 Escherichia virus PhAPEC2

Escherichia virus RB69

Escherichia virus STO

Shigella virus SHSML521

Shigella virus UTAM

Schizotequatrovirus

Vibrio virus KVP40

Vibrio virus ntl

Vibrio virus ValKK3

Slopekvirus

Enterobacter virus Eap3

Klebsiella virus KPI 5

Klebsiella virus KP27

Klebsiella virus Matisse

Klebsiella virus Miro

Klebsiella virus PMBT1

Tequatrovirus

Enterobacteria phage T4 sensu lato

Escherichia virus ARI

Escherichia virus C40

Escherichia virus CF2

Escherichia virus El 12

Escherichia virus ECML134

Escherichia virus HY01

Escherichia virus HY03

Escherichia virus Ime09

Escherichia virus RB 14

Escherichia virus RB3

Escherichia virus slur03

Escherichia virus slur04

Escherichia virus SV14

Escherichia virus T4 Shigella virus Pssl

Shigella virus Sf21

Shigella virus Sf22

Shigella virus Sf24

Shigella virus SHBML501

Shigella virus Shfl2

Vibrio phage nt-1 sensu lato

Yersinia virus DI

Yersinia virus PST unclassified Tevenvirinae

Acinetobacter phage AbTZAl

Acinetobacter phage Ac42

Acinetobacter phage Acj61

Acinetobacter phage Acj9

Acinetobacter phage AMI 01

Acinetobacter phage Henu6

Acinetobacter phage KARL-1

Acinetobacter phage vB_AbaM_PhT2

Acinetobacter phage vB AbaP Abraxas

Acinetobacter phage vB ApiM fHyAciO3

Acinetobacter phage ZZ1

Aeromonas phage 65,2

Aeromonas phage Ahl

Aeromonas phage AS-sw

Aeromonas phage AS-szw

Aeromonas phage AS-yi

Aeromonas phage AS-zj

Aeromonas phage AsFcp 1

Aeromonas phage AsFcp_2

Aeromonas phage AsFcp_4

Aeromonas phage Assk

Aeromonas phage Asswx 1

Aeromonas phage AsSzw2

Aeromonas phage Aswh l Aeromonas phage Aszh-1

Aeromonas phage CC2

Aeromonas phage phiAS5

Aeromonas phage PX29

Buttiauxella phage vb_ButM_GuL6

Citrobacter phage IME-CF2

Citrobacter phage Margaery

Citrobacter phage Maroon

Citrobacter phage Miller

Citrobacter phage vB CfrM CfPl

Cronobacter phage S13

Cronobacter phage vB CsaM GAP161

Cronobacter phage vB CsaM leB

Cronobacter phage vB CsaM leE

Cronobacter phage vB CsaM leN

Edwardsiella phage PEi20

Edwardsiella phage PEi26

Enterobacter phage EBPL

Enterobacter phage EC-F1

Enterobacter phage EC-F2

Enterobacter phage EC-W 1

Enterobacter phage EC-W2

Enterobacter phage vB_EclM_CIP9

Erwinia phage Cronus

Escherichia phage Lwl

Escherichia phage RDN37

Klebsiella phage AmPh_EK29

Klebsiella phage El

Klebsiella phage K0X11

Klebsiella phage K0X8

Klebsiella phage KPN1

Klebsiella phage Marfa

Klebsiella phage PhiKpNIH-6

Klebsiella phage vB_Kpn_F48

Klebsiella phage vB_Kpn_P545 Klebsiella phage vB KpnM Pottsl

Morganella phage vB MmoM MPl Panteoa phage Phynn

Pectobacterium bacteriophage PM2

Proteus phage phiP4-3

Proteus phage PM2

Proteus phage vB PmiM Pm5461

Pseudomonas phage PspYZU05

Serratia phage Muldoon

Serratia phage PS2

Shewanella phage Thanatos- 1

Shewanella phage Thanatos-2

Shigella phage vB _SdyM_006

Sinorhizobium phage vB SmelM phiMIO

Sinorhizobium phage vB SmelM phiM14 Vibrio phage vB VmeM-32

Yersinia phage JC221

TABLE E: Medicaments

Appendix I: Sequence of Lambda phage reboot Design I (virulent lambda) (SEQ ID NO: 2)

(sequence begins at COS site)

These sequences are the referred to in the methods section under "Phage lambda engineering" and are used in the experiment for Figure 4. gggcggcgacctcgcgggttttcgctatttatgaaaattttccggtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaataccctct gaaaagaaaggaaacgacaggtgctgaaagcgaggctttttggcctctgtcgtttcctttctctgtttttgtccgtggaatgaacaatggaagtcaaca aaaagcagctggctgacattttcggtgcgagtatccgtaccattcagaactggcaggaacagggaatgcccgttctgcgaggcggtggcaagggtaa tgaggtgctttatgactctgccgccgtcataaaatggtatgccgaaagggatgctgaaattgagaacgaaaagctgcgccgggaggttgaagaactg cggcaggccagcgaggcagatctccagccaggaactattgagtacgaacgccatcgacttacgcgtgcgcaggccgacgcacaggaactgaagaat gccagagactccgctgaagtggtggaaaccgcattctgtactttcgtgctgtcgcggatcgcaggtgaaattgccagtattctcgacgggctccccctgt cggtgcagcggcgttttccggaactggaaaaccgacatgttgatttcctgaaacgggatatcatcaaagccatgaacaaagcagccgcgctggatga actgataccggggttgctgagtgaatatatcgaacagtcaggttaacaggctgcggcattttgtccgcgccgggcttcgctcactgttcaggccggagc cacagaccgccgttgaatgggcggatgctaattactatctcccgaaagaatccgcataccaggaagggcgctgggaaacactgccctttcagcgggc catcatgaatgcgatgggcagcgactacatccgtgaggtgaatgtggtgaagtctgcccgtgtcggttattccaaaatgctgctgggtgtttatgcctac tttatagagcataagcagcgcaacacccttatctggttgccgacggatggtgatgccgagaactttatgaaaacccacgttgagccgactattcgtgat attccgtcgctgctggcgctggccccgtggtatggcaaaaagcaccgggataacacgctcaccatgaagcgtttcactaatgggcgtggcttctggtgc ctgggcggtaaagcggcaaaaaactaccgtgaaaagtcggtggatgtggcgggttatgatgaacttgctgcttttgatgatgatattgaacaggaagg ctctccgacgttcctgggtgacaagcgtattgaaggctcggtctggccaaagtccatccgtggctccacgccaaaagtgagaggcacctgtcagattg agcgtgcagccagtgaatccccgcattttatgcgttttcatgttgcctgcccgcattgcggggaggagcagtatcttaaatttggcgacaaagagacgc cgtttggcctcaaatggacgccggatgacccctccagcgtgttttatctctgcgagcataatgcctgcgtcatccgccagcaggagctggactttactga tgcccgttatatctgcgaaaagaccgggatctggacccgtgatggcattctctggttttcgtcatccggtgaagagattgagccacctgacagtgtgacc tttcacatctggacagcgtacagcccgttcaccacctgggtgcagattgtcaaagactggatgaaaacgaaaggggatacgggaaaacgtaaaacct tcgtaaacaccacgctcggtgagacgtgggaggcgaaaattggcgaacgtccggatgctgaagtgatggcagagcggaaagagcattattcagcgc ccgttcctgaccgtgtggcttacctgaccgccggtatcgactcccagctggaccgctacgaaatgcgcgtatggggatgggggccgggtgaggaaagc tggctgattgaccggcagattattatgggccgccacgacgatgaacagacgctgctgcgtgtggatgaggccatcaataaaacctatacccgccgga atggtgcagaaatgtcgatatcccgtatctgctgggatactggcgggattgacccgaccattgtgtatgaacgctcgaaaaaacatgggctgttccgg gtgatccccattaaaggggcatccgtctacggaaagccggtggccagcatgccacgtaagcgaaacaaaaacggggtttaccttaccgaaatcggta cggataccgcgaaagagcagatttataaccgcttcacactgacgccggaaggggatgaaccgcttcccggtgccgttcacttcccgaataacccggat atttttgatctgaccgaagcgcagcagctgactgctgaagagcaggtcgaaaaatgggtggatggcaggaaaaaaatactgtgggacagcaaaaag cgacgcaatgaggcactcgactgcttcgtttatgcgctggcggcgctgcgcatcagtatttcccgctggcagctggatctcagtgcgctgctggcgagc ctgcaggaagaggatggtgcagcaaccaacaagaaaacactggcagattacgcccgtgccttatccggagaggatgaatgacgcgacaggaagaa cttgccgctgcccgtgcggcactgcatgacctgatgacaggtaaacgggtggcaacagtacagaaagacggacgaagggtggagtttacggccactt ccgtgtctgacctgaaaaaatatattgcagagctggaagtgcagaccggcatgacacagcgacgcaggggacctgcaggattttatgtatgaaaacg cccaccattcccacccttctggggccggacggcatgacatcgctgcgcgaatatgccggttatcacggcggtggcagcggatttggagggcagttgcg gtcgtggaacccaccgagtgaaagtgtggatgcagccctgttgcccaactttacccgtggcaatgcccgcgcagacgatctggtacgcaataacggct atgccgccaacgccatccagctgcatcaggatcatatcgtcgggtcttttttccggctcagtcatcgcccaagctggcgctatctgggcatcggggagg aagaagcccgtgccttttcccgcgaggttgaagcggcatggaaagagtttgccgaggatgactgctgctgcattgacgttgagcgaaaacgcacgttt accatgatgattcgggaaggtgtggccatgcacgcctttaacggtgaactgttcgttcaggccacctgggataccagttcgtcgcggcttttccggaca cagttccggatggtcagcccgaagcgcatcagcaacccgaacaataccggcgacagccggaactgccgtgccggtgtgcagattaatgacagcggt gcggcgctgggatattacgtcagcgaggacgggtatcctggctggatgccgcagaaatggacatggataccccgtgagttacccggcgggcgcgcct cgttcattcacgtttttgaacccgtggaggacgggcagactcgcggtgcaaatgtgttttacagcgtgatggagcagatgaagatgctcgacacgctgc agaacacgcagctgcagagcgccattgtgaaggcgatgtatgccgccaccattgagagtgagctggatacgcagtcagcgatggattttattctgggc gcgaacagtcaggagcagcgggaaaggctgaccggctggattggtgaaattgccgcgtattacgccgcagcgccggtccggctgggaggcgcaaaa gtaccgcacctgatgccgggtgactcactgaacctgcagacggctcaggatacggataacggctactccgtgtttgagcagtcactgctgcggtatatc gctgccgggctgggtgtctcgtatgagcagctttcccggaattacgcccagatgagctactccacggcacgggccagtgcgaacgagtcgtgggcgta ctttatggggcggcgaaaattcgtcgcatcccgtcaggcgagccagatgtttctgtgctggctggaagaggccatcgttcgccgcgtggtgacgttacc ttcaaaagcgcgcttcagttttcaggaagcccgcagtgcctgggggaactgcgactggataggctccggtcgtatggccatcgatggtctgaaagaag ttcaggaagcggtgatgctgatagaagccggactgagtacctacgagaaagagtgcgcaaaacgcggtgacgactatcaggaaatttttgcccagca ggtccgtgaaacgatggagcgccgtgcagccggtcttaaaccgcccgcctgggcggctgcagcatttgaatccgggctgcgacaatcaacagaggag gagaagagtgacagcagagctgcgtaatctcccgcatattgccagcatggcctttaatgagccgctgatgcttgaacccgcctatgcgcgggttttcttt tgtgcgcttgcaggccagcttgggatcagcagcctgacggatgcggtgtccggcgacagcctgactgcccaggaggcactcgcgacgctggcattatc cggtgatgatgacggaccacgacaggcccgcagttatcaggtcatgaacggcatcgccgtgctgccggtgtccggcacgctggtcagccggacgcgg gcgctgcagccgtactcggggatgaccggttacaacggcattatcgcccgtctgcaacaggctgccagcgatccgatggtggacggcattctgctcga tatggacacgcccggcgggatggtggcgggggcatttgactgcgctgacatcatcgcccgtgtgcgtgacataaaaccggtatgggcgcttgccaacg acatgaactgcagtgcaggtcagttgcttgccagtgccgcctcccggcgtctggtcacgcagaccgcccggacaggctccatcggcgtcatgatggctc acagtaattacggtgctgcgctggagaaacagggtgtggaaatcacgctgatttacagcggcagccataaggtggatggcaacccctacagccatct tccggatgacgtccgggagacactgcagtcccggatggacgcaacccgccagatgtttgcgcagaaggtgtcggcatataccggcctgtccgtgcag gttgtgctggataccgaggctgcagtgtacagcggtcaggaggccattgatgccggactggctgatgaacttgttaacagcaccgatgcgatcaccgt catgcgtgatgcactggatgcacgtaaatcccgtctctcaggagggcgaatgaccaaagagactcaatcaacaactgtttcagccactgcttcgcagg ctgacgttactgacgtggtgccagcgacggagggcgagaacgccagcgcggcgcagccggacgtgaacgcgcagatcaccgcagcggttgcggca gaaaacagccgcattatggggatcctcaactgtgaggaggctcacggacgcgaagaacaggcacgcgtgctggcagaaacccccggtatgaccgtg aaaacggcccgccgcattctggccgcagcaccacagagtgcacaggcgcgcagtgacactgcgctggatcgtctgatgcagggggcaccggcaccg ctggctgcaggtaacccggcatctgatgccgttaacgatttgctgaacacaccagtgtaagggatgtttatgacgagcaaagaaacctttacccattac cagccgcagggcaacagtgacccggctcataccgcaaccgcgcccggcggattgagtgcgaaagcgcctgcaatgaccccgctgatgctggacacc tccagccgtaagctggttgcgtgggatggcaccaccgacggtgctgccgttggcattcttgcggttgctgctgaccagaccagcaccacgctgacgttc tacaagtccggcacgttccgttatgaggatgtgctctggccggaggctgccagcgacgagacgaaaaaacggaccgcgtttgccggaacggcaatca gcatcgtttaactttacccttcatcactaaaggccgcctgtgcggctttttttacgggatttttttatgtcgatgtacacaaccgcccaactgctggcggca aatgagcagaaatttaagtttgatccgctgtttctgcgtctctttttccgtgagagctatcccttcaccacggagaaagtctatctctcacaaattccggg actggtaaacatggcgctgtacgtttcgccgattgtttccggtgaggttatccgttcccgtggcggctccacctctgaatttacgccgggatatgtcaagc cgaagcatgaagtgaatccgcagatgaccctgcgtcgcctgccggatgaagatccgcagaatctggcggacccggcttaccgccgccgtcgcatcat catgcagaacatgcgtgacgaagagctggccattgctcaggtcgaagagatgcaggcagtttctgccgtgcttaagggcaaatacaccatgaccggt gaagccttcgatccggttgaggtggatatgggccgcagtgaggagaataacatcacgcagtccggcggcacggagtggagcaagcgtgacaagtcc acgtatgacccgaccgacgatatcgaagcctacgcgctgaacgccagcggtgtggtgaatatcatcgtgttcgatccgaaaggctgggcgctgttccg ttccttcaaagccgtcaaggagaagctggatacccgtcgtggctctaattccgagctggagacagcggtgaaagacctgggcaaagcggtgtcctata aggggatgtatggcgatgtggccatcgtcgtgtattccggacagtacgtggaaaacggcgtcaaaaagaacttcctgccggacaacacgatggtgct ggggaacactcaggcacgcggtctgcgcacctatggctgcattcaggatgcggacgcacagcgcgaaggcattaacgcctctgcccgttacccgaaa aactgggtgaccaccggcgatccggcgcgtgagttcaccatgattcagtcagcaccgctgatgctgctggctgaccctgatgagttcgtgtccgtacaa ctggcgtaatcatggcccttcggggccattgtttctctgtggaggagtccatgacgaaagatgaactgattgcccgtctccgctcgctgggtgaacaact gaaccgtgatgtcagcctgacggggacgaaagaagaactggcgctccgtgtggcagagctgaaagaggagcttgatgacacggatgaaactgccg gtcaggacacccctctcagccgggaaaatgtgctgaccggacatgaaaatgaggtgggatcagcgcagccggataccgtgattctggatacgtctga actggtcacggtcgtggcactggtgaagctgcatactgatgcacttcacgccacgcgggatgaacctgtggcatttgtgctgccgggaacggcgtttcg tgtctctgccggtgtggcagccgaaatgacagagcgcggcctggccagaatgcaataacgggaggcgctgtggctgatttcgataacctgttcgatgc tgccattgcccgcgccgatgaaacgatacgcgggtacatgggaacgtcagccaccattacatccggtgagcagtcaggtgcggtgatacgtggtgttt ttgatgaccctgaaaatatcagctatgccggacagggcgtgcgcgttgaaggctccagcccgtccctgtttgtccggactgatgaggtgcggcagctgc ggcgtggagacacgctgaccatcggtgaggaaaatttctgggtagatcgggtttcgccggatgatggcggaagttgtcatctctggcttggacggggc gtaccgcctgccgttaaccgtcgccgctgaaagggggatgtatggccataaaaggtcttgagcaggccgttgaaaacctcagccgtatcagcaaaac ggcggtgcctggtgccgccgcaatggccattaaccgcgttgcttcatccgcgatatcgcagtcggcgtcacaggttgcccgtgagacaaaggtacgcc ggaaactggtaaaggaaagggccaggctgaaaagggccacggtcaaaaatccgcaggccagaatcaaagttaaccggggggatttgcccgtaatc aagctgggtaatgcgcgggttgtcctttcgcgccgcaggcgtcgtaaaaaggggcagcgttcatccctgaaaggtggcggcagcgtgcttgtggtggg taaccgtcgtattcccggcgcgtttattcagcaactgaaaaatggccggtggcatgtcatgcagcgtgtggctgggaaaaaccgttaccccattgatgt ggtgaaaatcccgatggcggtgccgctgaccacggcgtttaaacaaaatattgagcggatacggcgtgaacgtcttccgaaagagctgggctatgcg ctgcagcatcaactgaggatggtaataaagcgatgaaacatactgaactccgtgcagccgtactggatgcactggagaagcatgacaccggggcga cgttttttgatggtcgccccgctgtttttgatgaggcggattttccggcagttgccgtttatctcaccggcgctgaatacacgggcgaagagctggacag cgatacctggcaggcggagctgcatatcgaagttttcctgcctgctcaggtgccggattcagagctggatgcgtggatggagtcccggatttatccggt gatgagcgatatcccggcactgtcagatttgatcaccagtatggtggccagcggctatgactaccggcgcgacgatgatgcgggcttgtggagttcag ccgatctgacttatgtcattacctatgaaatgtgaggacgctatgcctgtaccaaatcctacaatgccggtgaaaggtgccgggaccaccctgtgggtt tataaggggagcggtgacccttacgcgaatccgctttcagacgttgactggtcgcgtctggcaaaagttaaagacctgacgcccggcgaactgaccgc tgagtcctatgacgacagctatctcgatgatgaagatgcagactggactgcgaccgggcaggggcagaaatctgccggagataccagcttcacgctg gcgtggatgcccggagagcaggggcagcaggcgctgctggcgtggtttaatgaaggcgatacccgtgcctataaaatccgcttcccgaacggcacgg tcgatgtgttccgtggctgggtcagcagtatcggtaaggcggtgacggcgaaggaagtgatcacccgcacggtgaaagtcaccaatgtgggacgtcc gtcgatggcagaagatcgcagcacggtaacagcggcaaccggcatgaccgtgacgcctgccagcacctcggtggtgaaagggcagagcaccacgct gaccgtggccttccagccggagggcgtaaccgacaagagctttcgtgcggtgtctgcggataaaacaaaagccaccgtgtcggtcagtggtatgacc atcaccgtgaacggcgttgctgcaggcaaggtcaacattccggttgtatccggtaatggtgagtttgctgcggttgcagaaattaccgtcaccgccagt taatccggagagtcagcgatgttcctgaaaaccgaatcatttgaacataacggtgtgaccgtcacgctttctgaactgtcagccctgcagcgcattgag catctcgccctgatgaaacggcaggcagaacaggcggagtcagacagcaaccggaagtttactgtggaagacgccatcagaaccggcgcgtttctg gtggcgatgtccctgtggcataaccatccgcagaagacgcagatgccgtccatgaatgaagccgttaaacagattgagcaggaagtgcttaccacct ggcccacggaggcaatttctcatgctgaaaacgtggtgtaccggctgtctggtatgtatgagtttgtggtgaataatgcccctgaacagacagaggac gccgggcccgcagagcctgtttctgcgggaaagtgttcgacggtgagctgagttttgccctgaaactggcgcgtgagatggggcgacccgactggcgt gccatgcttgccgggatgtcatccacggagtatgccgactggcaccgcttttacagtacccattattttcatgatgttctgctggatatgcacttttccggg ctgacgtacaccgtgctcagcctgtttttcagcgatccggatatgcatccgctggatttcagtctgctgaaccggcgcgaggctgacgaagagcctgaa gatgatgtgctgatgcagaaagcggcagggcttgccggaggtgtccgctttggcccggacgggaatgaagttatccccgcttccccggatgtggcgga catgacggaggatgacgtaatgctgatgacagtatcagaagggatcgcaggaggagtccggtatggctgaaccggtaggcgatctggtcgttgattt gagtctggatgcggccagatttgacgagcagatggccagagtcaggcgtcatttttctggtacggaaagtgatgcgaaaaaaacagcggcagtcgtt gaacagtcgctgagccgacaggcgctggctgcacagaaagcggggatttccgtcgggcagtataaagccgccatgcgtatgctgcctgcacagttca ccgacgtggccacgcagcttgcaggcgggcaaagtccgtggctgatcctgctgcaacagggggggcaggtgaaggactccttcggcgggatgatccc catgttcagggggcttgccggtgcgatcaccctgccgatggtgggggccacctcgctggcggtggcgaccggtgcgctggcgtatgcctggtatcagg gcaactcaaccctgtccgatttcaacaaaacgctggtcctttccggcaatcaggcgggactgacggcagatcgtatgctggtcctgtccagagccggg caggcggcagggctgacgtttaaccagaccagcgagtcactcagcgcactggttaaggcgggggtaagcggtgaggctcagattgcgtccatcagcc agagtgtggcgcgtttctcctctgcatccggcgtggaggtggacaaggtcgctgaagccttcgggaagctgaccacagacccgacgtcggggctgac ggcgatggctcgccagttccataacgtgtcggcggagcagattgcgtatgttgctcagttgcagcgttccggcgatgaagccggggcattgcaggcgg cgaacgaggccgcaacgaaagggtttgatgaccagacccgccgcctgaaagagaacatgggcacgctggaaacctgggcagacaggactgcgcgg gcattcaaatccatgtgggatgcggtgctggatattggtcgtcctgataccgcgcaggagatgctgattaaggcagaggctgcgtataagaaagcaga cgacatctggaatctgcgcaaggatgattattttgttaacgatgaagcgcgggcgcgttactgggatgatcgtgaaaaggcccgtcttgcgcttgaagc cgcccgaaagaaggctgagcagcagactcaacaggacaaaaatgcgcagcagcagagcgataccgaagcgtcacggctgaaatataccgaagag gcgcagaaggcttacgaacggctgcagacgccgctggagaaatataccgcccgtcaggaagaactgaacaaggcactgaaagacgggaaaatcct gcaggcggattacaacacgctgatggcggcggcgaaaaaggattatgaagcgacgctgaaaaagccgaaacagtccagcgtgaaggtgtctgcgg gcgatcgtcaggaagacagtgctcatgctgccctgctgacgcttcaggcagaactccggacgctggagaagcatgccggagcaaatgagaaaatca gccagcagcgccgggatttgtggaaggcggagagtcagttcgcggtactggaggaggcggcgcaacgtcgccagctgtctgcacaggagaaatccc tgctggcgcataaagatgagacgctggagtacaaacgccagctggctgcacttggcgacaaggttacgtatcaggagcgcctgaacgcgctggcgca gcaggcggataaattcgcacagcagcaacgggcaaaacgggccgccattgatgcgaaaagccgggggctgactgaccggcaggcagaacgggaa gccacggaacagcgcctgaaggaacagtatggcgataatccgctggcgctgaataacgtcatgtcagagcagaaaaagacctgggcggctgaaga ccagcttcgcgggaactggatggcaggcctgaagtccggctggagtgagtgggaagagagcgccacggacagtatgtcgcaggtaaaaagtgcagc cacgcagacctttgatggtattgcacagaatatggcggcgatgctgaccggcagtgagcagaactggcgcagcttcacccgttccgtgctgtccatgat gacagaaattctgcttaagcaggcaatggtggggattgtcgggagtatcggcagcgccattggcggggctgttggtggcggcgcatccgcgtcaggc ggtacagccattcaggccgctgcggcgaaattccattttgcaaccggaggatttacgggaaccggcggcaaatatgagccagcggggattgttcacc gtggtgagtttgtcttcacgaaggaggcaaccagccggattggcgtggggaatctttaccggctgatgcgcggctatgccaccggcggttatgtcggta caccgggcagcatggcagacagccggtcgcaggcgtccgggacgtttgagcagaataaccatgtggtgattaacaacgacggcacgaacgggcag ataggtccggctgctctgaaggcggtgtatgacatggcccgcaagggtgcccgtgatgaaattcagacacagatgcgtgatggtggcctgttctccgg aggtggacgatgaagaccttccgctggaaagtgaaacccggtatggatgtggcttcggtcccttctgtaagaaaggtgcgctttggtgatggctattct cagcgagcgcctgccgggctgaatgccaacctgaaaacgtacagcgtgacgctttctgtcccccgtgaggaggccacggtactggagtcgtttctgga agagcacgggggctggaaatcctttctgtggacgccgccttatgagtggcggcagataaaggtgacctgcgcaaaatggtcgtcgcgggtcagtatg ctgcgtgttgagttcagcgcagagtttgaacaggtggtgaactgatgcaggatatccggcaggaaacactgaatgaatgcacccgtgcggagcagtc ggccagcgtggtgctctgggaaatcgacctgacagaggtcggtggagaacgttattttttctgtaatgagcagaacgaaaaaggtgagccggtcacct ggcaggggcgacagtatcagccgtatcccattcaggggagcggttttgaactgaatggcaaaggcaccagtacgcgccccacgctgacggtttctaa cctgtacggtatggtcaccgggatggcggaagatatgcagagtctggtcggcggaacggtggtccggcgtaaggtttacgcccgttttctggatgcggt gaacttcgtcaacggaaacagttacgccgatccggagcaggaggtgatcagccgctggcgcattgagcagtgcagcgaactgagcgcggtgagtgc ctcctttgtactgtccacgccgacggaaacggatggcgctgtttttccgggacgtatcatgctggccaacacctgcacctggacctatcgcggtgacga gtgcggttatagcggtccggctgtcgcggatgaatatgaccagccaacgtccgatatcacgaaggataaatgcagcaaatgcctgagcggttgtaagt tccgcaataacgtcggcaactttggcggcttcctttccattaacaaactttcgcagtaaatcccatgacacagacagaatcagcgattctggcgcacgc ccggcgatgtgcgccagcggagtcgtgcggcttcgtggtaagcacgccggagggggaaagatatttcccctgcgtgaatatctccggtgagccggag gctatttccgtatgtcgccggaagactggctgcaggcagaaatgcagggtgagattgtggcgctggtccacagccaccccggtggtctgccctggctg agtgaggccgaccggcggctgcaggtgcagagtgatttgccgtggtggctggtctgccgggggacgattcataagttccgctgtgtgccgcatctcacc gggcggcgctttgagcacggtgtgacggactgttacacactgttccgggatgcttatcatctggcggggattgagatgccggactttcatcgtgaggat gactggtggcgtaacggccagaatctctatctggataatctggaggcgacggggctgtatcaggtgccgttgtcagcggcacagccgggcgatgtgct gctgtgctgttttggttcatcagtgccgaatcacgccgcaatttactgcggcgacggcgagctgctgcaccatattcctgaacaactgagcaaacgaga gaggtacaccgacaaatggcagcgacgcacacactccctctggcgtcaccgggcatggcgcgcatctgcctttacggggatttacaacgatttggtcg ccgcatcgaccttcgtgtgaaaacgggggctgaagccatccgggcactggccacacagctcccggcgtttcgtcagaaactgagcgacggctggtatc aggtacggattgccgggcgggacgtcagcacgtccgggttaacggcgcagttacatgagactctgcctgatggcgctgtaattcatattgttcccagag tcgccggggccaagtcaggtggcgtattccagattgtcctgggggctgccgccattgccggatcattctttaccgccggagccacccttgcagcatggg gggcagccattggggccggtggtatgaccggcatcctgttttctctcggtgccagtatggtgctcggtggtgtggcgcagatgctggcaccgaaagcca gaactccccgtatacagacaacggataacggtaagcagaacacctatttctcctcactggataacatggttgcccagggcaatgttctgcctgttctgt acggggaaatgcgcgtggggtcacgcgtggtttctcaggagatcagcacggcagacgaaggggacggtggtcaggttgtggtgattggtcgctgatg caaaatgttttatgtgaaaccgcctgcgggcggttttgtcatttatggagcgtgaggaatgggtaaaggaagcagtaaggggcataccccgcgcgaa gcgaaggacaacctgaagtccacgcagttgctgagtgtgatcgatgccatcagcgaagggccgattgaaggtccggtggatggcttaaaaagcgtgc tgctgaacagtacgccggtgctggacactgaggggaataccaacatatccggtgtcacggtggtgttccgggctggtgagcaggagcagactccgcc ggagggatttgaatcctccggctccgagacggtgctgggtacggaagtgaaatatgacacgccgatcacccgcaccattacgtctgcaaacatcgac cgtctgcgctttaccttcggtgtacaggcactggtggaaaccacctcaaagggtgacaggaatccgtcggaagtccgcctgctggttcagatacaacgt aacggtggctgggtgacggaaaaagacatcaccattaagggcaaaaccacctcgcagtatctggcctcggtggtgatgggtaacctgccgccgcgcc cgtttaatatccggatgcgcaggatgacgccggacagcaccacagaccagctgcagaacaaaacgctctggtcgtcatacactgaaatcatcgatgt gaaacagtgctacccgaacacggcactggtcggcgtgcaggtggactcggagcagttcggcagccagcaggtgagccgtaattatcatctgcgcggg cgtattctgcaggtgccgtcgaactataacccgcagacgcggcaatacagcggtatctgggacggaacgtttaaaccggcatacagcaacaacatgg cctggtgtctgtgggatatgctgacccatccgcgctacggcatggggaaacgtcttggtgcggcggatgtggataaatgggcgctgtatgtcatcggcc agtactgcgaccagtcagtgccggacggctttggcggcacggagccgcgcatcacctgtaatgcgtacctgaccacacagcgtaaggcgtgggatgt gctcagcgatttctgctcggcgatgcgctgtatgccggtatggaacgggcagacgctgacgttcgtgcaggaccgaccgtcggataagacgtggacct ataaccgcagtaatgtggtgatgccggatgatggcgcgccgttccgctacagcttcagcgccctgaaggaccgccataatgccgttgaggtgaactgg attgacccgaacaacggctgggagacggcgacagagcttgttgaagatacgcaggccattgcccgttacggtcgtaatgttacgaagatggatgcctt tggctgtaccagccgggggcaggcacaccgcgccgggctgtggctgattaaaacagaactgctggaaacgcagaccgtggatttcagcgtcggcgca gaagggcttcgccatgtaccgggcgatgttattgaaatctgcgatgatgactatgccggtatcagcaccggtggtcgtgtgctggcggtgaacagcca gacccggacgctgacgctcgaccgtgaaatcacgctgccatcctccggtaccgcgctgataagcctggttgacggaagtggcaatccggtcagcgtgg aggttcagtccgtcaccgacggcgtgaaggtaaaagtgagccgtgttcctgacggtgttgctgaatacagcgtatgggagctgaagctgccgacgctg cgccagcgactgttccgctgcgtgagtatccgtgagaacgacgacggcacgtatgccatcaccgccgtgcagcatgtgccggaaaaagaggccatcg tggataacggggcgcactttgacggcgaacagagtggcacggtgaatggtgtcacgccgccagcggtgcagcacctgaccgcagaagtcactgcag acagcggggaatatcaggtgctggcgcgatgggacacaccgaaggtggtgaagggcgtgagtttcctgctccgtctgaccgtaacagcggacgacg gcagtgagcggctggtcagcacggcccggacgacggaaaccacataccgcttcacgcaactggcgctggggaactacaggctgacagtccgggcgg taaatgcgtgggggcagcagggcgatccggcgtcggtatcgttccggattgccgcaccggcagcaccgtcgaggattgagctgacgccgggctatttt cagataaccgccacgccgcatcttgccgtttatgacccgacggtacagtttgagttctggttctcggaaaagcagattgcggatatcagacaggttgaa accagcacgcgttatcttggtacggcgctgtactggatagccgccagtatcaatatcaaaccgggccatgattattacttttatatccgcagtgtgaaca ccgttggcaaatcggcattcgtggaggccgtcggtcgggcgagcgatgatgcggaaggttacctggattttttcaaaggcaagataaccgaatcccat ctcggcaaggagctgctggaaaaagtcgagctgacggaggataacgccagcagactggaggagttttcgaaagagtggaaggatgccagtgataa gtggaatgccatgtgggctgtcaaaattgagcagaccaaagacggcaaacattatgtcgcgggtattggcctcagcatggaggacacggaggaagg caaactgagccagtttctggttgccgccaatcgtatcgcatttattgacccggcaaacgggaatgaaacgccgatgtttgtggcgcagggcaaccaga tattcatgaacgacgtgttcctgaagcgcctgacggcccccaccattaccagcggcggcaatcctccggccttttccctgacaccggacggaaagctga ccgctaaaaatgcggatatcagtggcagtgtgaatgcgaactccgggacgctcagtaatgtgacgatagctgaaaactgtacgataaacggtacgct gagggcggaaaaaatcgtcggggacattgtaaaggcggcgagcgcggcttttccgcgccagcgtgaaagcagtgtggactggccgtcaggtacccg tactgtcaccgtgaccgatgaccatccttttgatcgccagatagtggtgcttccgctgacgtttcgcggaagtaagcgtactgtcagcggcaggacaac gtattcgatgtgttatctgaaagtactgatgaacggtgcggtgatttatgatggcgcggcgaacgaggcggtacaggtgttctcccgtattgttgacatg ccagcgggtcggggaaacgtgatcctgacgttcacgcttacgtccacacggcattcggcagatattccgccgtatacgtttgccagcgatgtgcaggtt atggtgattaagaaacaggcgctgggcatcagcgtggtctgagtgtgttacagaggttcgtccgggaacgggcgttttattataaaacagtgagaggt gaacgatgcgtaatgtgtgtattgccgttgctgtctttgccgcacttgcggtgacagtcactccggcccgtgcggaaggtggacatggtacgtttacggt gggctattttcaagtgaaaccgggtacattgccgtcgttgtcgggcggggataccggtgtgagtcatctgaaagggattaacgtgaagtaccgttatga gctgacggacagtgtgggggtgatggcttccctggggttcgccgcgtcgaaaaagagcagcacagtgatgaccggggaggatacgtttcactatgag agcctgcgtggacgttatgtgagcgtgatggccggaccggttttacaaatcagtaagcaggtcagtgcgtacgccatggccggagtggctcacagtcg gtggtccggcagtacaatggattaccgtaagacggaaatcactcccgggtatatgaaagagacgaccactgccagggacgaaagtgcaatgcggca tacctcagtggcgtggagtgcaggtatacagattaatccggcagcgtccgtcgttgttgatattgcttatgaaggctccggcagtggcgactggcgtact gacggattcatcgttggggtcggttataaattctgattagccaggtaacacagtgttatgacagcccgccggaaccggtgggcttttttgtggggtgaat atggcagtaaagatttcaggagtcctgaaagacggcacaggaaaaccggtacagaactgcaccattcagctgaaagccagacgtaacagcaccac ggtggtggtgaacacggtgggctcagagaatccggatgaagccgggcgttacagcatggatgtggagtacggtcagtacagtgtcatcctgcaggtt gacggttttccaccatcgcacgccgggaccatcaccgtgtatgaagattcacaaccggggacgctgaatgattttctctgtgccatgacggaggatgat gcccggccggaggtgctgcgtcgtcttgaactgatggtggaagaggtggcgcgtaacgcgtccgtggtggcacagagtacggcagacgcgaagaaa tcagccggcgatgccagtgcatcagctgctcaggtcgcggcccttgtgactgatgcaactgactcagcacgcgccgccagcacgtccgccggacaggc tgcatcgtcagctcaggaagcgtcctccggcgcagaagcggcatcagcaaaggccactgaagcggaaaaaagtgccgcagccgcagagtcctcaa aaaacgcggcggccaccagtgccggtgcggcgaaaacgtcagaaacgaatgctgcagcgtcacaacaatcagccgccacgtctgcctccaccgcgg ccacgaaagcgtcagaggccgccacttcagcacgagatgcggtggcctcaaaagaggcagcaaaatcatcagaaacgaacgcatcatcaagtgcc ggtcgtgcagcttcctcggcaacggcggcagaaaattctgccagggcggcaaaaacgtccgagacgaatgccaggtcatctgaaacagcagcggaa cggagcgcctctgccgcggcagacgcaaaaacagcggcggcggggagtgcgtcaacggcatccacgaaggcgacagaggctgcgggaagtgcggt atcagcatcgcagagcaaaagtgcggcagaagcggcggcaatacgtgcaaaaaattcggcaaaacgtgcagaagatatagcttcagctgtcgcgct tgaggatgcggacacaacgagaaaggggatagtgcagctcagcagtgcaaccaacagcacgtctgaaacgcttgctgcaacgccaaaggcggtta aggtggtaatggatgaaacgaacagaaaagcccactggacagtccggcactgaccggaacgccaacagcaccaaccgcgctcaggggaacaaac aatacccagattgcgaacaccgcttttgtactggccgcgattgcagatgttatcgacgcgtcacctgacgcactgaatacgctgaatgaactggccgca gcgctcgggaatgatccagattttgctaccaccatgactaacgcgcttgcgggtaaacaaccgaagaatgcgacactgacggcgctggcagggctttc cacggcgaaaaataaattaccgtattttgcggaaaatgatgccgccagcctgactgaactgactcaggttggcagggatattctggcaaaaaattccg ttgcagatgttcttgaataccttggggccggtgagaattcggcctttccggcaggtgcgccgatcccgtggccatcagatatcgttccgtctggctacgtc ctgatgcaggggcaggcgtttgacaaatcagcctacccaaaacttgctgtcgcgtatccatcgggtgtgcttcctgatatgcgaggctggacaatcaag gggaaacccgccagcggtcgtgctgtattgtctcaggaacaggatggaattaagtcgcacacccacagtgccagtgcatccggtacggatttgggga cgaaaaccacatcgtcgtttgattacgggacgaaaacaacaggcagtttcgattacggcaccaaatcgacgaataacacgggggctcatgctcacag tctgagcggttcaacaggggccgcgggtgctcatgcccacacaagtggtttaaggatgaacagttctggctggagtcagtatggaacagcaaccatta caggaagtttatccacagttaaaggaaccagcacacagggtattgcttatttatcgaaaacggacagtcagggcagccacagtcactcattgtccggt acagccgtgagtgccggtgcacatgcgcatacagttggtattggtgcgcaccagcatccggttgttatcggtgctcatgcccattctttcagtattggttc acacggacacaccatcaccgttaacgctgcgggtaacgcggaaaacaccgtcaaaaacattgcatttaactatattgtgaggcttgcataatggcatt cagaatgagtgaacaaccacggaccataaaaatttataatctgctggccggaactaatgaatttattggtgaaggtgacgcatatattccgcctcata ccggtctgcctgcaaacagtaccgatattgcaccgccagatattccggctggctttgtggctgttttcaacagtgatgaggcatcgtggcatctcgttga agaccatcggggtaaaaccgtctatgacgtggcttccggcgacgcgttatttatttctgaactcggtccgttaccggaaaattttacctggttatcgccg ggaggggaatatcagaagtggaacggcacagcctgggtgaaggatacggaagcagaaaaactgttccggatccgggaggcggaagaaacaaaaa aaagcctgatgcaggtagccagtgagcatattgcgccgcttcaggatgctgcagatctggaaattgcaacgaaggaagaaacctcgttgctggaagc ctggaagaagtatcgggtgttgctgaaccgtgttgatacatcaactgcacctgatattgagtggcctgctgtccctgttatggagtaatcgttttgtgata tgccgcagaaacgttgtatgaaataacgttctgcggttagttagtatattgtaaagctgagtattggtttatttggcgattattatcttcaggagaataat ggaagttctatgactcaattgttcatagtgtttacatcaccgccaattgcttttaagactgaacgcatgaaatatggtttttcgtcatgttttgagtctgct gttgatatttctaaagtcggttttttttcttcgttttctctaactattttccatgaaatacatttttgattattatttgaatcaattccaattacctgaagtcttt catctataattggcattgtatgtattggtttattggagtagatgcttgcttttctgagccatagctctgatatccaaatgaagccataggcatttgttatttt ggctctgtcagctgcataacgccaaaaaatatatttatctgcttgatcttcaaatgttgtattgattaaatcaattggatggaattgtttatcataaaaaa ttaatgtttgaatgtgataaccgtcctttaaaaaagtcgtttctgcaagcttggctgtatagtcaactaactcttctgtcgaagtgatatttttaggcttatc taccagttttagacgctctttaatatcttcaggaattattttattgtcatattgtatcatgctaaatgacaatttgcttatggagtaatcttttaattttaaat aagttattctcctggcttcatcaaataaagagtcgaatgatgttggcgaaatcacatcgtcacccattggattgtttatttgtatgccaagagagttaca gcagttatacattctgccatagattatagctaaggcatgtaataattcgtaatcttttagcgtattagcgacccatcgtctttctgatttaataatagatga ttcagttaaatatgaaggtaatttcttttgtgcaagtctgactaacttttttataccaatgtttaacatactttcatttgtaataaactcaatgtcattttctt caatgtaagatgaaataagagtagcctttgcctcgctatacatttctaaatcgccttgtttttctatcgtattgcgagaatttttagcccaagccattaatg gatcatttttccatttttcaataacattattgttataccaaatgtcatatcctataatctggtttttgtttttttgaataataaatgttactgttcttgcggtttg gaggaattgattcaaattcaagcgaaataattcagggtcaaaatatgtatcaatgcagcatttgagcaagtgcgataaatctttaagtcttctttcccat ggttttttagtcataaaactctccattttgataggttgcatgctagatgctgatatattttagaggtgataaaattaactgcttaactgtcaatgtaataca agttgtttgatctttgcaatgattcttatcagaaaccatatagtaaattagttacacaggaaatttttaatattattattatcattcattatgtattaaaatt agagttgtggcttggctctgctaacacgttgctcataggagatatggtagagccgcagacacgtcgtatgcaggaacgtgctgcggctggctggtgaa cttccgatagtgcgggtgttgaatgatttccagttgctaccgattttacatattttttgcatgagagaatttgtaccacctcccaccgaccatctatgactg tacgccactgtccctaggactgctatgtgccggagcggacattacaaacgtccttctcggtgcatgccactgttgccaatgacctgcctaggaattggtt agcaagttactaccggattttgtaaaaacagccctcctcatataaaaagtattcgttcacttccgataagcgtcgtaattttctatctttcatcatattcta gatccctctgaaaaaatcttccgagtttgctaggcactgatacataactcttttccaataattggggaagtcattcaaatctataataggtttcagatttg cttcaataaattctgactgtagctgctgaaacgttgcggttgaactatatttccttataacttttacgaaagagtttctttgagtaatcacttcactcaagt gcttccctgcctccaaacgatacctgttagcaatatttaatagcttgaaatgatgaagagctctgtgtttgtcttcctgcctccagttcgccgggcattca acataaaaactgatagcacccggagttccggaaacgaaatttgcatatacccattgctcacgaaaaaaaatgtccttgtcgatatagggatgaatcgc ttggtgtacctcatctactgcgaaaacttgacctttctctcccatattgcagtcgcggcacgatggaactaaattaataggcatcaccgaaaattcagga taatgtgcaataggaagaaaatgatctatattttttgtctgtcctatatcaccacaaaatggacatttttcacctgatgaaacaagcatgtcatcgtaat atgttctagcgggtttgtttttatctcggagattattttcataaagcttttctaatttaacctttgtcaggttaccaactactaaggttgtaggctcaagagg gtgtgtcctgtcgtaggtaaataactgacctgtcgagcttaatattctatattgttgttctttctgcaaaaaagtggggaagtgagtaatgaaattatttct aacatttatctgcatcataccttccgagcatttattaagcatttcgctataagttctcgctggaagaggtagttttttcattgtactttaccttcatctctgtt cattatcatcgcttttaaaacggttcgaccttctaatcctatctgaccattataattttttagaatggtttcataagaaagctctgaatcaacggactgcga taataagtggtggtatccagaatttgtcacttcaagtaaaaacacctcacgagttaaaacacctaagttctcaccgaatgtctcaatatccggacggat aatatttattgcttctcttgaccgtaggactttccacatgcaggattttggaacctcttgcagtactactggggaatgagttgcaattattgcta caeca tt gcgtgcatcgagtaagtcgcttaatgttcgtaaaaaagcagagagcaaaggtggatgcagatgaacctctggttcatcgaataaaactaatgactttt cgccaacgacatctactaatcttgtgatagtaaataaaacaattgcatgtccagagctcattcgaagcagatatttctggatattgtcataaaacaattt agtgaatttatcatcgtccacttgaatctgtggttcattacgtcttaactcttcatatttagaaatgaggctgatgagttccatatttgaaaagttttcatca ctacttagttttttgatagcttcaagccagagttgtctttttctatctactctcatacaaccaataaatgctgaaatgaattctaagcggagatcgcctagt gattttaaactattgctggcagcattcttgagtccaatataaaagtattgtgtaccttttgctgggtcaggttgttctttaggaggagtaaaaggatcaaa tgcactaaacgaaactgaaacaagcgatcgaaaatatccctttgggattcttgactcgataagtctattattttcagagaaaaaatattcattgttttct gggttggtgattgcaccaatcattccattcaaaattgttgttttaccacacccattccgcccgataaaagcatgaatgttcgtgctgggcatagaattaa ccgtcacctcaaaaggtatagttaaatcactgaatccgggagcactttttctattaaatgaaaagtggaaatctgacaattctggcaaaccatttaaca cacgtgcgaactgtccatgaatttctgaaagagttacccctctaagtaatgaggtgttaaggacgctttcattttcaatgtcggctaatcgatttggccat actactaaatcctgaatagctttaagaaggttatgtttaaaaccatcgcttaatttgctgagattaacatagtagtcaatgctttcacctaaggaaaaaa acatttcagggagttgactgaattttttatctattaatgaataagtgcttacttcttctttttgacctacaaaaccaattttaacatttccgatatcgcatttt tcaccatgctcatcaaagacagtaagataaaacattgtaacaaaggaatagtcattccaaccatctgctcgtaggaatgccttatttttttctactgcag gaatatacccgcctctttcaataacactaaactccaacatatagtaacccttaattttattaaaataaccgcaatttatttggcggcaacacaggatctc tcttttaagttactctctattacatacgttttccatctaaaaattagtagtattgaacttaacggggcatcgtattgtagttttccatatttagctttctgcttc cttttggataacccactgttattcatgttgcatggtgcactgtttataccaacgatatagtctattaatgcatatatagtatcgccgaacgattagctcttc aggcttctgaagaagcgtttcaagtactaataagccgatagatagccacggacttcgtagccatttttcataagtgttaacttccgctcctcgctcataa cagacattcactacagttatggcggaaaggtatgcatgctgggtgtggggaagtcgtgaaagaaaagaagtcagctgcgtcgtttgacatcactgcta tcttcttactggttatgcaggtcgtagtgggtggcacacaaagctttgcactggattgcgaggctttgtgcttctctggagtgcgacaggtttgatgacaa aaaattagcgcaagaagacaaaaatcaccttgcgctaatgctctgttacaggtcactaataccatctaagtagttgattcatagtgactgcatatgttgt gttttacagtattatgtagtctgttttttatgcaaaatctaatttaatatattgatatttatatcattttacgtttctcgttcagcttttttatactaagttggca ttataaaaaagcattgcttatcaatttgttgcaacgaacaggtcactatcagtcaaaataaaatcattatttgatttcaattttgtcccactccctgcctct gtcatcacgatactgtgatgccatggtgtccgacttatgcccgagaagatgttgagcaaacttatcgcttatctgcttctcatagagtcttgcagacaaa ctgcgcaactcgtgaaaggtaggcggatccccttcgaaggaaagacctgatgcttttcgtgcgcgcataaaataccttgatactgtgccggatgaaag cggttcgcgacgagtagatgcaattatggtttctccgccaagaatctctttgcatttatcaagtgtttccttcattgatattccgagagcatcaatatgca atgctgttgggatggcaatttttacgcctgttttgctttgctcgacataaagatatccatctacgatatcagaccacttcatttcgcataaatcaccaactc gttgcccggtaacaacagccagttccattgcaagtctgagccaacatggtgatgattctgctgcttgataaattttcaggtattcgtcagccgtaagtctt gatctccttacctctgattttgctgcgcgagtggcagcgacatggtttgttgttatatggccttcagctattgcctctcggaatgcatcgctcagtgttgat ctgattaacttggctgacgccgccttgccctcgtctatgtatccattgagcattgccgcaatttcttttgtggtgatgtcttcaagtggagcatcaggcaga cccctccttattgctttaattttgctcatgtaatttatgagtgtcttctgcttgattcctctgctggccaggattttttcgtagcgatcaagccatgaatgtaa cgtaacggaattatcactgttgattctcgctgtcagaggcttgtgtttgtgtcctgaaaataactcaatgttggcctgtatagcttcagtgattgcgattcg cctgtctctgcctaatccaaactctttacccgtccttgggtccctgtagcagtaatatccattgtttcttatataaaggttagggggtaaatcccggcgctc atgacttcgccttcttcccatttctgatcctcttcaaaaggccacctgttactggtcgatttaagtcaacctttaccgctgattcgtggaacagatactctct tccatccttaaccggaggtgggaatatcctgcattcccgaacccatcgacgaactgtttcaaggcttcttggacgtcgctggcgtgcgttccactcctga agtgtcaagtacatcgcaaagtctccgcaattacacgcaagaaaaaaccgccatcaggcggcttggtgttctttcagttcttcaattcgaatattggtta cgtctgcatgtgctatctgcgcccatatcatccagtggtcgtagcagtcgttgatgttctccgcttcgataactctgttgaatggctctccattccattctcc tgtgactcggaagtgcatttatcatctccataaaacaaaacccgccgtagcgagttcagataaaataaatccccgcgagtgcgaggattgttatgtaat attgggtttaatcatctatatgttttgtacagagagggcaagtatcgtttccaccgtactcgtgataataattttgcacggtatcagtcatttctcgcacat tgcagaatggggatttgtcttcattagacttataaaccttcatggaatatttgtatgccgactctatatctataccttcatctacataaacaccttcgtgat gtctgcatggagacaagacaccggatctgcacaacattgataacgcccaatctttttgctcagactctaactcattgatactcatttataaactccttgc aatgtatgtcgtttcagctaaacggtatcagcaatgtttatgtaaagaaacagtaagataatactcaacccgatgtttgagtacggtcatcatctgaca ctacagactctggcatcgctgtgaagacgacgcgaaattcagcattttcacaagcgttatcttttacaaaaccgatctcactctcctttgatgcgaatgc cagcgtcagacatcatatgcagatactcacctgcatcctgaacccattgacctccaaccccgtaatagcgatgcgtaatgatgtcgatagttactaacg ggtcttgttcgattaactgccgcagaaactcttccaggtcaccagtgcagtgcttgataacaggagtcttcccaggatggcgaacaacaagaaactgg tttccgtcttcacggacttcgttgctttccagtttagcaatacgcttactcccatccgagataacaccttcgtaatactcacgctgctcgttgagttttgatt ttgctgtttcaagctcaacacgcagtttccctactgttagcgcaatatcctcgttctcctggtcgcggcgtttgatgtattgctggtttctttcccgttcatcc agcagttccagcacaatcgatggtgttaccaattcatggaaaaggtctgcgtcaaatccccagtcgtcatgcattgcctgctctgccgcttcacgcagtg cctgagagttaatttcgctcacttcgaacctctctgtttactgataagttccagatcctcctggcaacttgcacaagtccgacaaccctgaacgaccagg cgtcttcgttcatctatcggatcgccacactcacaacaatgagtggcagatatagcctggtggttcaggcggcgcatttttattgctgtgttgcgctgtaa ttcttctatttctgatgctgaatcaatgatgtctgccatctttcattaatccctgaactgttggttaatacgcttgagggtgaatgcgaataataaaaaag gagcctgtagctccctgatgattttgcttttcatgttcatcgttccttaaagacgccgtttaacatgccgattgccaggcttaaatgagtcggtgtgaatcc catcagcgttaccgtttcgcggtgcttcttcagtacgctacggcaaatgtcatcgacgtttttatccggaaactgctgtctggctttttttgatttcagaatt agcctgacgggcaatgctgcgaagggcgttttcctgctgaggtgtcattgaacaagtcccatgtcggcaagcataagcacacagaatatgaagcccg ctgccagaaaaatgcattccgtggttgtcatacctggtttctctcatctgcttctgctttcgccaccatcatttccagcttttgtgaaagggatgcggctaa cgtatgaaattcttcgtctgtttctactggtattggcacaaacctgattccaatttgagcaaggctatgtgccatctcgatactcgttcttaactcaacaga agatgctttgtgcatacagcccctcgtttattatttatctcctcagccagccgctgtgctttcagtggatttcggataacagaaaggccgggaaataccc agcctcgctttgtaacggagtagacgaaagtgattgcgcctacccggatattatcgtgaggatgcgtcatcgccattgctccccaaatacaaaaccaat ttcagccagtgcctcgtccattttttcgatgaactccggcacgatctcgtcaaaactcgccatgtacttttcatcccgctcaatcacgacataatgcaggc cttcacgcttcatacgcgggtcatagttggcaaagtaccaggcattttttcgcgtcacccacatgctgtactgcacctgggccatgtaagctgactttatg gcctcgaaaccaccgagccggaacttcatgaaatcccgggaggtaaacgggcatttcagttcaaggccgttgccgtcactgcataaaccatcgggag agcaggcggtacgcatactttcgtcgcgatagatgatcggggattcagtaacattcacgccggaagtgaattcaaacagggttctggcgtcgttctcgt actgttttccccaggccagtgctttagcgttaacttccggagccacaccggtgcaaacctcagcaagcagggtgtggaagtaggacattttcatgtcag gccacttctttccggagcggggttttgctatcacgttgtgaacttctgaagcggtgatgacgccgagccgtaatttgtgccacgcatcatccccctgttcg acagctctcacatcgatcccggtacgctgcaggataatgtccggtgtcatgctgccaccttctgctctgcggctttctgtttcaggaatccaagagctttt actgcttcggcctgtgtcagttctgacgatgcacgaatgtcgcggcgaaatatctgggaacagagcggcaataagtcgtcatcccatgttttatccagg gcgatcagcagagtgttaatctcctgcatggtttcatcgttaaccggagtgatgtcgcgttccggctgacgttctgcagtgtatgcagtattttcgacaat gcgctcggcttcatccttgtcatagataccagcaaatccgaaggccagacgggcacactgaatcatggctttatgacgtaacatccgtttgggatgcga ctgccacggccccgtgatttctctgccttcgcgagttttgaatggttcgcggcggcattcatccatccattcggtaacgcagatcggatgattacggtcct tgcggtaaatccggcatgtacaggattcattgtcctgctcaaagtccatgccatcaaactgctggttttcattgatgatgcgggaccagccatcaacgcc caccaccggaacgatgccattctgcttatcaggaaaggcgtaaatttctttcgtccacggattaaggccgtactggttggcaacgatcagtaatgcgat gaactgcgcatcgctggcatcacctttaaatgccgtctggcgaagagtggtgatcagttcctgtgggtcgacagaatccatgccgacacgttcagccag cttcccagccagcgttgcgagtgcagtactcattcgttttatacctctgaatcaatatcaacctggtggtgagcaatggtttcaaccatgtaccggatgtg ttctgccatgcgctcctgaaactcaacatcgtcatcaaacgcacgggtaatggattttttgctggccccgtggcgttgcaaatgatcgatgcatagcgat tcaaacaggtgctggggcaggcctttttccatgtcgtctgccagttctgcctctttctcttcacgggcgagctgctggtagtgacgcgcccagctctgagc ctcaagacgatcctgaatgtaataagcgttcatggctgaactcctgaaatagctgtgaaaatatcgcccgcgaaatgccgggctgattaggaaaaca ggaaagggggttagtgaatgcttttgcttgatctcagtttcagtattaatatccattttttataagcgtcgacggcttcacgaaacatcttttcatcgccaa taaaagtggcgatagtgaatttagtctggatagccataagtgtttgatccattctttgggactcctggctgattaagtatgtcgataaggcgtttccatcc gtcacgtaatttacgggtgattcgttcaagtaaagattcggaagggcagccagcaacaggccaccctgcaatggcatattgcatggtgtgctccttatt tatacataacgaaaaacgcctcgagtgaagcgttattggtatgcggtaaaaccgcactcaggcggccttgatagtcatatcatctgaatcaaatattcc tgatgtatcgatatcggtaattcttattccttcgctaccatccattggaggccatccttcctgaccatttccatcattccagtcgaactcacacacaacacc atatgcatttaagtcgcttgaaattgctataagcagagcatgttgcgccagcatgattaatacagcatttaatacagagccgtgtttattgagtcggtat tcagagtctgaccagaaattattaatctggtgaagtttttcctctgtcattacgtcatggtcgatttcaatttctattgatgctttccagtcgtaatcaatga tgtattttttgatgtttgacatctgttcatatcctcacagataaaaaatcgccctcacactggagggcaaagaagatttccaataatcagaacaagtcgg ctcctgtttagttacgagcgacattgctccgtgtattcactcgttggaatgaatacacagtgcagtgtttattctgttatttatgccaaaaataaaggcca ctatcaggcagctttgttgttctgtttaccaagttctctggcaatcattgccgtcgttcgtattgcccatttatcgacatatttcccatcttccattacagga aacatttcttcaggcttaaccatgcattccgattgcagcttgcatccattgcatcgcttgaattgtccacaccattgatttttatcaatagtcgtagtcata cggatagtcctggtattgttccatcacatcctgaggatgctcttcgaactcttcaaattcttcttccatatatcaccttaaatagtggattgcggtagtaaa gattgtgcctgtcttttaaccacatcaggctcggtggttctcgtgtacccctacagcgagaaatcggataaactattacaacccctacagtttgatgagt atagaaatggatccactcgttattctcggacgagtgttcagtaatgaacctctggagagaaccatgtatatgatcgttatctgggttggacttctgctttt aagcccagataactggcctgaatatgttaatgagagaatcggtattcctcatgtgtggcatgttttcgtctttgctcttgcattttcgctagcaattaatgt gcatcgattatcagctattgccagcgccagatataagcgatttaagctaagaaaacgcattaagatgcaaaacgataaagtgcgatcagtaattcaa aaccttacagaagagcaatctatggttttgtgcgcagcccttaatgaaggcaggaagtatgtggttacatcaaaacaattcccatacattagtgagttg attgagcttggtgtgttgaacaaaactttttcccgatggaatggaaagcatatattattccctattgaggatatttactggactgaattagttgccagcta tgatccatataatattgagataaagccaaggccaatatctaagtaactagataagaggaatcgattttcccttaattttctggcgtccactgcatgttat gccgcgttcgccaggcttgctgtaccatgtgcgctgattcttgcgctcaatacgttgcaggttgctttcaatctgtttgtggtattcagccagcactgtaag gtctatcggatttagtgcgctttctactcgtgatttcggtttgcgattcagcgagagaatagggcggttaactggttttgcgcttaccccaaccaacaggg gatttgctgctttccattgagcctgtttctctgcgcgacgttcgcggcggcgtgtttgtgcatccatctggattctcctgtcagttagctttggtggtgtgtg gcagttgtagtcctgaacgaaaaccccccgcgattggcacattggcagctaatccggaatcgcacttacggccaatgcttcgtttcgtatcacacaccc caaagccttctgctttgaatgctgcccttcttcagggcttaatttttaagagcgtcaccttcatggtggtcagtgcgtcctgctgatgtgctcagtatcacc gccagtggtatttatgtcaacaccgccagagataatttatcaccgcagatggttatctgtatgttttttatatgaatttattttttgcaggggggcattgttt ggtaggtgagagatctgaattgctatgtttagtgagttgtatctatttatttttcaataaatacaattggttatgtgttttgggggcgatcgtgaggcaaa gaaaacccggcgctgaggccgggacgttaaatctatcaccgcaagggataaatatctaacaccgtgcgtgttgactattttacctctggcggtgataat ggttgcatgtactaaggaggttgtatggaacaacgcataaccctgaaagattatgcaatgcgctttgggcaaaccaagacagctaaagatctcggcgt atatcaaagcgcgatcaacaaggccattcatgcaggccgaaagatttttttaactataaacgctgatggaagcgtttatgcggaagaggtaaagccct tcccgagtaacaaaaaaacaacagcataaataaccccgctcttacacattccagccctgaaaaagggcatcaaattaaaccacacctatggtgtatg catttatttgcatacattcaatcaattgttatctaaggaaatacttacatatggttcgtgcaaacaaacgcaacgaggctctacgaatcgagagtgcgtt gcttaacaaaatcgcaatgcttggaactgagaagacagcggaagctgtgggcgttgataagtcgcagatcagcaggtggaagagggactggattcc aaagttctcaatgctgcttgctgttcttgaatggggggtcgttgacgacgacatggctcgattggcgcgacaagttgctgcgattctcaccaataaaaa acgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctatcaacaggagtcattatgacaaatacagcaa aaatactcaacttcggcagaggtaactttgccggacaggagcgtaatgtggcagatctcgatgatggttacgccagactatcaaatatgctgcttgag gcttattcgggcgcagatctgaccaagcgacagtttaaagtgctgcttgccattctgcgtaaaacctatgggtggaataaaccaatggacagaatcac cgattctcaacttagcgagattacaaagttacctgtcaaacggtgcaatgaagccaagttagaactcgtcagaatgaatattatcaagcagcaaggcg gcatgtttggaccaaataaaaacatctcagaatggtgcatccctcaaaacgagggaaaatcccctaaaacgagggataaaacatccctcaaattggg ggattgctatccctcaaaacagggggacacaaaagacactattacaaaagaaaaaagaaaagattattcgtcagagaattctggcgaatcctctgac cagccagaaaacgacctttctgtggtgaaaccggatgctgcaattcagagcggcagcaagtgggggacagcagaagacctgaccgccgcagagtgg atgtttgacatggtgaagactatcgcaccatcagccagaaaaccgaattttgctgggtgggctaacgatatccgcctgatgcgtgaacgtgacggacg taaccaccgcgacatgtgtgtgctgttccgctgggcatgccaggacaacttctggtccggtaacgtgctgagcccggccaaactccgcgataagtgga cccaactcgaaatcaaccgtaacaagcaacaggcaggcgtgacagccagcaaaccaaaactcgacctgacaaacacagactggatttacggggtg gatctatgaaaaacatcgccgcacagatggttaactttgaccgtgagcagatgcgtcggatcgccaacaacatgccggaacagtacgacgaaaagc cgcaggtacagcaggtagcgcagatcatcaacggtgtgttcagccagttactggcaactttcccggcgagcctggctaaccgtgaccagaacgaagt gaacgaaatccgtcgccagtgggttctggcttttcgggaaaacgggatcaccacgatggaacaggttaacgcaggaatgcgcgtagcccgtcggcag aatcgaccatttctgccatcacccgggcagtttgttgcatggtgccgggaagaagcatccgttaccgccggactgccaaacgtcagcgagctggttgat atggtttacgagtattgccggaagcgaggcctgtatccggatgcggagtcttatccgtggaaatcaaacgcgcactactggctggttaccaacctgtat cagaacatgcgggccaatgcgcttactgatgcggaattacgccgtaaggccgcagatgagcttgtccatatgactgcgagaattaaccgtggtgaggc gatccctgaaccagtaaaacaacttcctgtcatgggcggtagacctctaaatcgtgcacaggctctggcgaagatcgcagaaatcaaagctaagttcg gactgaaaggagcaagtgtatgacgggcaaagaggcaattattcattacctggggacgcataatagcttctgtgcgccggacgttgccgcgctaaca ggcgcaacagtaaccagcataaatcaggccgcggctaaaatggcacgggcaggtcttctggttatcgaaggtaaggtctggcgaacggtgtattacc ggtttgctaccagggaagaacgggaaggaaagatgagcacgaacctggtttttaaggagtgtcgccagagtgccgcgatgaaacgggtattggcgg tatatggagttaaaagatgaccatctacattactgagctaataacaggcctgctggtaatcgcaggcctttttatttgggggagagggaagtcatgaaa aaactaacctttgaaattcgatctccagcacatcagcaaaacgctattcacgcagtacagcaaatccttccagacccaaccaaaccaatcgtagtaac cattcaggaacgcaaccgcagcttagaccaaaacaggaagctatgggcctgcttaggtgacgtctctcgtcaggttgaatggcatggtcgctggctgg atgcagaaagctggaagtgtgtgtttaccgcagcattaaagcagcaggatgttgttcctaaccttgccgggaatggctttgtggtaataggccagtcaa ccagcaggatgcgtgtaggcgaatttgcggagctattagagcttatacaggcattcggtacagagcgtggcgttaagtggtcagacgaagcgagact ggctctggagtggaaagcgagatggggagacagggctgcatgataaatgtcgttagtttctccggtggcaggacgtcagcatatttgctctggctaat ggagcaaaagcgacgggcaggtaaagacgtgcattacgttttcatggatacaggttgtgaacatccaatgacatatcggtttgtcagggaagttgtga agttctgggatataccgctcaccgtattgcaggttgatatcaacccggagcttggacagccaaatggttatacggtatgggaaccaaaggatattcag acgcgaatgcctgttctgaagccatttatcgatatggtaaagaaatatggcactccatacgtcggcggcgcgttctgcactgacagattaaaactcgtt cccttcaccaaatactgtgatgaccatttcgggcgagggaattacaccacgtggattggcatcagagctgatgaaccgaagcggctaaagccaaagc ctggaatcagatatcttgctgaactgtcagactttgagaaggaagatatcctcgcatggtggaagcaacaaccattcgatttgcaaataccggaacat ctcggtaactgcatattctgcattaaaaaatcaacgcaaaaaatcggacttgcctgcaaagatgaggagggattgcagcgtgtttttaatgaggtcatc acgggatcccatgtgcgtgacggacatcgggaaacgccaaaggagattatgtaccgaggaagaatgtcgctggacggtatcgcgaaaatgtattca gaaaatgattatcaagccctgtatcaggacatggtacgagctaaaagattcgataccggctcttgttctgagtcatgcgaaatatttggagggcagctt gatttcgacttcgggagggaagctgcatgatgcgatgttatcggtgcggtgaatgcaaagaagataaccgcttccgaccaaatcaaccttactggaat cgatggtgtctccggtgtgaaagaacaccaacaggggtgttaccactaccgcaggaaaaggaggacgtgtggcgagacagcgacgaagtatcaccg acataatctgcgaaaactgcaaataccttccaacgaaacgcaccagaaataaacccaagccaatcccaaaagaatctgacgtaaaaaccttcaact acacggctcacctgtgggatatccggtggctaagacgtcgtgcgaggaaaacaaggtgattgaccaaaatcgaagttacgaacaagaaagcgtcga gcgagctttaacgtgcgctaactgcggtcagaagctgcatgtgctggaagttcacgtgtgtgagcactgctgcgcagaactgatgagcgatccgaata gctcgatgcacgaggaagaagatgatggctaaaccagcgcgaagacgatgtaaaaacgatgaatgccgggaatggtttcaccctgcattcgctaatc agtggtggtgctctccagagtgtggaaccaagatagcactcgaacgacgaagtaaagaacgcgaaaaagcggaaaaagcagcagagaagaaacg acgacgagaggagcagaaacagaaagataaacttaagattcgaaaactcgccttaaagccccgcagttactggattaaacaagcccaacaagccgt aaacgccttcatcagagaaagggaccgcgacttaccatgtatctcgtgcggaacgctcacgtctgctcagtgggatgccggacattaccggacaactg ctgcggcacctcaactccgatttaatgaacgcaatattcacaagcaatgcgtggtgtgcaaccagcacaaaagcggaaatctcgttccgtatcgcgtc gaactgattagccgcatcgggcaggaagcagtagacgaaatcgaatcaaaccataaccgccatcgctggactatcgaagagtgcaaggcgatcaag gcagagtaccaacagaaactcaaagacctgcgaaatagcagaagtgaggccgcatgacgttctcagtaaaaaccattccagacatgctcgttgaag catacggaaatcagacagaagtagcacgcagactgaaatgtagtcgcggtacggtcagaaaatacgttgatgataaagacgggaaaatgcacgcc atcgtcaacgacgttctcatggttcatcgcggatggagtgaaagagatgcgctattacgaaaaaattgatggcagcaaataccgaaatatttgggtag ttggcgatctgcacggatgctacacgaacctgatgaacaaactggatacgattggattcgacaacaaaaaagacctgcttatctcggtgggcgatttg gttgatcgtggtgcagagaacgttgaatgcctggaattaatcacattcccctggttcagagctgtacgtggaaaccatgagcaaatgatgattgatggc ttatcagagcgtggaaacgttaatcactggctgcttaatggcggtggctggttctttaatctcgattacgacaaagaaattctggctaaagctcttgccc ataaagcagatgaacttccgttaatcatcgaactggtgagcaaagataaaaaatatgttatctgccacgccgattatccctttgacgaatacgagtttg gaaagccagttgatcatcagcaggtaatctggaaccgcgaacgaatcagcaactcacaaaacgggatcgtgaaagaaatcaaaggcgcggacacg ttcatctttggtcatacgccagcagtgaaaccactcaagtttgccaaccaaatgtatatcgataccggcgcagtgttctgcggaaacctaacattgattc aggtacagggagaaggcgcatgagactcgaaagcgtagctaaatttcattcgccaaaaagcccgatgatgagcgactcaccacgggccacggcttc tgactctctttccggtactgatgtgatggctgctatggggatggcgcaatcacaagccggattcggtatggctgcattctgcggtaagcacgaactcag ccagaacgacaaacaaaaggctatcaactatctgatgcaatttgcacacaaggtatcggggaaataccgtggtgtggcaaagcttgaaggaaatact aaggcaaaggtactgcaagtgctcgcaacattcgcttatgcggattattgccgtagtgccgcgacgccgggggcaagatgcagagattgccatggtac aggccgtgcggttgatattgccaaaacagagctgtgggggagagttgtcgagaaagagtgcggaagatgcaaaggcgtcggctattcaaggatgcc agcaagcgcagcatatcgcgctgtgacgatgctaatcccaaaccttacccaacccacctggtcacgcactgttaagccgctgtatgacgctctggtggt gcaatgccacaaagaagagtcaatcgcagacaacattttgaatgcggtcacacgttagcagcatgattgccacggatggcaacatattaacggcatg atattgacttattgaataaaattgggtaaatttgactcaacgatgggttaattcgctcgttgtggtagtgagatgaaaagaggcggcgcttactaccgat tccgcctagttggtcacttcgacgtatcgtctggaactccaaccatcgcaggcagagaggtctgcaaaatgcaatcccgaaacagttcgcaggtaata gttagagcctgcataacggtttcgggattttttatatctgcacaacaggtaagagcattgagtcgataatcgtgaagagtcggcgagcctggttagcca gtgctctttccgttgtgctgaattaagcgaataccggaagcagaaccggatcaccaaatgcgtacaggcgtcatcgccgcccagcaacagcacaacc caaactgagccgtagccactgtctgtcctgaattcattagtaatagttacgctgcggccttttacacatgaccttcgtgaaagcgggtggcaggaggtc gcgctaacaacctcctgccgttttgcccgtgcatatcggtcacgaacaaatctgattactaaacacagtagcctggatttgttctatcagtaatcgacctt attcctaattaaatagagcaaatccccttattgggggtaagacatgaagatgccagaaaaacatgacctgttggccgccattctcgcggcaaaggaac aaggcatcggggcaatccttgcgtttgcaatggcgtaccttcgcggcagatataatggcggtgcgtttacaaaaacagtaatcgacgcaacgatgtgc gccattatcgcctggttcattcgtgaccttctcgacttcgccggactaagtagcaatctcgcttatataacgagcgtgtttatcggctacatcggtactga ctcgattggttcgcttatcaaacgcttcgctgctaaaaaagccggagtagaagatggtagaaatcaataatcaacgtaaggcgttcctcgatatgctgg cgtggtcggagggaactgataacggacgtcagaaaaccagaaatcatggttatgacgtcattgtaggcggagagctatttactgattactccgatcac cctcgcaaacttgtcacgctaaacccaaaactcaaatcaacaggcgccggacgctaccagcttctttcccgttggtgggatgcctaccgcaagcagctt ggcctgaaagacttctctccgaaaagtcaggacgctgtggcattgcagcagattaaggagcgtggcgctttacctatgattgatcgtggtgatatccgt caggcaatcgaccgttgcagcaatatctgggcttcactgccgggcgctggttatggtcagttcgagcataaggctgacagcctgattgcaaaattcaa agaagcgggcggaacggtcagagagattgatgtatgagcagagtcaccgcgattatctccgctctggttatctgcatcatcgtctgcctgtcatgggct gttaatcattaccgtgataacgccattacctacaaagcccagcgcgacaaaaatgccagagaactgaagctggcgaacgcggcaattactgacatgc agatgcgtcagcgtgatgttgctgcgctcgatgcaaaatacacgaaggagttagctgatgctaaagctgaaaatgatgctctgcgtgatgatgttgcc gctggtcgtcgtcggttgcacatcaaagcagtctgtcagtcagtgcgtgaagccaccaccgcctccggcgtggataatgcagcctccccccgactggc agacaccgctgaacgggattatttcaccctcagagagaggctgatcactatgcaaaaacaactggaaggaacccagaagtatattaatgagcagtgc agatagagttgcccatatcgatgggcaactcatgcaattattgtgagcaatacacacgcgcttccagcggagtataaatgcctaaagtaataaaaccg agcaatccatttacgaatgtttgctgggtttctgttttaacaacattttctgcgccgccacaaattttggctgcatcgacagttttcttctgcccaattccag aaacgaagaaatgatgggtgatggtttcctttggtgctactgctgccggtttgttttgaacagtaaacgtctgttgagcacatcctgtaataagcagggc cagcgcagtagcgagtagcatttttttcatggtgttattcccgatgctttttgaagttcgcagaatcgtatgtgtagaaaattaaacaaaccctaaacaa tgagttgaaatttcatattgttaatatttattaatgtatgtcaggtgcgatgaatcgtcattgtattcccggattaactatgtccacagccctgacgggga acttctctgcgggagtgtccgggaataattaaaacgatgcacacagggtttagcgcgtacacgtattgcattatgccaacgccccggtgctgacacgg aagaaaccggacgttatgatttagcgtggaaagatttgtgtagtgttctgaatgctctcagtaaatagtaatgaattatcaaaggtatagtaatatcttt tatgttcatggatatttgtaacccatcggaaaactcctgctttagcaagattttccctgtattgctgaaatgtgatttctcttgatttcaacctatcatagga cgtttctataagatgcgtgtttcttgagaatttaacatttacaacctttttaagtccttttattaacacggtgttatcgttttctaacacgatgtgaatattat ctgtggctagatagtaaatataatgtgagacgttgtgacgttttagttcagaataaaacaattcacagtctaaatcttttcgcacttgatcgaatatttct ttaaaaatggcaacctgagccattggtaaaaccttccatgtgatacgagggcgcgtagtttgcattatcgtttttatcgtttcaatctggtctgacctcctt gtgttttgttgatgatttatgtcaaatattaggaatgttttcacttaatagtattggttgcgtaacaaagtgcggtcctgctggcattctggagggaaatac aaccgacagatgtatgtaaggccaacgtgctcaaatcttcatacagaaagatttgaagtaatattttaaccgctagatgaagagcaagcgcatggag cgacaaaatgaataaagaacaatctgctgatgatccctccgtggatctgattcgtgtaaaaaatatgcttaatagcaccatttctatgagttaccctgat gttgtaattgcatgtatagaacataaggtgtctctggaagcattcagagcaattgaggcagcgttggtgaagcacgataataatatgaaggattattcc ctggtggttgactgatcaccataactgctaatcattcaaactatttagtctgtgacagagccaacacgcagtctgtcactgtcaggaaagtggtaaaac tgcaactcaattactgcaatgccctcgtaattaagtgaatttacaatatcgtcctgttcggagggaagaacgcgggatgttcattcttcatcacttttaat tgatgtatatgctctcttttctgacgttagtctccgacggcaggcttcaatgacccaggctgagaaattcccggaccctttttgctcaagagcgatgttaa tttgttcaatcatttggttaggaaagcggatgttgcgggttgttgttctgcgggttctgttcttcgttgacatgaggttgccccgtattcagtgtcgctgatt tgtattgtctgaagttgtttttacgttaagttgatgcagatcaattaatacgatacctgcgtcataattgattatttgacgtggtttgatggcctccacgca cgttgtgatatgtagatgataatcattatcactttacgggtcctttccggtgatccgacaggttacg

Appendix II: Sequence of Lambda phage reboot Design II (virulent lambda, EcoKI MTase) (SEQ ID NO: 3)

(sequence begins at COS site) gggcggcgacctcgcgggttttcgctatttatgaaaattttccggtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaataccctct gaaaagaaaggaaacgacaggtgctgaaagcgaggctttttggcctctgtcgtttcctttctctgtttttgtccgtggaatgaacaatggaagtcaaca aaaagcagctggctgacattttcggtgcgagtatccgtaccattcagaactggcaggaacagggaatgcccgttctgcgaggcggtggcaagggtaa tgaggtgctttatgactctgccgccgtcataaaatggtatgccgaaagggatgctgaaattgagaacgaaaagctgcgccgggaggttgaagaactg cggcaggccagcgaggcagatctccagccaggaactattgagtacgaacgccatcgacttacgcgtgcgcaggccgacgcacaggaactgaagaat gccagagactccgctgaagtggtggaaaccgcattctgtactttcgtgctgtcgcggatcgcaggtgaaattgccagtattctcgacgggctccccctgt cggtgcagcggcgttttccggaactggaaaaccgacatgttgatttcctgaaacgggatatcatcaaagccatgaacaaagcagccgcgctggatga actgataccggggttgctgagtgaatatatcgaacagtcaggttaacaggctgcggcattttgtccgcgccgggcttcgctcactgttcaggccggagc cacagaccgccgttgaatgggcggatgctaattactatctcccgaaagaatccgcataccaggaagggcgctgggaaacactgccctttcagcgggc catcatgaatgcgatgggcagcgactacatccgtgaggtgaatgtggtgaagtctgcccgtgtcggttattccaaaatgctgctgggtgtttatgcctac tttatagagcataagcagcgcaacacccttatctggttgccgacggatggtgatgccgagaactttatgaaaacccacgttgagccgactattcgtgat attccgtcgctgctggcgctggccccgtggtatggcaaaaagcaccgggataacacgctcaccatgaagcgtttcactaatgggcgtggcttctggtgc ctgggcggtaaagcggcaaaaaactaccgtgaaaagtcggtggatgtggcgggttatgatgaacttgctgcttttgatgatgatattgaacaggaagg ctctccgacgttcctgggtgacaagcgtattgaaggctcggtctggccaaagtccatccgtggctccacgccaaaagtgagaggcacctgtcagattg agcgtgcagccagtgaatccccgcattttatgcgttttcatgttgcctgcccgcattgcggggaggagcagtatcttaaatttggcgacaaagagacgc cgtttggcctcaaatggacgccggatgacccctccagcgtgttttatctctgcgagcataatgcctgcgtcatccgccagcaggagctggactttactga tgcccgttatatctgcgaaaagaccgggatctggacccgtgatggcattctctggttttcgtcatccggtgaagagattgagccacctgacagtgtgacc tttcacatctggacagcgtacagcccgttcaccacctgggtgcagattgtcaaagactggatgaaaacgaaaggggatacgggaaaacgtaaaacct tcgtaaacaccacgctcggtgagacgtgggaggcgaaaattggcgaacgtccggatgctgaagtgatggcagagcggaaagagcattattcagcgc ccgttcctgaccgtgtggcttacctgaccgccggtatcgactcccagctggaccgctacgaaatgcgcgtatggggatgggggccgggtgaggaaagc tggctgattgaccggcagattattatgggccgccacgacgatgaacagacgctgctgcgtgtggatgaggccatcaataaaacctatacccgccgga atggtgcagaaatgtcgatatcccgtatctgctgggatactggcgggattgacccgaccattgtgtatgaacgctcgaaaaaacatgggctgttccgg gtgatccccattaaaggggcatccgtctacggaaagccggtggccagcatgccacgtaagcgaaacaaaaacggggtttaccttaccgaaatcggta cggataccgcgaaagagcagatttataaccgcttcacactgacgccggaaggggatgaaccgcttcccggtgccgttcacttcccgaataacccggat atttttgatctgaccgaagcgcagcagctgactgctgaagagcaggtcgaaaaatgggtggatggcaggaaaaaaatactgtgggacagcaaaaag cgacgcaatgaggcactcgactgcttcgtttatgcgctggcggcgctgcgcatcagtatttcccgctggcagctggatctcagtgcgctgctggcgagc ctgcaggaagaggatggtgcagcaaccaacaagaaaacactggcagattacgcccgtgccttatccggagaggatgaatgacgcgacaggaagaa cttgccgctgcccgtgcggcactgcatgacctgatgacaggtaaacgggtggcaacagtacagaaagacggacgaagggtggagtttacggccactt ccgtgtctgacctgaaaaaatatattgcagagctggaagtgcagaccggcatgacacagcgacgcaggggacctgcaggattttatgtatgaaaacg cccaccattcccacccttctggggccggacggcatgacatcgctgcgcgaatatgccggttatcacggcggtggcagcggatttggagggcagttgcg gtcgtggaacccaccgagtgaaagtgtggatgcagccctgttgcccaactttacccgtggcaatgcccgcgcagacgatctggtacgcaataacggct atgccgccaacgccatccagctgcatcaggatcatatcgtcgggtcttttttccggctcagtcatcgcccaagctggcgctatctgggcatcggggagg aagaagcccgtgccttttcccgcgaggttgaagcggcatggaaagagtttgccgaggatgactgctgctgcattgacgttgagcgaaaacgcacgttt accatgatgattcgggaaggtgtggccatgcacgcctttaacggtgaactgttcgttcaggccacctgggataccagttcgtcgcggcttttccggaca cagttccggatggtcagcccgaagcgcatcagcaacccgaacaataccggcgacagccggaactgccgtgccggtgtgcagattaatgacagcggt gcggcgctgggatattacgtcagcgaggacgggtatcctggctggatgccgcagaaatggacatggataccccgtgagttacccggcgggcgcgcct cgttcattcacgtttttgaacccgtggaggacgggcagactcgcggtgcaaatgtgttttacagcgtgatggagcagatgaagatgctcgacacgctgc agaacacgcagctgcagagcgccattgtgaaggcgatgtatgccgccaccattgagagtgagctggatacgcagtcagcgatggattttattctgggc gcgaacagtcaggagcagcgggaaaggctgaccggctggattggtgaaattgccgcgtattacgccgcagcgccggtccggctgggaggcgcaaaa gtaccgcacctgatgccgggtgactcactgaacctgcagacggctcaggatacggataacggctactccgtgtttgagcagtcactgctgcggtatatc gctgccgggctgggtgtctcgtatgagcagctttcccggaattacgcccagatgagctactccacggcacgggccagtgcgaacgagtcgtgggcgta ctttatggggcggcgaaaattcgtcgcatcccgtcaggcgagccagatgtttctgtgctggctggaagaggccatcgttcgccgcgtggtgacgttacc ttcaaaagcgcgcttcagttttcaggaagcccgcagtgcctgggggaactgcgactggataggctccggtcgtatggccatcgatggtctgaaagaag ttcaggaagcggtgatgctgatagaagccggactgagtacctacgagaaagagtgcgcaaaacgcggtgacgactatcaggaaatttttgcccagca ggtccgtgaaacgatggagcgccgtgcagccggtcttaaaccgcccgcctgggcggctgcagcatttgaatccgggctgcgacaatcaacagaggag gagaagagtgacagcagagctgcgtaatctcccgcatattgccagcatggcctttaatgagccgctgatgcttgaacccgcctatgcgcgggttttcttt tgtgcgcttgcaggccagcttgggatcagcagcctgacggatgcggtgtccggcgacagcctgactgcccaggaggcactcgcgacgctggcattatc cggtgatgatgacggaccacgacaggcccgcagttatcaggtcatgaacggcatcgccgtgctgccggtgtccggcacgctggtcagccggacgcgg gcgctgcagccgtactcggggatgaccggttacaacggcattatcgcccgtctgcaacaggctgccagcgatccgatggtggacggcattctgctcga tatggacacgcccggcgggatggtggcgggggcatttgactgcgctgacatcatcgcccgtgtgcgtgacataaaaccggtatgggcgcttgccaacg acatgaactgcagtgcaggtcagttgcttgccagtgccgcctcccggcgtctggtcacgcagaccgcccggacaggctccatcggcgtcatgatggctc acagtaattacggtgctgcgctggagaaacagggtgtggaaatcacgctgatttacagcggcagccataaggtggatggcaacccctacagccatct tccggatgacgtccgggagacactgcagtcccggatggacgcaacccgccagatgtttgcgcagaaggtgtcggcatataccggcctgtccgtgcag gttgtgctggataccgaggctgcagtgtacagcggtcaggaggccattgatgccggactggctgatgaacttgttaacagcaccgatgcgatcaccgt catgcgtgatgcactggatgcacgtaaatcccgtctctcaggagggcgaatgaccaaagagactcaatcaacaactgtttcagccactgcttcgcagg ctgacgttactgacgtggtgccagcgacggagggcgagaacgccagcgcggcgcagccggacgtgaacgcgcagatcaccgcagcggttgcggca gaaaacagccgcattatggggatcctcaactgtgaggaggctcacggacgcgaagaacaggcacgcgtgctggcagaaacccccggtatgaccgtg aaaacggcccgccgcattctggccgcagcaccacagagtgcacaggcgcgcagtgacactgcgctggatcgtctgatgcagggggcaccggcaccg ctggctgcaggtaacccggcatctgatgccgttaacgatttgctgaacacaccagtgtaagggatgtttatgacgagcaaagaaacctttacccattac cagccgcagggcaacagtgacccggctcataccgcaaccgcgcccggcggattgagtgcgaaagcgcctgcaatgaccccgctgatgctggacacc tccagccgtaagctggttgcgtgggatggcaccaccgacggtgctgccgttggcattcttgcggttgctgctgaccagaccagcaccacgctgacgttc tacaagtccggcacgttccgttatgaggatgtgctctggccggaggctgccagcgacgagacgaaaaaacggaccgcgtttgccggaacggcaatca gcatcgtttaactttacccttcatcactaaaggccgcctgtgcggctttttttacgggatttttttatgtcgatgtacacaaccgcccaactgctggcggca aatgagcagaaatttaagtttgatccgctgtttctgcgtctctttttccgtgagagctatcccttcaccacggagaaagtctatctctcacaaattccggg actggtaaacatggcgctgtacgtttcgccgattgtttccggtgaggttatccgttcccgtggcggctccacctctgaatttacgccgggatatgtcaagc cgaagcatgaagtgaatccgcagatgaccctgcgtcgcctgccggatgaagatccgcagaatctggcggacccggcttaccgccgccgtcgcatcat catgcagaacatgcgtgacgaagagctggccattgctcaggtcgaagagatgcaggcagtttctgccgtgcttaagggcaaatacaccatgaccggt gaagccttcgatccggttgaggtggatatgggccgcagtgaggagaataacatcacgcagtccggcggcacggagtggagcaagcgtgacaagtcc acgtatgacccgaccgacgatatcgaagcctacgcgctgaacgccagcggtgtggtgaatatcatcgtgttcgatccgaaaggctgggcgctgttccg ttccttcaaagccgtcaaggagaagctggatacccgtcgtggctctaattccgagctggagacagcggtgaaagacctgggcaaagcggtgtcctata aggggatgtatggcgatgtggccatcgtcgtgtattccggacagtacgtggaaaacggcgtcaaaaagaacttcctgccggacaacacgatggtgct ggggaacactcaggcacgcggtctgcgcacctatggctgcattcaggatgcggacgcacagcgcgaaggcattaacgcctctgcccgttacccgaaa aactgggtgaccaccggcgatccggcgcgtgagttcaccatgattcagtcagcaccgctgatgctgctggctgaccctgatgagttcgtgtccgtacaa ctggcgtaatcatggcccttcggggccattgtttctctgtggaggagtccatgacgaaagatgaactgattgcccgtctccgctcgctgggtgaacaact gaaccgtgatgtcagcctgacggggacgaaagaagaactggcgctccgtgtggcagagctgaaagaggagcttgatgacacggatgaaactgccg gtcaggacacccctctcagccgggaaaatgtgctgaccggacatgaaaatgaggtgggatcagcgcagccggataccgtgattctggatacgtctga actggtcacggtcgtggcactggtgaagctgcatactgatgcacttcacgccacgcgggatgaacctgtggcatttgtgctgccgggaacggcgtttcg tgtctctgccggtgtggcagccgaaatgacagagcgcggcctggccagaatgcaataacgggaggcgctgtggctgatttcgataacctgttcgatgc tgccattgcccgcgccgatgaaacgatacgcgggtacatgggaacgtcagccaccattacatccggtgagcagtcaggtgcggtgatacgtggtgttt ttgatgaccctgaaaatatcagctatgccggacagggcgtgcgcgttgaaggctccagcccgtccctgtttgtccggactgatgaggtgcggcagctgc ggcgtggagacacgctgaccatcggtgaggaaaatttctgggtagatcgggtttcgccggatgatggcggaagttgtcatctctggcttggacggggc gtaccgcctgccgttaaccgtcgccgctgaaagggggatgtatggccataaaaggtcttgagcaggccgttgaaaacctcagccgtatcagcaaaac ggcggtgcctggtgccgccgcaatggccattaaccgcgttgcttcatccgcgatatcgcagtcggcgtcacaggttgcccgtgagacaaaggtacgcc ggaaactggtaaaggaaagggccaggctgaaaagggccacggtcaaaaatccgcaggccagaatcaaagttaaccggggggatttgcccgtaatc aagctgggtaatgcgcgggttgtcctttcgcgccgcaggcgtcgtaaaaaggggcagcgttcatccctgaaaggtggcggcagcgtgcttgtggtggg taaccgtcgtattcccggcgcgtttattcagcaactgaaaaatggccggtggcatgtcatgcagcgtgtggctgggaaaaaccgttaccccattgatgt ggtgaaaatcccgatggcggtgccgctgaccacggcgtttaaacaaaatattgagcggatacggcgtgaacgtcttccgaaagagctgggctatgcg ctgcagcatcaactgaggatggtaataaagcgatgaaacatactgaactccgtgcagccgtactggatgcactggagaagcatgacaccggggcga cgttttttgatggtcgccccgctgtttttgatgaggcggattttccggcagttgccgtttatctcaccggcgctgaatacacgggcgaagagctggacag cgatacctggcaggcggagctgcatatcgaagttttcctgcctgctcaggtgccggattcagagctggatgcgtggatggagtcccggatttatccggt gatgagcgatatcccggcactgtcagatttgatcaccagtatggtggccagcggctatgactaccggcgcgacgatgatgcgggcttgtggagttcag ccgatctgacttatgtcattacctatgaaatgtgaggacgctatgcctgtaccaaatcctacaatgccggtgaaaggtgccgggaccaccctgtgggtt tataaggggagcggtgacccttacgcgaatccgctttcagacgttgactggtcgcgtctggcaaaagttaaagacctgacgcccggcgaactgaccgc tgagtcctatgacgacagctatctcgatgatgaagatgcagactggactgcgaccgggcaggggcagaaatctgccggagataccagcttcacgctg gcgtggatgcccggagagcaggggcagcaggcgctgctggcgtggtttaatgaaggcgatacccgtgcctataaaatccgcttcccgaacggcacgg tcgatgtgttccgtggctgggtcagcagtatcggtaaggcggtgacggcgaaggaagtgatcacccgcacggtgaaagtcaccaatgtgggacgtcc gtcgatggcagaagatcgcagcacggtaacagcggcaaccggcatgaccgtgacgcctgccagcacctcggtggtgaaagggcagagcaccacgct gaccgtggccttccagccggagggcgtaaccgacaagagctttcgtgcggtgtctgcggataaaacaaaagccaccgtgtcggtcagtggtatgacc atcaccgtgaacggcgttgctgcaggcaaggtcaacattccggttgtatccggtaatggtgagtttgctgcggttgcagaaattaccgtcaccgccagt taatccggagagtcagcgatgttcctgaaaaccgaatcatttgaacataacggtgtgaccgtcacgctttctgaactgtcagccctgcagcgcattgag catctcgccctgatgaaacggcaggcagaacaggcggagtcagacagcaaccggaagtttactgtggaagacgccatcagaaccggcgcgtttctg gtggcgatgtccctgtggcataaccatccgcagaagacgcagatgccgtccatgaatgaagccgttaaacagattgagcaggaagtgcttaccacct ggcccacggaggcaatttctcatgctgaaaacgtggtgtaccggctgtctggtatgtatgagtttgtggtgaataatgcccctgaacagacagaggac gccgggcccgcagagcctgtttctgcgggaaagtgttcgacggtgagctgagttttgccctgaaactggcgcgtgagatggggcgacccgactggcgt gccatgcttgccgggatgtcatccacggagtatgccgactggcaccgcttttacagtacccattattttcatgatgttctgctggatatgcacttttccggg ctgacgtacaccgtgctcagcctgtttttcagcgatccggatatgcatccgctggatttcagtctgctgaaccggcgcgaggctgacgaagagcctgaa gatgatgtgctgatgcagaaagcggcagggcttgccggaggtgtccgctttggcccggacgggaatgaagttatccccgcttccccggatgtggcgga catgacggaggatgacgtaatgctgatgacagtatcagaagggatcgcaggaggagtccggtatggctgaaccggtaggcgatctggtcgttgattt gagtctggatgcggccagatttgacgagcagatggccagagtcaggcgtcatttttctggtacggaaagtgatgcgaaaaaaacagcggcagtcgtt gaacagtcgctgagccgacaggcgctggctgcacagaaagcggggatttccgtcgggcagtataaagccgccatgcgtatgctgcctgcacagttca ccgacgtggccacgcagcttgcaggcgggcaaagtccgtggctgatcctgctgcaacagggggggcaggtgaaggactccttcggcgggatgatccc catgttcagggggcttgccggtgcgatcaccctgccgatggtgggggccacctcgctggcggtggcgaccggtgcgctggcgtatgcctggtatcagg gcaactcaaccctgtccgatttcaacaaaacgctggtcctttccggcaatcaggcgggactgacggcagatcgtatgctggtcctgtccagagccggg caggcggcagggctgacgtttaaccagaccagcgagtcactcagcgcactggttaaggcgggggtaagcggtgaggctcagattgcgtccatcagcc agagtgtggcgcgtttctcctctgcatccggcgtggaggtggacaaggtcgctgaagccttcgggaagctgaccacagacccgacgtcggggctgac ggcgatggctcgccagttccataacgtgtcggcggagcagattgcgtatgttgctcagttgcagcgttccggcgatgaagccggggcattgcaggcgg cgaacgaggccgcaacgaaagggtttgatgaccagacccgccgcctgaaagagaacatgggcacgctggaaacctgggcagacaggactgcgcgg gcattcaaatccatgtgggatgcggtgctggatattggtcgtcctgataccgcgcaggagatgctgattaaggcagaggctgcgtataagaaagcaga cgacatctggaatctgcgcaaggatgattattttgttaacgatgaagcgcgggcgcgttactgggatgatcgtgaaaaggcccgtcttgcgcttgaagc cgcccgaaagaaggctgagcagcagactcaacaggacaaaaatgcgcagcagcagagcgataccgaagcgtcacggctgaaatataccgaagag gcgcagaaggcttacgaacggctgcagacgccgctggagaaatataccgcccgtcaggaagaactgaacaaggcactgaaagacgggaaaatcct gcaggcggattacaacacgctgatggcggcggcgaaaaaggattatgaagcgacgctgaaaaagccgaaacagtccagcgtgaaggtgtctgcgg gcgatcgtcaggaagacagtgctcatgctgccctgctgacgcttcaggcagaactccggacgctggagaagcatgccggagcaaatgagaaaatca gccagcagcgccgggatttgtggaaggcggagagtcagttcgcggtactggaggaggcggcgcaacgtcgccagctgtctgcacaggagaaatccc tgctggcgcataaagatgagacgctggagtacaaacgccagctggctgcacttggcgacaaggttacgtatcaggagcgcctgaacgcgctggcgca gcaggcggataaattcgcacagcagcaacgggcaaaacgggccgccattgatgcgaaaagccgggggctgactgaccggcaggcagaacgggaa gccacggaacagcgcctgaaggaacagtatggcgataatccgctggcgctgaataacgtcatgtcagagcagaaaaagacctgggcggctgaaga ccagcttcgcgggaactggatggcaggcctgaagtccggctggagtgagtgggaagagagcgccacggacagtatgtcgcaggtaaaaagtgcagc cacgcagacctttgatggtattgcacagaatatggcggcgatgctgaccggcagtgagcagaactggcgcagcttcacccgttccgtgctgtccatgat gacagaaattctgcttaagcaggcaatggtggggattgtcgggagtatcggcagcgccattggcggggctgttggtggcggcgcatccgcgtcaggc ggtacagccattcaggccgctgcggcgaaattccattttgcaaccggaggatttacgggaaccggcggcaaatatgagccagcggggattgttcacc gtggtgagtttgtcttcacgaaggaggcaaccagccggattggcgtggggaatctttaccggctgatgcgcggctatgccaccggcggttatgtcggta caccgggcagcatggcagacagccggtcgcaggcgtccgggacgtttgagcagaataaccatgtggtgattaacaacgacggcacgaacgggcag ataggtccggctgctctgaaggcggtgtatgacatggcccgcaagggtgcccgtgatgaaattcagacacagatgcgtgatggtggcctgttctccgg aggtggacgatgaagaccttccgctggaaagtgaaacccggtatggatgtggcttcggtcccttctgtaagaaaggtgcgctttggtgatggctattct cagcgagcgcctgccgggctgaatgccaacctgaaaacgtacagcgtgacgctttctgtcccccgtgaggaggccacggtactggagtcgtttctgga agagcacgggggctggaaatcctttctgtggacgccgccttatgagtggcggcagataaaggtgacctgcgcaaaatggtcgtcgcgggtcagtatg ctgcgtgttgagttcagcgcagagtttgaacaggtggtgaactgatgcaggatatccggcaggaaacactgaatgaatgcacccgtgcggagcagtc ggccagcgtggtgctctgggaaatcgacctgacagaggtcggtggagaacgttattttttctgtaatgagcagaacgaaaaaggtgagccggtcacct ggcaggggcgacagtatcagccgtatcccattcaggggagcggttttgaactgaatggcaaaggcaccagtacgcgccccacgctgacggtttctaa cctgtacggtatggtcaccgggatggcggaagatatgcagagtctggtcggcggaacggtggtccggcgtaaggtttacgcccgttttctggatgcggt gaacttcgtcaacggaaacagttacgccgatccggagcaggaggtgatcagccgctggcgcattgagcagtgcagcgaactgagcgcggtgagtgc ctcctttgtactgtccacgccgacggaaacggatggcgctgtttttccgggacgtatcatgctggccaacacctgcacctggacctatcgcggtgacga gtgcggttatagcggtccggctgtcgcggatgaatatgaccagccaacgtccgatatcacgaaggataaatgcagcaaatgcctgagcggttgtaagt tccgcaataacgtcggcaactttggcggcttcctttccattaacaaactttcgcagtaaatcccatgacacagacagaatcagcgattctggcgcacgc ccggcgatgtgcgccagcggagtcgtgcggcttcgtggtaagcacgccggagggggaaagatatttcccctgcgtgaatatctccggtgagccggag gctatttccgtatgtcgccggaagactggctgcaggcagaaatgcagggtgagattgtggcgctggtccacagccaccccggtggtctgccctggctg agtgaggccgaccggcggctgcaggtgcagagtgatttgccgtggtggctggtctgccgggggacgattcataagttccgctgtgtgccgcatctcacc gggcggcgctttgagcacggtgtgacggactgttacacactgttccgggatgcttatcatctggcggggattgagatgccggactttcatcgtgaggat gactggtggcgtaacggccagaatctctatctggataatctggaggcgacggggctgtatcaggtgccgttgtcagcggcacagccgggcgatgtgct gctgtgctgttttggttcatcagtgccgaatcacgccgcaatttactgcggcgacggcgagctgctgcaccatattcctgaacaactgagcaaacgaga gaggtacaccgacaaatggcagcgacgcacacactccctctggcgtcaccgggcatggcgcgcatctgcctttacggggatttacaacgatttggtcg ccgcatcgaccttcgtgtgaaaacgggggctgaagccatccgggcactggccacacagctcccggcgtttcgtcagaaactgagcgacggctggtatc aggtacggattgccgggcgggacgtcagcacgtccgggttaacggcgcagttacatgagactctgcctgatggcgctgtaattcatattgttcccagag tcgccggggccaagtcaggtggcgtattccagattgtcctgggggctgccgccattgccggatcattctttaccgccggagccacccttgcagcatggg gggcagccattggggccggtggtatgaccggcatcctgttttctctcggtgccagtatggtgctcggtggtgtggcgcagatgctggcaccgaaagcca gaactccccgtatacagacaacggataacggtaagcagaacacctatttctcctcactggataacatggttgcccagggcaatgttctgcctgttctgt acggggaaatgcgcgtggggtcacgcgtggtttctcaggagatcagcacggcagacgaaggggacggtggtcaggttgtggtgattggtcgctgatg caaaatgttttatgtgaaaccgcctgcgggcggttttgtcatttatggagcgtgaggaatgggtaaaggaagcagtaaggggcataccccgcgcgaa gcgaaggacaacctgaagtccacgcagttgctgagtgtgatcgatgccatcagcgaagggccgattgaaggtccggtggatggcttaaaaagcgtgc tgctgaacagtacgccggtgctggacactgaggggaataccaacatatccggtgtcacggtggtgttccgggctggtgagcaggagcagactccgcc ggagggatttgaatcctccggctccgagacggtgctgggtacggaagtgaaatatgacacgccgatcacccgcaccattacgtctgcaaacatcgac cgtctgcgctttaccttcggtgtacaggcactggtggaaaccacctcaaagggtgacaggaatccgtcggaagtccgcctgctggttcagatacaacgt aacggtggctgggtgacggaaaaagacatcaccattaagggcaaaaccacctcgcagtatctggcctcggtggtgatgggtaacctgccgccgcgcc cgtttaatatccggatgcgcaggatgacgccggacagcaccacagaccagctgcagaacaaaacgctctggtcgtcatacactgaaatcatcgatgt gaaacagtgctacccgaacacggcactggtcggcgtgcaggtggactcggagcagttcggcagccagcaggtgagccgtaattatcatctgcgcggg cgtattctgcaggtgccgtcgaactataacccgcagacgcggcaatacagcggtatctgggacggaacgtttaaaccggcatacagcaacaacatgg cctggtgtctgtgggatatgctgacccatccgcgctacggcatggggaaacgtcttggtgcggcggatgtggataaatgggcgctgtatgtcatcggcc agtactgcgaccagtcagtgccggacggctttggcggcacggagccgcgcatcacctgtaatgcgtacctgaccacacagcgtaaggcgtgggatgt gctcagcgatttctgctcggcgatgcgctgtatgccggtatggaacgggcagacgctgacgttcgtgcaggaccgaccgtcggataagacgtggacct ataaccgcagtaatgtggtgatgccggatgatggcgcgccgttccgctacagcttcagcgccctgaaggaccgccataatgccgttgaggtgaactgg attgacccgaacaacggctgggagacggcgacagagcttgttgaagatacgcaggccattgcccgttacggtcgtaatgttacgaagatggatgcctt tggctgtaccagccgggggcaggcacaccgcgccgggctgtggctgattaaaacagaactgctggaaacgcagaccgtggatttcagcgtcggcgca gaagggcttcgccatgtaccgggcgatgttattgaaatctgcgatgatgactatgccggtatcagcaccggtggtcgtgtgctggcggtgaacagcca gacccggacgctgacgctcgaccgtgaaatcacgctgccatcctccggtaccgcgctgataagcctggttgacggaagtggcaatccggtcagcgtgg aggttcagtccgtcaccgacggcgtgaaggtaaaagtgagccgtgttcctgacggtgttgctgaatacagcgtatgggagctgaagctgccgacgctg cgccagcgactgttccgctgcgtgagtatccgtgagaacgacgacggcacgtatgccatcaccgccgtgcagcatgtgccggaaaaagaggccatcg tggataacggggcgcactttgacggcgaacagagtggcacggtgaatggtgtcacgccgccagcggtgcagcacctgaccgcagaagtcactgcag acagcggggaatatcaggtgctggcgcgatgggacacaccgaaggtggtgaagggcgtgagtttcctgctccgtctgaccgtaacagcggacgacg gcagtgagcggctggtcagcacggcccggacgacggaaaccacataccgcttcacgcaactggcgctggggaactacaggctgacagtccgggcgg taaatgcgtgggggcagcagggcgatccggcgtcggtatcgttccggattgccgcaccggcagcaccgtcgaggattgagctgacgccgggctatttt cagataaccgccacgccgcatcttgccgtttatgacccgacggtacagtttgagttctggttctcggaaaagcagattgcggatatcagacaggttgaa accagcacgcgttatcttggtacggcgctgtactggatagccgccagtatcaatatcaaaccgggccatgattattacttttatatccgcagtgtgaaca ccgttggcaaatcggcattcgtggaggccgtcggtcgggcgagcgatgatgcggaaggttacctggattttttcaaaggcaagataaccgaatcccat ctcggcaaggagctgctggaaaaagtcgagctgacggaggataacgccagcagactggaggagttttcgaaagagtggaaggatgccagtgataa gtggaatgccatgtgggctgtcaaaattgagcagaccaaagacggcaaacattatgtcgcgggtattggcctcagcatggaggacacggaggaagg caaactgagccagtttctggttgccgccaatcgtatcgcatttattgacccggcaaacgggaatgaaacgccgatgtttgtggcgcagggcaaccaga tattcatgaacgacgtgttcctgaagcgcctgacggcccccaccattaccagcggcggcaatcctccggccttttccctgacaccggacggaaagctga ccgctaaaaatgcggatatcagtggcagtgtgaatgcgaactccgggacgctcagtaatgtgacgatagctgaaaactgtacgataaacggtacgct gagggcggaaaaaatcgtcggggacattgtaaaggcggcgagcgcggcttttccgcgccagcgtgaaagcagtgtggactggccgtcaggtacccg tactgtcaccgtgaccgatgaccatccttttgatcgccagatagtggtgcttccgctgacgtttcgcggaagtaagcgtactgtcagcggcaggacaac gtattcgatgtgttatctgaaagtactgatgaacggtgcggtgatttatgatggcgcggcgaacgaggcggtacaggtgttctcccgtattgttgacatg ccagcgggtcggggaaacgtgatcctgacgttcacgcttacgtccacacggcattcggcagatattccgccgtatacgtttgccagcgatgtgcaggtt atggtgattaagaaacaggcgctgggcatcagcgtggtctgagtgtgttacagaggttcgtccgggaacgggcgttttattataaaacagtgagaggt gaacgatgcgtaatgtgtgtattgccgttgctgtctttgccgcacttgcggtgacagtcactccggcccgtgcggaaggtggacatggtacgtttacggt gggctattttcaagtgaaaccgggtacattgccgtcgttgtcgggcggggataccggtgtgagtcatctgaaagggattaacgtgaagtaccgttatga gctgacggacagtgtgggggtgatggcttccctggggttcgccgcgtcgaaaaagagcagcacagtgatgaccggggaggatacgtttcactatgag agcctgcgtggacgttatgtgagcgtgatggccggaccggttttacaaatcagtaagcaggtcagtgcgtacgccatggccggagtggctcacagtcg gtggtccggcagtacaatggattaccgtaagacggaaatcactcccgggtatatgaaagagacgaccactgccagggacgaaagtgcaatgcggca tacctcagtggcgtggagtgcaggtatacagattaatccggcagcgtccgtcgttgttgatattgcttatgaaggctccggcagtggcgactggcgtact gacggattcatcgttggggtcggttataaattctgattagccaggtaacacagtgttatgacagcccgccggaaccggtgggcttttttgtggggtgaat atggcagtaaagatttcaggagtcctgaaagacggcacaggaaaaccggtacagaactgcaccattcagctgaaagccagacgtaacagcaccac ggtggtggtgaacacggtgggctcagagaatccggatgaagccgggcgttacagcatggatgtggagtacggtcagtacagtgtcatcctgcaggtt gacggttttccaccatcgcacgccgggaccatcaccgtgtatgaagattcacaaccggggacgctgaatgattttctctgtgccatgacggaggatgat gcccggccggaggtgctgcgtcgtcttgaactgatggtggaagaggtggcgcgtaacgcgtccgtggtggcacagagtacggcagacgcgaagaaa tcagccggcgatgccagtgcatcagctgctcaggtcgcggcccttgtgactgatgcaactgactcagcacgcgccgccagcacgtccgccggacaggc tgcatcgtcagctcaggaagcgtcctccggcgcagaagcggcatcagcaaaggccactgaagcggaaaaaagtgccgcagccgcagagtcctcaa aaaacgcggcggccaccagtgccggtgcggcgaaaacgtcagaaacgaatgctgcagcgtcacaacaatcagccgccacgtctgcctccaccgcgg ccacgaaagcgtcagaggccgccacttcagcacgagatgcggtggcctcaaaagaggcagcaaaatcatcagaaacgaacgcatcatcaagtgcc ggtcgtgcagcttcctcggcaacggcggcagaaaattctgccagggcggcaaaaacgtccgagacgaatgccaggtcatctgaaacagcagcggaa cggagcgcctctgccgcggcagacgcaaaaacagcggcggcggggagtgcgtcaacggcatccacgaaggcgacagaggctgcgggaagtgcggt atcagcatcgcagagcaaaagtgcggcagaagcggcggcaatacgtgcaaaaaattcggcaaaacgtgcagaagatatagcttcagctgtcgcgct tgaggatgcggacacaacgagaaaggggatagtgcagctcagcagtgcaaccaacagcacgtctgaaacgcttgctgcaacgccaaaggcggtta aggtggtaatggatgaaacgaacagaaaagcccactggacagtccggcactgaccggaacgccaacagcaccaaccgcgctcaggggaacaaac aatacccagattgcgaacaccgcttttgtactggccgcgattgcagatgttatcgacgcgtcacctgacgcactgaatacgctgaatgaactggccgca gcgctcgggaatgatccagattttgctaccaccatgactaacgcgcttgcgggtaaacaaccgaagaatgcgacactgacggcgctggcagggctttc cacggcgaaaaataaattaccgtattttgcggaaaatgatgccgccagcctgactgaactgactcaggttggcagggatattctggcaaaaaattccg ttgcagatgttcttgaataccttggggccggtgagaattcggcctttccggcaggtgcgccgatcccgtggccatcagatatcgttccgtctggctacgtc ctgatgcaggggcaggcgtttgacaaatcagcctacccaaaacttgctgtcgcgtatccatcgggtgtgcttcctgatatgcgaggctggacaatcaag gggaaacccgccagcggtcgtgctgtattgtctcaggaacaggatggaattaagtcgcacacccacagtgccagtgcatccggtacggatttgggga cgaaaaccacatcgtcgtttgattacgggacgaaaacaacaggcagtttcgattacggcaccaaatcgacgaataacacgggggctcatgctcacag tctgagcggttcaacaggggccgcgggtgctcatgcccacacaagtggtttaaggatgaacagttctggctggagtcagtatggaacagcaaccatta caggaagtttatccacagttaaaggaaccagcacacagggtattgcttatttatcgaaaacggacagtcagggcagccacagtcactcattgtccggt acagccgtgagtgccggtgcacatgcgcatacagttggtattggtgcgcaccagcatccggttgttatcggtgctcatgcccattctttcagtattggttc acacggacacaccatcaccgttaacgctgcgggtaacgcggaaaacaccgtcaaaaacattgcatttaactatattgtgaggcttgcataatggcatt cagaatgagtgaacaaccacggaccataaaaatttataatctgctggccggaactaatgaatttattggtgaaggtgacgcatatattccgcctcata ccggtctgcctgcaaacagtaccgatattgcaccgccagatattccggctggctttgtggctgttttcaacagtgatgaggcatcgtggcatctcgttga agaccatcggggtaaaaccgtctatgacgtggcttccggcgacgcgttatttatttctgaactcggtccgttaccggaaaattttacctggttatcgccg ggaggggaatatcagaagtggaacggcacagcctgggtgaaggatacggaagcagaaaaactgttccggatccgggaggcggaagaaacaaaaa aaagcctgatgcaggtagccagtgagcatattgcgccgcttcaggatgctgcagatctggaaattgcaacgaaggaagaaacctcgttgctggaagc ctggaagaagtatcgggtgttgctgaaccgtgttgatacatcaactgcacctgatattgagtggcctgctgtccctgttatggagtaatcgttttgtgata tgccgcagaaacgttgtatgaaataacgttctgcggttagttagtatattgtaaagctgagtattggtttatttggcgattattatcttcaggagaataat ggaagttctatgactcaattgttcatagtgtttacatcaccgccaattgcttttaagactgaacgcatgaaatatggtttttcgtcatgttttgagtctgct gttgatatttctaaagtcggttttttttcttcgttttctctaactattttccatgaaatacatttttgattattatttgaatcaattccaattacctgaagtcttt catctataattggcattgtatgtattggtttattggagtagatgcttgcttttctgagccatagctctgatatccaaatgaagccataggcatttgttatttt ggctctgtcagctgcataacgccaaaaaatatatttatctgcttgatcttcaaatgttgtattgattaaatcaattggatggaattgtttatcataaaaaa ttaatgtttgaatgtgataaccgtcctttaaaaaagtcgtttctgcaagcttggctgtatagtcaactaactcttctgtcgaagtgatatttttaggcttatc taccagttttagacgctctttaatatcttcaggaattattttattgtcatattgtatcatgctaaatgacaatttgcttatggagtaatcttttaattttaaat aagttattctcctggcttcatcaaataaagagtcgaatgatgttggcgaaatcacatcgtcacccattggattgtttatttgtatgccaagagagttaca gcagttatacattctgccatagattatagctaaggcatgtaataattcgtaatcttttagcgtattagcgacccatcgtctttctgatttaataatagatga ttcagttaaatatgaaggtaatttcttttgtgcaagtctgactaacttttttataccaatgtttaacatactttcatttgtaataaactcaatgtcattttctt caatgtaagatgaaataagagtagcctttgcctcgctatacatttctaaatcgccttgtttttctatcgtattgcgagaatttttagcccaagccattaatg gatcatttttccatttttcaataacattattgttataccaaatgtcatatcctataatctggtttttgtttttttgaataataaatgttactgttcttgcggtttg gaggaattgattcaaattcaagcgaaataattcagggtcaaaatatgtatcaatgcagcatttgagcaagtgcgataaatctttaagtcttctttcccat ggttttttagtcataaaactctccattttgataggttgcatgctagatgctgatatattttagaggtgataaaattaactgcttaactgtcaatgtaataca agttgtttgatctttgcaatgattcttatcagaaaccatatagtaaattagttacacaggaaatttttaatattattattatcattcattatgtattaaaatt agagttgtggcttggctctgctaacacgttgctcataggagatatggtagagccgcagacacgtcgtatgcaggaacgtgctgcggctggctggtgaa cttccgatagtgcgggtgttgaatgatttccagttgctaccgattttacatattttttgcatgagagaatttgtaccacctcccaccgaccatctatgactg tacgccactgtccctaggactgctatgtgccggagcggacattacaaacgtccttctcggtgcatgccactgttgccaatgacctgcctaggaattggtt agcaagttactaccggattttgtaaaaacagccctcctcatataaaaagtattcgttcacttccgataagcgtcgtaattttctatctttcatcatattcta gatccctctgaaaaaatcttccgagtttgctaggcactgatacataactcttttccaataattggggaagtcattcaaatctataataggtttcagatttg cttcaataaattctgactgtagctgctgaaacgttgcggttgaactatatttccttataacttttacgaaagagtttctttgagtaatcacttcactcaagt gcttccctgcctccaaacgatacctgttagcaatatttaatagcttgaaatgatgaagagctctgtgtttgtcttcctgcctccagttcgccgggcattca acataaaaactgatagcacccggagttccggaaacgaaatttgcatatacccattgctcacgaaaaaaaatgtccttgtcgatatagggatgaatcgc ttggtgtacctcatctactgcgaaaacttgacctttctctcccatattgcagtcgcggcacgatggaactaaattaataggcatcaccgaaaattcagga taatgtgcaataggaagaaaatgatctatattttttgtctgtcctatatcaccacaaaatggacatttttcacctgatgaaacaagcatgtcatcgtaat atgttctagcgggtttgtttttatctcggagattattttcataaagcttttctaatttaacctttgtcaggttaccaactactaaggttgtaggctcaagagg gtgtgtcctgtcgtaggtaaataactgacctgtcgagcttaatattctatattgttgttctttctgcaaaaaagtggggaagtgagtaatgaaattatttct aacatttatctgcatcataccttccgagcatttattaagcatttcgctataagttctcgctggaagaggtagttttttcattgtactttaccttcatctctgtt cattatcatcgcttttaaaacggttcgaccttctaatcctatctgaccattataattttttagaatggtttcataagaaagctctgaatcaacggactgcga taataagtggtggtatccagaatttgtcacttcaagtaaaaacacctcacgagttaaaacacctaagttctcaccgaatgtctcaatatccggacggat aatatttattgcttctcttgaccgtaggactttccacatgcaggattttggaacctcttgcagtactactggggaatgagttgcaattattgcta caeca tt gcgtgcatcgagtaagtcgcttaatgttcgtaaaaaagcagagagcaaaggtggatgcagatgaacctctggttcatcgaataaaactaatgactttt cgccaacgacatctactaatcttgtgatagtaaataaaacaattgcatgtccagagctcattcgaagcagatatttctggatattgtcataaaacaattt agtgaatttatcatcgtccacttgaatctgtggttcattacgtcttaactcttcatatttagaaatgaggctgatgagttccatatttgaaaagttttcatca ctacttagttttttgatagcttcaagccagagttgtctttttctatctactctcatacaaccaataaatgctgaaatgaattctaagcggagatcgcctagt gattttaaactattgctggcagcattcttgagtccaatataaaagtattgtgtaccttttgctgggtcaggttgttctttaggaggagtaaaaggatcaaa tgcactaaacgaaactgaaacaagcgatcgaaaatatccctttgggattcttgactcgataagtctattattttcagagaaaaaatattcattgttttct gggttggtgattgcaccaatcattccattcaaaattgttgttttaccacacccattccgcccgataaaagcatgaatgttcgtgctgggcatagaattaa ccgtcacctcaaaaggtatagttaaatcactgaatccgggagcactttttctattaaatgaaaagtggaaatctgacaattctggcaaaccatttaaca cacgtgcgaactgtccatgaatttctgaaagagttacccctctaagtaatgaggtgttaaggacgctttcattttcaatgtcggctaatcgatttggccat actactaaatcctgaatagctttaagaaggttatgtttaaaaccatcgcttaatttgctgagattaacatagtagtcaatgctttcacctaaggaaaaaa acatttcagggagttgactgaattttttatctattaatgaataagtgcttacttcttctttttgacctacaaaaccaattttaacatttccgatatcgcatttt tcaccatgctcatcaaagacagtaagataaaacattgtaacaaaggaatagtcattccaaccatctgctcgtaggaatgccttatttttttctactgcag gaatatacccgcctctttcaataacactaaactccaacatatagtaacccttaattttattaaaataaccgcaatttatttggcggcaacacaggatctc tcttttaagttactctctattacatacgttttccatctaaaaattagtagtattgaacttaacggggcatcgtattgtagttttccatatttagctttctgcttc cttttggataacccactgttattcatgttgcatggtgcactgtttataccaacgatatagtctattaatgcatatatagtatcgccgaacgattagctcttc aggcttctgaagaagcgtttcaagtactaataagccgatagatagccacggacttcgtagccatttttcataagtgttaacttccgctcctcgctcataa cagacattcactacagttatggcggaaaggtatgcatgctgggtgtggggaagtcgtgaaagaaaagaagtcagctgcgtcgtttgacatcactgcta tcttcttactggttatgcaggtcgtagtgggtggcacacaaagctttgcactggattgcgaggctttgtgcttctctggagtgcgacaggtttgatgacaa aaaattagcgcaagaagacaaaaatcaccttgcgctaatgctctgttacaggtcactaataccatctaagtagttgattcatagtgactgcatatgttgt gttttacagtattatgtagtctgttttttatgcaaaatctaatttaatatattgatatttatatcattttacgtttctcgttcagcttttttatactaagttggca ttataaaaaagcattgcttatcaatttgttgcaacgaacaggtcactatcagtcaaaataaaatcattatttgatttcaattttgtcccactccctgcctct gtcatcacgatactgtgatgccatggtgtccgacttatgcccgagaagatgttgagcaaacttatcgcttatctgcttctcatagagtcttgcagacaaa ctgcgcaactcgtgaaaggtaggcggatccccttcgaaggaaagacctgatgcttttcgtgcgcgcataaaataccttgatactgtgccggatgaaag cggttcgcgacgagtagatgcaattatggtttctccgccaagaatctctttgcatttatcaagtgtttccttcattgatattccgagagcatcaatatgca atgctgttgggatggcaatttttacgcctgttttgctttgctcgacataaagatatccatctacgatatcagaccacttcatttcgcataaatcaccaactc gttgcccggtaacaacagccagttccattgcaagtctgagccaacatggtgatgattctgctgcttgataaattttcaggtattcgtcagccgtaagtctt gatctccttacctctgattttgctgcgcgagtggcagcgacatggtttgttgttatatggccttcagctattgcctctcggaatgcatcgctcagtgttgat ctgattaacttggctgacgccgccttgccctcgtctatgtatccattgagcattgccgcaatttcttttgtggtgatgtcttcaagtggagcatcaggcaga cccctccttattgctttaattttgctcatgtaatttatgagtgtcttctgcttgattcctctgctggccaggattttttcgtagcgatcaagccatgaatgtaa cgtaacggaattatcactgttgattctcgctgtcagaggcttgtgtttgtgtcctgaaaataactcaatgttggcctgtatagcttcagtgattgcgattcg cctgtctctgcctaatccaaactctttacccgtccttgggtccctgtagcagtaatatccattgtttcttatataaaggttagggggtaaatcccggcgctc atgacttcgccttcttcccatttctgatcctcttcaaaaggccacctgttactggtcgatttaagtcaacctttaccgctgattcgtggaacagatactctct tccatccttaaccggaggtgggaatatcctgcattcccgaacccatcgacgaactgtttcaaggcttcttggacgtcgctggcgtgcgttccactcctga agtgtcaagtacatcgcaaagtctccgcaattacacgcaagaaaaaaccgccatcaggcggcttggtgttctttcagttcttcaattcgaatattggtta cgtctgcatgtgctatctgcgcccatatcatccagtggtcgtagcagtcgttgatgttctccgcttcgataactctgttgaatggctctccattccattctcc tgtgactcggaagtgcatttatcatctccataaaacaaaacccgccgtagcgagttcagataaaataaatccccgcgagtgcgaggattgttatgtaat attgggtttaatcatctatatgttttgtacagagagggcaagtatcgtttccaccgtactcgtgataataattttgcacggtatcagtcatttctcgcacat tgcagaatggggatttgtcttcattagacttataaaccttcatggaatatttgtatgccgactctatatctataccttcatctacataaacaccttcgtgat gtctgcatggagacaagacaccggatctgcacaacattgataacgcccaatctttttgctcagactctaactcattgatactcatttataaactccttgc aatgtatgtcgtttcagctaaacggtatcagcaatgtttatgtaaagaaacagtaagataatactcaacccgatgtttgagtacggtcatcatctgaca ctacagactctggcatcgctgtgaagacgacgcgaaattcagcattttcacaagcgttatcttttacaaaaccgatctcactctcctttgatgcgaatgc cagcgtcagacatcatatgcagatactcacctgcatcctgaacccattgacctccaaccccgtaatagcgatgcgtaatgatgtcgatagttactaacg ggtcttgttcgattaactgccgcagaaactcttccaggtcaccagtgcagtgcttgataacaggagtcttcccaggatggcgaacaacaagaaactgg tttccgtcttcacggacttcgttgctttccagtttagcaatacgcttactcccatccgagataacaccttcgtaatactcacgctgctcgttgagttttgatt ttgctgtttcaagctcaacacgcagtttccctactgttagcgcaatatcctcgttctcctggtcgcggcgtttgatgtattgctggtttctttcccgttcatcc agcagttccagcacaatcgatggtgttaccaattcatggaaaaggtctgcgtcaaatccccagtcgtcatgcattgcctgctctgccgcttcacgcagtg cctgagagttaatttcgctcacttcgaacctctctgtttactgataagttccagatcctcctggcaacttgcacaagtccgacaaccctgaacgaccagg cgtcttcgttcatctatcggatcgccacactcacaacaatgagtggcagatatagcctggtggttcaggcggcgcatttttattgctgtgttgcgctgtaa ttcttctatttctgatgctgaatcaatgatgtctgccatctttcattaatccctgaactgttggttaatacgcttgagggtgaatgcgaataataaaaaag gagcctgtagctccctgatgattttgcttttcatgttcatcgttccttaaagacgccgtttaacatgccgattgccaggcttaaatgagtcggtgtgaatcc catcagcgttaccgtttcgcggtgcttcttcagtacgctacggcaaatgtcatcgacgtttttatccggaaactgctgtctggctttttttgatttcagaatt agcctgacgggcaatgctgcgaagggcgttttcctgctgaggtgtcattgaacaagtcccatgtcggcaagcataagcacacagaatatgaagcccg ctgccagaaaaatgcattccgtggttgtcatacctggtttctctcatctgcttctgctttcgccaccatcatttccagcttttgtgaaagggatgcggctaa cgtatgaaattcttcgtctgtttctactggtattggcacaaacctgattccaatttgagcaaggctatgtgccatctcgatactcgttcttaactcaacaga agatgctttgtgcatacagcccctcgtttattatttatctcctcagccagccgctgtgctttcagtggatttcggataacagaaaggccgggaaataccc agcctcgctttgtaacggagtagacgaaagtgattgcgcctacccggatattatcgtgaggatgcgtcatcgccattgctccccaaatacaaaaccaat ttcagccagtgcctcgtccattttttcgatgaactccggcacgatctcgtcaaaactcgccatgtacttttcatcccgctcaatcacgacataatgcaggc cttcacgcttcatacgcgggtcatagttggcaaagtaccaggcattttttcgcgtcacccacatgctgtactgcacctgggccatgtaagctgactttatg gcctcgaaaccaccgagccggaacttcatgaaatcccgggaggtaaacgggcatttcagttcaaggccgttgccgtcactgcataaaccatcgggag agcaggcggtacgcatactttcgtcgcgatagatgatcggggattcagtaacattcacgccggaagtgaattcaaacagggttctggcgtcgttctcgt actgttttccccaggccagtgctttagcgttaacttccggagccacaccggtgcaaacctcagcaagcagggtgtggaagtaggacattttcatgtcag gccacttctttccggagcggggttttgctatcacgttgtgaacttctgaagcggtgatgacgccgagccgtaatttgtgccacgcatcatccccctgttcg acagctctcacatcgatcccggtacgctgcaggataatgtccggtgtcatgctgccaccttctgctctgcggctttctgtttcaggaatccaagagctttt actgcttcggcctgtgtcagttctgacgatgcacgaatgtcgcggcgaaatatctgggaacagagcggcaataagtcgtcatcccatgttttatccagg gcgatcagcagagtgttaatctcctgcatggtttcatcgttaaccggagtgatgtcgcgttccggctgacgttctgcagtgtatgcagtattttcgacaat gcgctcggcttcatccttgtcatagataccagcaaatccgaaggccagacgggcacactgaatcatggctttatgacgtaacatccgtttgggatgcga ctgccacggccccgtgatttctctgccttcgcgagttttgaatggttcgcggcggcattcatccatccattcggtaacgcagatcggatgattacggtcct tgcggtaaatccggcatgtacaggattcattgtcctgctcaaagtccatgccatcaaactgctggttttcattgatgatgcgggaccagccatcaacgcc caccaccggaacgatgccattctgcttatcaggaaaggcgtaaatttctttcgtccacggattaaggccgtactggttggcaacgatcagtaatgcgat gaactgcgcatcgctggcatcacctttaaatgccgtctggcgaagagtggtgatcagttcctgtgggtcgacagaatccatgccgacacgttcagccag cttcccagccagcgttgcgagtgcagtactcattcgttttatacctctgaatcaatatcaacctggtggtgagcaatggtttcaaccatgtaccggatgtg ttctgccatgcgctcctgaaactcaacatcgtcatcaaacgcacgggtaatggattttttgctggccccgtggcgttgcaaatgatcgatgcatagcgat tcaaacaggtgctggggcaggcctttttccatgtcgtctgccagttctgcctctttctcttcacgggcgagctgctggtagtgacgcgcccagctctgagc ctcaagacgatcctgaatgtaataagcgttcatggctgaactcctgaaatagctgtgaaaatatcgcccgcgaaatgccgggctgattaggaaaaca ggaaagggggttagtgaatgcttttgcttgatctcagtttcagtattaatatccattttttataagcgtcgacggcttcacgaaacatcttttcatcgccaa taaaagtggcgatagtgaatttagtctggatagccataagtgtttgatccattctttgggactcctggctgattaagtatgtcgataaggcgtttccatcc gtcacgtaatttacgggtgattcgttcaagtaaagattcggaagggcagccagcaacaggccaccctgcaatggcatattgcatggtgtgctccttatt tatacataacgaaaaacgcctcgagtgaagcgttattggtatgcggtaaaaccgcactcaggcggccttgatagtcatatcatctgaatcaaatattcc tgatgtatcgatatcggtaattcttattccttcgctaccatccattggaggccatccttcctgaccatttccatcattccagtcgaactcacacacaacacc atatgcatttaagtcgcttgaaattgctataagcagagcatgttgcgccagcatgattaatacagcatttaatacagagccgtgtttattgagtcggtat tcagagtctgaccagaaattattaatctggtgaagtttttcctctgtcattacgtcatggtcgatttcaatttctattgatgctttccagtcgtaatcaatga tgtattttttgatgtttgacatctgttcatatcctcacagataaaaaatcgccctcacactggagggcaaagaagatttccaataatcagaacaagtcgg ctcctgtttagttacgagcgacattgctccgtgtattcactcgttggaatgaatacacagtgcagtgtttattctgttatttatgccaaaaataaaggcca ctatcaggcagctttgttgttctgtttaccaagttctctggcaatcattgccgtcgttcgtattgcccatttatcgacatatttcccatcttccattacagga aacatttcttcaggcttaaccatgcattccgattgcagcttgcatccattgcatcgcttgaattgtccacaccattgatttttatcaatagtcgtagtcata cggatagtcctggtattgttccatcacatcctgaggatgctcttcgaactcttcaaattcttcttccatatatcaccttaaatagtggattgcggtagtaaa gattgtgcctgtcttttaaccacatcaggctcggtggttctcgtgtacccctacagcgagaaatcggataaactattacaacccctacagtttgatgagt atagaaatggatccactcgttattctcggacgagtgttcagtaatgaacctctggagagaaccatgtatatgatcgttatctgggttggacttctgctttt aagcccagataactggcctgaatatgttaatgagagaatcggtattcctcatgtgtggcatgttttcgtctttgctcttgcattttcgctagcaattaatgt gcatcgattatcagctattgccagcgccagatataagcgatttaagctaagaaaacgcattaagatgcaaaacgataaagtgcgatcagtaattcaa aaccttacagaagagcaatctatggttttgtgcgcagcccttaatgaaggcaggaagtatgtggttacatcaaaacaattcccatacattagtgagttg attgagcttggtgtgttgaacaaaactttttcccgatggaatggaaagcatatattattccctattgaggatatttactggactgaattagttgccagcta tgatccatataatattgagataaagccaaggccaatatctaagtaactagataagaggaatcgattttcccttaattttctggcgtccactgcatgttat gccgcgttcgccaggcttgctgtaccatgtgcgctgattcttgcgctcaatacgttgcaggttgctttcaatctgtttgtggtattcagccagcactgtaag gtctatcggatttagtgcgctttctactcgtgatttcggtttgcgattcagcgagagaatagggcggttaactggttttgcgcttaccccaaccaacaggg gatttgctgctttccattgagcctgtttctctgcgcgacgttcgcggcggcgtgtttgtgcatccatctggattctcctgtcagttagctttggtggtgtgtg gcagttgtagtcctgaacgaaaaccccccgcgattggcacattggcagctaatccggaatcgcacttacggccaatgcttcgtttcgtatcacacaccc caaagccttctgctttgaatgctgcccttcttcagggcttaatttttaagagcgtcaccttcatggtggtcagtgcgtcctgctgatgtgctcagtatcacc gccagtggtatttatgtcaacaccgccagagataatttatcaccgcagatggttatctgtatgttttttatatgaatttattttttgcaggggggcattgttt ggtaggtgagagatctgaattgctatgtttagtgagttgtatctatttatttttcaataaatacaattggttatgtgttttgggggcgatcgtgaggcaaa gaaaacccggcgctgaggccgggtcaggattttttacgtgaggcttttttacccccgctagctgcgcgttcagctttgattttttccagcaacgcggcgg cgctgttttctccgctgatcaaatccgggttttcggcccgccactgggcggtaagttcaccacggaacgcttttgccaggatggattgcgtcaggttgttg acgcgggctaaggcgttgttgacctgtttttctatggtgtcggcgtaggcgaagagttgctcgacgcggcgaacgatttcggcttgttcttttactggagg taataaaacaacttgggatttgatatcttttcctgaaatacctttttgaccagaagttgttttcacgcagttcatcattgcatttcgtgctgagggggatga aaaaaatatttcgatatattctggtaaagcatctttggttaatcgagctcgaataagtttatcaggatatagcaaattttgatgttgtaattttttcaataa cccacaaacaccaacaaattctaaacttccgttatagcgagtaaataaaagatctccatcttgtaatttgtggcggtttagttcactttctgaacattcta gaaaccgaatatcgttttgatctacatggccagcacgtacagaactaatgcgtagtattggatgaccaacaccactttcatttggctttgatgaaagac cattacgtaattcagttaagatagattcaaaatttaacttcttaaatacagaatgttgcggctcaaaattacgccatttttctgtcaattttccattaactg cgccccccaataccgcttgacgaaaacgtttcaggatttgtgggatttgctcaaaacgtgctttggtgctgtctacctgcgccagcagcgtatcgagtttt tcagcgatgattttttgttcggcaagtggtgggattggtatatttatcaaatcaaagcttgccggcttaatattattaatatttgcaccagcagaaagtga tgaaattttgtttcgataaagagaagattttgtgaaatgagcaataaaaccagaaaatataagtttttcaggacgtaatacaccgcaaaatgcgccga aactacattcaaatggtagatgctgatgtgcggatttaccaactacggatttgctccctgatgacattgcaataacaatatcttcaggagatattttttga ttttctttaacaagatttttaggaacaaaaaccaagtccgtagtatcaaacttgccattctgaatattgttcgcacggataagaggcaaataatcatcttt tagataatttattgcctgctcttttttatacgttactcctcggattagagttgtgaccgtagatactggggcgataacccacccctccggcaatttccccgc actcattccttcaccccaccaaacgcttcttccagcaactgacgctgcaaatcggcctcatcgctcgcccccagttcacgcatcagcgcatccagttcag acagcgcctgtaccagttcgcccatcgcttctgccgctaatacatccggctccggcaggctgtcggcatcaatactgtctttatctttcagccaggagat atccagcgaatcggattttgcggtgcggatccactcacggctgaacttgcgccagcggctggtagcaagatgctggtcggtgtttttgttctcttcgctgt cggcaacttccgtctcttcggcgttaaaactccattcaccttcagtgcgcgggcttaaaccgtgcgggtcttcgccatacacgcgctcaaacggctgcaa atgctcgtcggtaaacggtgtgcgcttgccgaaactcggcatattggtacgcaggtcatacacccacacgtcgtcggtgcagttcttatcctgatgcgg gttcgccactgtgcctttggtaaagaacagcacgttggtcttcacgccctgcgcgtaaaaaataccggtcggcagacgcagaatggtgtgcagatgac acttatccatcaggtcacgacggatgtcggtgcctttgccgccttcaaacaacacgttatctggcaccaccaccgccgcacgaccgccgggatgcagc gtttcgataatatgctgcataaagcacagctgtttgttgctggtcgggtgaacaaaggtgcgggtaatgttggtgcctgcggcgctgccaaacggcggg ttagtggcgacaatatgcgccttcggcaggttttcaccgtcgctgcccagggtgttgcccagacggattgcgccgccgtggtcgaggttgccttcaatat cgtgcagcaggcagttcatcagtgccagacgacgggtgccgggcaccagttcgaggccgataaacgcgcggtggatctggaaatcctgcgtgtcgcc atcaaggtcgtccagatcattggtttgcgacttaacatagcggtcggcttcaatcaaaaagcccgccgtacctgccgccgggtcctgcaccacttcacg cggctgcggtttcagcagatgaataatggttttaatcagcggacgcggggtgaagtactggcctgcaccagacttggtttcattcgcgttcttctgcaac agcccttcgtacatatcgccgaagtcatcgcgcgacttaccgtgcgcgccgttgtaccagtccagcgaatccatattgctgaccagtgcggttatttgttt cggctcggtgatggtggtactaacattatgaaaaactgcctgtaccagctttttgtcatcttcgcctaaatgcacgagcatttttcggtagaactgcaact gctcctggccgatgcgggatttcaggtcatcccagcggtaaccttccggcaggtattccgcttcctgacccgtctctttacacattttcaaaaacagcag cgaggcgagttcattgacgtagttttgataggaaacgccgccatcgcgcaggttgtcgcacagcttccacagcttcgcgaccagatcgttattgttcatt ctagatctcctctttaatgctcgctagcactgtacctaggactgagctagccgtcaaacgttaaatctatcaccgcaagggataaatatctaacaccgtg cgtgttgactattttacctctggcggtgataatggttgcatgtactaaggaggttgtatggaacaacgcataaccctgaaagattatgcaatgcgctttg ggcaaaccaagacagctaaagatctcggcgtatatcaaagcgcgatcaacaaggccattcatgcaggccgaaagatttttttaactataaacgctgat ggaagcgtttatgcggaagaggtaaagcccttcccgagtaacaaaaaaacaacagcataaataaccccgctcttacacattccagccctgaaaaag ggcatcaaattaaaccacacctatggtgtatgcatttatttgcatacattcaatcaattgttatctaaggaaatacttacatatggttcgtgcaaacaaa cgcaacgaggctctacgaatcgagagtgcgttgcttaacaaaatcgcaatgcttggaactgagaagacagcggaagctgtgggcgttgataagtcgc agatcagcaggtggaagagggactggattccaaagttctcaatgctgcttgctgttcttgaatggggggtcgttgacgacgacatggctcgattggcgc gacaagttgctgcgattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctgaggtcattactggatctat caacaggagtcattatgacaaatacagcaaaaatactcaacttcggcagaggtaactttgccggacaggagcgtaatgtggcagatctcgatgatgg ttacgccagactatcaaatatgctgcttgaggcttattcgggcgcagatctgaccaagcgacagtttaaagtgctgcttgccattctgcgtaaaacctat gggtggaataaaccaatggacagaatcaccgattctcaacttagcgagattacaaagttacctgtcaaacggtgcaatgaagccaagttagaactcg tcagaatgaatattatcaagcagcaaggcggcatgtttggaccaaataaaaacatctcagaatggtgcatccctcaaaacgagggaaaatcccctaa aacgagggataaaacatccctcaaattgggggattgctatccctcaaaacagggggacacaaaagacactattacaaaagaaaaaagaaaagatt attcgtcagagaattctggcgaatcctctgaccagccagaaaacgacctttctgtggtgaaaccggatgctgcaattcagagcggcagcaagtgggg gacagcagaagacctgaccgccgcagagtggatgtttgacatggtgaagactatcgcaccatcagccagaaaaccgaattttgctgggtgggctaac gatatccgcctgatgcgtgaacgtgacggacgtaaccaccgcgacatgtgtgtgctgttccgctgggcatgccaggacaacttctggtccggtaacgtg ctgagcccggccaaactccgcgataagtggacccaactcgaaatcaaccgtaacaagcaacaggcaggcgtgacagccagcaaaccaaaactcga cctgacaaacacagactggatttacggggtggatctatgaaaaacatcgccgcacagatggttaactttgaccgtgagcagatgcgtcggatcgcca acaacatgccggaacagtacgacgaaaagccgcaggtacagcaggtagcgcagatcatcaacggtgtgttcagccagttactggcaactttcccggc gagcctggctaaccgtgaccagaacgaagtgaacgaaatccgtcgccagtgggttctggcttttcgggaaaacgggatcaccacgatggaacaggtt aacgcaggaatgcgcgtagcccgtcggcagaatcgaccatttctgccatcacccgggcagtttgttgcatggtgccgggaagaagcatccgttaccgc cggactgccaaacgtcagcgagctggttgatatggtttacgagtattgccggaagcgaggcctgtatccggatgcggagtcttatccgtggaaatcaa acgcgcactactggctggttaccaacctgtatcagaacatgcgggccaatgcgcttactgatgcggaattacgccgtaaggccgcagatgagcttgtc catatgactgcgagaattaaccgtggtgaggcgatccctgaaccagtaaaacaacttcctgtcatgggcggtagacctctaaatcgtgcacaggctct ggcgaagatcgcagaaatcaaagctaagttcggactgaaaggagcaagtgtatgacgggcaaagaggcaattattcattacctggggacgcataat agcttctgtgcgccggacgttgccgcgctaacaggcgcaacagtaaccagcataaatcaggccgcggctaaaatggcacgggcaggtcttctggttat cgaaggtaaggtctggcgaacggtgtattaccggtttgctaccagggaagaacgggaaggaaagatgagcacgaacctggtttttaaggagtgtcgc cagagtgccgcgatgaaacgggtattggcggtatatggagttaaaagatgaccatctacattactgagctaataacaggcctgctggtaatcgcaggc ctttttatttgggggagagggaagtcatgaaaaaactaacctttgaaattcgatctccagcacatcagcaaaacgctattcacgcagtacagcaaatc cttccagacccaaccaaaccaatcgtagtaaccattcaggaacgcaaccgcagcttagaccaaaacaggaagctatgggcctgcttaggtgacgtct ctcgtcaggttgaatggcatggtcgctggctggatgcagaaagctggaagtgtgtgtttaccgcagcattaaagcagcaggatgttgttcctaaccttg ccgggaatggctttgtggtaataggccagtcaaccagcaggatgcgtgtaggcgaatttgcggagctattagagcttatacaggcattcggtacagag cgtggcgttaagtggtcagacgaagcgagactggctctggagtggaaagcgagatggggagacagggctgcatgataaatgtcgttagtttctccgg tggcaggacgtcagcatatttgctctggctaatggagcaaaagcgacgggcaggtaaagacgtgcattacgttttcatggatacaggttgtgaacatc caatgacatatcggtttgtcagggaagttgtgaagttctgggatataccgctcaccgtattgcaggttgatatcaacccggagcttggacagccaaatg gttatacggtatgggaaccaaaggatattcagacgcgaatgcctgttctgaagccatttatcgatatggtaaagaaatatggcactccatacgtcggc ggcgcgttctgcactgacagattaaaactcgttcccttcaccaaatactgtgatgaccatttcgggcgagggaattacaccacgtggattggcatcaga gctgatgaaccgaagcggctaaagccaaagcctggaatcagatatcttgctgaactgtcagactttgagaaggaagatatcctcgcatggtggaagc aacaaccattcgatttgcaaataccggaacatctcggtaactgcatattctgcattaaaaaatcaacgcaaaaaatcggacttgcctgcaaagatgag gagggattgcagcgtgtttttaatgaggtcatcacgggatcccatgtgcgtgacggacatcgggaaacgccaaaggagattatgtaccgaggaagaa tgtcgctggacggtatcgcgaaaatgtattcagaaaatgattatcaagccctgtatcaggacatggtacgagctaaaagattcgataccggctcttgtt ctgagtcatgcgaaatatttggagggcagcttgatttcgacttcgggagggaagctgcatgatgcgatgttatcggtgcggtgaatgcaaagaagata accgcttccgaccaaatcaaccttactggaatcgatggtgtctccggtgtgaaagaacaccaacaggggtgttaccactaccgcaggaaaaggagga cgtgtggcgagacagcgacgaagtatcaccgacataatctgcgaaaactgcaaataccttccaacgaaacgcaccagaaataaacccaagccaatc ccaaaagaatctgacgtaaaaaccttcaactacacggctcacctgtgggatatccggtggctaagacgtcgtgcgaggaaaacaaggtgattgacca aaatcgaagttacgaacaagaaagcgtcgagcgagctttaacgtgcgctaactgcggtcagaagctgcatgtgctggaagttcacgtgtgtgagcac tgctgcgcagaactgatgagcgatccgaatagctcgatgcacgaggaagaagatgatggctaaaccagcgcgaagacgatgtaaaaacgatgaat gccgggaatggtttcaccctgcattcgctaatcagtggtggtgctctccagagtgtggaaccaagatagcactcgaacgacgaagtaaagaacgcga aaaagcggaaaaagcagcagagaagaaacgacgacgagaggagcagaaacagaaagataaacttaagattcgaaaactcgccttaaagccccg cagttactggattaaacaagcccaacaagccgtaaacgccttcatcagagaaagggaccgcgacttaccatgtatctcgtgcggaacgctcacgtctg ctcagtgggatgccggacattaccggacaactgctgcggcacctcaactccgatttaatgaacgcaatattcacaagcaatgcgtggtgtgcaaccag cacaaaagcggaaatctcgttccgtatcgcgtcgaactgattagccgcatcgggcaggaagcagtagacgaaatcgaatcaaaccataaccgccatc gctggactatcgaagagtgcaaggcgatcaaggcagagtaccaacagaaactcaaagacctgcgaaatagcagaagtgaggccgcatgacgttctc agtaaaaaccattccagacatgctcgttgaagcatacggaaatcagacagaagtagcacgcagactgaaatgtagtcgcggtacggtcagaaaata cgttgatgataaagacgggaaaatgcacgccatcgtcaacgacgttctcatggttcatcgcggatggagtgaaagagatgcgctattacgaaaaaat tgatggcagcaaataccgaaatatttgggtagttggcgatctgcacggatgctacacgaacctgatgaacaaactggatacgattggattcgacaaca aaaaagacctgcttatctcggtgggcgatttggttgatcgtggtgcagagaacgttgaatgcctggaattaatcacattcccctggttcagagctgtacg tggaaaccatgagcaaatgatgattgatggcttatcagagcgtggaaacgttaatcactggctgcttaatggcggtggctggttctttaatctcgattac gacaaagaaattctggctaaagctcttgcccataaagcagatgaacttccgttaatcatcgaactggtgagcaaagataaaaaatatgttatctgcca cgccgattatccctttgacgaatacgagtttggaaagccagttgatcatcagcaggtaatctggaaccgcgaacgaatcagcaactcacaaaacggg atcgtgaaagaaatcaaaggcgcggacacgttcatctttggtcatacgccagcagtgaaaccactcaagtttgccaaccaaatgtatatcgataccgg cgcagtgttctgcggaaacctaacattgattcaggtacagggagaaggcgcatgagactcgaaagcgtagctaaatttcattcgccaaaaagcccga tgatgagcgactcaccacgggccacggcttctgactctctttccggtactgatgtgatggctgctatggggatggcgcaatcacaagccggattcggta tggctgcattctgcggtaagcacgaactcagccagaacgacaaacaaaaggctatcaactatctgatgcaatttgcacacaaggtatcggggaaata ccgtggtgtggcaaagcttgaaggaaatactaaggcaaaggtactgcaagtgctcgcaacattcgcttatgcggattattgccgtagtgccgcgacgc cgggggcaagatgcagagattgccatggtacaggccgtgcggttgatattgccaaaacagagctgtgggggagagttgtcgagaaagagtgcggaa gatgcaaaggcgtcggctattcaaggatgccagcaagcgcagcatatcgcgctgtgacgatgctaatcccaaaccttacccaacccacctggtcacgc actgttaagccgctgtatgacgctctggtggtgcaatgccacaaagaagagtcaatcgcagacaacattttgaatgcggtcacacgttagcagcatga ttgccacggatggcaacatattaacggcatgatattgacttattgaataaaattgggtaaatttgactcaacgatgggttaattcgctcgttgtggtagt gagatgaaaagaggcggcgcttactaccgattccgcctagttggtcacttcgacgtatcgtctggaactccaaccatcgcaggcagagaggtctgcaa aatgcaatcccgaaacagttcgcaggtaatagttagagcctgcataacggtttcgggattttttatatctgcacaacaggtaagagcattgagtcgata atcgtgaagagtcggcgagcctggttagccagtgctctttccgttgtgctgaattaagcgaataccggaagcagaaccggatcaccaaatgcgtacag gcgtcatcgccgcccagcaacagcacaacccaaactgagccgtagccactgtctgtcctgaattcattagtaatagttacgctgcggccttttacacat gaccttcgtgaaagcgggtggcaggaggtcgcgctaacaacctcctgccgttttgcccgtgcatatcggtcacgaacaaatctgattactaaacacag tagcctggatttgttctatcagtaatcgaccttattcctaattaaatagagcaaatccccttattgggggtaagacatgaagatgccagaaaaacatga cctgttggccgccattctcgcggcaaaggaacaaggcatcggggcaatccttgcgtttgcaatggcgtaccttcgcggcagatataatggcggtgcgtt tacaaaaacagtaatcgacgcaacgatgtgcgccattatcgcctggttcattcgtgaccttctcgacttcgccggactaagtagcaatctcgcttatata acgagcgtgtttatcggctacatcggtactgactcgattggttcgcttatcaaacgcttcgctgctaaaaaagccggagtagaagatggtagaaatcaa taatcaacgtaaggcgttcctcgatatgctggcgtggtcggagggaactgataacggacgtcagaaaaccagaaatcatggttatgacgtcattgtag gcggagagctatttactgattactccgatcaccctcgcaaacttgtcacgctaaacccaaaactcaaatcaacaggcgccggacgctaccagcttcttt cccgttggtgggatgcctaccgcaagcagcttggcctgaaagacttctctccgaaaagtcaggacgctgtggcattgcagcagattaaggagcgtggc gctttacctatgattgatcgtggtgatatccgtcaggcaatcgaccgttgcagcaatatctgggcttcactgccgggcgctggttatggtcagttcgagc ataaggctgacagcctgattgcaaaattcaaagaagcgggcggaacggtcagagagattgatgtatgagcagagtcaccgcgattatctccgctctg gttatctgcatcatcgtctgcctgtcatgggctgttaatcattaccgtgataacgccattacctacaaagcccagcgcgacaaaaatgccagagaactg aagctggcgaacgcggcaattactgacatgcagatgcgtcagcgtgatgttgctgcgctcgatgcaaaatacacgaaggagttagctgatgctaaag ctgaaaatgatgctctgcgtgatgatgttgccgctggtcgtcgtcggttgcacatcaaagcagtctgtcagtcagtgcgtgaagccaccaccgcctccg gcgtggataatgcagcctccccccgactggcagacaccgctgaacgggattatttcaccctcagagagaggctgatcactatgcaaaaacaactgga aggaacccagaagtatattaatgagcagtgcagatagagttgcccatatcgatgggcaactcatgcaattattgtgagcaatacacacgcgcttccag cggagtataaatgcctaaagtaataaaaccgagcaatccatttacgaatgtttgctgggtttctgttttaacaacattttctgcgccgccacaaattttgg ctgcatcgacagttttcttctgcccaattccagaaacgaagaaatgatgggtgatggtttcctttggtgctactgctgccggtttgttttgaacagtaaac gtctgttgagcacatcctgtaataagcagggccagcgcagtagcgagtagcatttttttcatggtgttattcccgatgctttttgaagttcgcagaatcgt atgtgtagaaaattaaacaaaccctaaacaatgagttgaaatttcatattgttaatatttattaatgtatgtcaggtgcgatgaatcgtcattgtattccc ggattaactatgtccacagccctgacggggaacttctctgcgggagtgtccgggaataattaaaacgatgcacacagggtttagcgcgtacacgtatt gcattatgccaacgccccggtgctgacacggaagaaaccggacgttatgatttagcgtggaaagatttgtgtagtgttctgaatgctctcagtaaatag taatgaattatcaaaggtatagtaatatcttttatgttcatggatatttgtaacccatcggaaaactcctgctttagcaagattttccctgtattgctgaaa tgtgatttctcttgatttcaacctatcataggacgtttctataagatgcgtgtttcttgagaatttaacatttacaacctttttaagtccttttattaacacgg tgttatcgttttctaacacgatgtgaatattatctgtggctagatagtaaatataatgtgagacgttgtgacgttttagttcagaataaaacaattcacag tctaaatcttttcgcacttgatcgaatatttctttaaaaatggcaacctgagccattggtaaaaccttccatgtgatacgagggcgcgtagtttgcattat cgtttttatcgtttcaatctggtctgacctccttgtgttttgttgatgatttatgtcaaatattaggaatgttttcacttaatagtattggttgcgtaacaaag tgcggtcctgctggcattctggagggaaatacaaccgacagatgtatgtaaggccaacgtgctcaaatcttcatacagaaagatttgaagtaatatttt aaccgctagatgaagagcaagcgcatggagcgacaaaatgaataaagaacaatctgctgatgatccctccgtggatctgattcgtgtaaaaaatatg cttaatagcaccatttctatgagttaccctgatgttgtaattgcatgtatagaacataaggtgtctctggaagcattcagagcaattgaggcagcgttgg tgaagcacgataataatatgaaggattattccctggtggttgactgatcaccataactgctaatcattcaaactatttagtctgtgacagagccaacac gcagtctgtcactgtcaggaaagtggtaaaactgcaactcaattactgcaatgccctcgtaattaagtgaatttacaatatcgtcctgttcggagggaa gaacgcgggatgttcattcttcatcacttttaattgatgtatatgctctcttttctgacgttagtctccgacggcaggcttcaatgacccaggctgagaaat tcccggaccctttttgctcaagagcgatgttaatttgttcaatcatttggttaggaaagcggatgttgcgggttgttgttctgcgggttctgttcttcgttga catgaggttgccccgtattcagtgtcgctgatttgtattgtctgaagttgtttttacgttaagttgatgcagatcaattaatacgatacctgcgtcataatt gattatttgacgtggtttgatggcctccacgcacgttgtgatatgtagatgataatcattatcactttacgggtcctttccggtgatccgacaggttacg

Appendix III: Sequence of Lambda phage reboot Design II (virulent lambda, Eco0015 MTase) (SEQ ID NO: 4)

(sequence begins at COS site) gggcggcgacctcgcgggttttcgctatttatgaaaattttccggtttaaggcgtttccgttcttcttcgtcataacttaatgtttttatttaaaataccctct gaaaagaaaggaaacgacaggtgctgaaagcgaggctttttggcctctgtcgtttcctttctctgtttttgtccgtggaatgaacaatggaagtcaaca aaaagcagctggctgacattttcggtgcgagtatccgtaccattcagaactggcaggaacagggaatgcccgttctgcgaggcggtggcaagggtaa tgaggtgctttatgactctgccgccgtcataaaatggtatgccgaaagggatgctgaaattgagaacgaaaagctgcgccgggaggttgaagaactg cggcaggccagcgaggcagatctccagccaggaactattgagtacgaacgccatcgacttacgcgtgcgcaggccgacgcacaggaactgaagaat gccagagactccgctgaagtggtggaaaccgcattctgtactttcgtgctgtcgcggatcgcaggtgaaattgccagtattctcgacgggctccccctgt cggtgcagcggcgttttccggaactggaaaaccgacatgttgatttcctgaaacgggatatcatcaaagccatgaacaaagcagccgcgctggatga actgataccggggttgctgagtgaatatatcgaacagtcaggttaacaggctgcggcattttgtccgcgccgggcttcgctcactgttcaggccggagc cacagaccgccgttgaatgggcggatgctaattactatctcccgaaagaatccgcataccaggaagggcgctgggaaacactgccctttcagcgggc catcatgaatgcgatgggcagcgactacatccgtgaggtgaatgtggtgaagtctgcccgtgtcggttattccaaaatgctgctgggtgtttatgcctac tttatagagcataagcagcgcaacacccttatctggttgccgacggatggtgatgccgagaactttatgaaaacccacgttgagccgactattcgtgat attccgtcgctgctggcgctggccccgtggtatggcaaaaagcaccgggataacacgctcaccatgaagcgtttcactaatgggcgtggcttctggtgc ctgggcggtaaagcggcaaaaaactaccgtgaaaagtcggtggatgtggcgggttatgatgaacttgctgcttttgatgatgatattgaacaggaagg ctctccgacgttcctgggtgacaagcgtattgaaggctcggtctggccaaagtccatccgtggctccacgccaaaagtgagaggcacctgtcagattg agcgtgcagccagtgaatccccgcattttatgcgttttcatgttgcctgcccgcattgcggggaggagcagtatcttaaatttggcgacaaagagacgc cgtttggcctcaaatggacgccggatgacccctccagcgtgttttatctctgcgagcataatgcctgcgtcatccgccagcaggagctggactttactga tgcccgttatatctgcgaaaagaccgggatctggacccgtgatggcattctctggttttcgtcatccggtgaagagattgagccacctgacagtgtgacc tttcacatctggacagcgtacagcccgttcaccacctgggtgcagattgtcaaagactggatgaaaacgaaaggggatacgggaaaacgtaaaacct tcgtaaacaccacgctcggtgagacgtgggaggcgaaaattggcgaacgtccggatgctgaagtgatggcagagcggaaagagcattattcagcgc ccgttcctgaccgtgtggcttacctgaccgccggtatcgactcccagctggaccgctacgaaatgcgcgtatggggatgggggccgggtgaggaaagc tggctgattgaccggcagattattatgggccgccacgacgatgaacagacgctgctgcgtgtggatgaggccatcaataaaacctatacccgccgga atggtgcagaaatgtcgatatcccgtatctgctgggatactggcgggattgacccgaccattgtgtatgaacgctcgaaaaaacatgggctgttccgg gtgatccccattaaaggggcatccgtctacggaaagccggtggccagcatgccacgtaagcgaaacaaaaacggggtttaccttaccgaaatcggta cggataccgcgaaagagcagatttataaccgcttcacactgacgccggaaggggatgaaccgcttcccggtgccgttcacttcccgaataacccggat atttttgatctgaccgaagcgcagcagctgactgctgaagagcaggtcgaaaaatgggtggatggcaggaaaaaaatactgtgggacagcaaaaag cgacgcaatgaggcactcgactgcttcgtttatgcgctggcggcgctgcgcatcagtatttcccgctggcagctggatctcagtgcgctgctggcgagc ctgcaggaagaggatggtgcagcaaccaacaagaaaacactggcagattacgcccgtgccttatccggagaggatgaatgacgcgacaggaagaa cttgccgctgcccgtgcggcactgcatgacctgatgacaggtaaacgggtggcaacagtacagaaagacggacgaagggtggagtttacggccactt ccgtgtctgacctgaaaaaatatattgcagagctggaagtgcagaccggcatgacacagcgacgcaggggacctgcaggattttatgtatgaaaacg cccaccattcccacccttctggggccggacggcatgacatcgctgcgcgaatatgccggttatcacggcggtggcagcggatttggagggcagttgcg gtcgtggaacccaccgagtgaaagtgtggatgcagccctgttgcccaactttacccgtggcaatgcccgcgcagacgatctggtacgcaataacggct atgccgccaacgccatccagctgcatcaggatcatatcgtcgggtcttttttccggctcagtcatcgcccaagctggcgctatctgggcatcggggagg aagaagcccgtgccttttcccgcgaggttgaagcggcatggaaagagtttgccgaggatgactgctgctgcattgacgttgagcgaaaacgcacgttt accatgatgattcgggaaggtgtggccatgcacgcctttaacggtgaactgttcgttcaggccacctgggataccagttcgtcgcggcttttccggaca cagttccggatggtcagcccgaagcgcatcagcaacccgaacaataccggcgacagccggaactgccgtgccggtgtgcagattaatgacagcggt gcggcgctgggatattacgtcagcgaggacgggtatcctggctggatgccgcagaaatggacatggataccccgtgagttacccggcgggcgcgcct cgttcattcacgtttttgaacccgtggaggacgggcagactcgcggtgcaaatgtgttttacagcgtgatggagcagatgaagatgctcgacacgctgc agaacacgcagctgcagagcgccattgtgaaggcgatgtatgccgccaccattgagagtgagctggatacgcagtcagcgatggattttattctgggc gcgaacagtcaggagcagcgggaaaggctgaccggctggattggtgaaattgccgcgtattacgccgcagcgccggtccggctgggaggcgcaaaa gtaccgcacctgatgccgggtgactcactgaacctgcagacggctcaggatacggataacggctactccgtgtttgagcagtcactgctgcggtatatc gctgccgggctgggtgtctcgtatgagcagctttcccggaattacgcccagatgagctactccacggcacgggccagtgcgaacgagtcgtgggcgta ctttatggggcggcgaaaattcgtcgcatcccgtcaggcgagccagatgtttctgtgctggctggaagaggccatcgttcgccgcgtggtgacgttacc ttcaaaagcgcgcttcagttttcaggaagcccgcagtgcctgggggaactgcgactggataggctccggtcgtatggccatcgatggtctgaaagaag ttcaggaagcggtgatgctgatagaagccggactgagtacctacgagaaagagtgcgcaaaacgcggtgacgactatcaggaaatttttgcccagca ggtccgtgaaacgatggagcgccgtgcagccggtcttaaaccgcccgcctgggcggctgcagcatttgaatccgggctgcgacaatcaacagaggag gagaagagtgacagcagagctgcgtaatctcccgcatattgccagcatggcctttaatgagccgctgatgcttgaacccgcctatgcgcgggttttcttt tgtgcgcttgcaggccagcttgggatcagcagcctgacggatgcggtgtccggcgacagcctgactgcccaggaggcactcgcgacgctggcattatc cggtgatgatgacggaccacgacaggcccgcagttatcaggtcatgaacggcatcgccgtgctgccggtgtccggcacgctggtcagccggacgcgg gcgctgcagccgtactcggggatgaccggttacaacggcattatcgcccgtctgcaacaggctgccagcgatccgatggtggacggcattctgctcga tatggacacgcccggcgggatggtggcgggggcatttgactgcgctgacatcatcgcccgtgtgcgtgacataaaaccggtatgggcgcttgccaacg acatgaactgcagtgcaggtcagttgcttgccagtgccgcctcccggcgtctggtcacgcagaccgcccggacaggctccatcggcgtcatgatggctc acagtaattacggtgctgcgctggagaaacagggtgtggaaatcacgctgatttacagcggcagccataaggtggatggcaacccctacagccatct tccggatgacgtccgggagacactgcagtcccggatggacgcaacccgccagatgtttgcgcagaaggtgtcggcatataccggcctgtccgtgcag gttgtgctggataccgaggctgcagtgtacagcggtcaggaggccattgatgccggactggctgatgaacttgttaacagcaccgatgcgatcaccgt catgcgtgatgcactggatgcacgtaaatcccgtctctcaggagggcgaatgaccaaagagactcaatcaacaactgtttcagccactgcttcgcagg ctgacgttactgacgtggtgccagcgacggagggcgagaacgccagcgcggcgcagccggacgtgaacgcgcagatcaccgcagcggttgcggca gaaaacagccgcattatggggatcctcaactgtgaggaggctcacggacgcgaagaacaggcacgcgtgctggcagaaacccccggtatgaccgtg aaaacggcccgccgcattctggccgcagcaccacagagtgcacaggcgcgcagtgacactgcgctggatcgtctgatgcagggggcaccggcaccg ctggctgcaggtaacccggcatctgatgccgttaacgatttgctgaacacaccagtgtaagggatgtttatgacgagcaaagaaacctttacccattac cagccgcagggcaacagtgacccggctcataccgcaaccgcgcccggcggattgagtgcgaaagcgcctgcaatgaccccgctgatgctggacacc tccagccgtaagctggttgcgtgggatggcaccaccgacggtgctgccgttggcattcttgcggttgctgctgaccagaccagcaccacgctgacgttc tacaagtccggcacgttccgttatgaggatgtgctctggccggaggctgccagcgacgagacgaaaaaacggaccgcgtttgccggaacggcaatca gcatcgtttaactttacccttcatcactaaaggccgcctgtgcggctttttttacgggatttttttatgtcgatgtacacaaccgcccaactgctggcggca aatgagcagaaatttaagtttgatccgctgtttctgcgtctctttttccgtgagagctatcccttcaccacggagaaagtctatctctcacaaattccggg actggtaaacatggcgctgtacgtttcgccgattgtttccggtgaggttatccgttcccgtggcggctccacctctgaatttacgccgggatatgtcaagc cgaagcatgaagtgaatccgcagatgaccctgcgtcgcctgccggatgaagatccgcagaatctggcggacccggcttaccgccgccgtcgcatcat catgcagaacatgcgtgacgaagagctggccattgctcaggtcgaagagatgcaggcagtttctgccgtgcttaagggcaaatacaccatgaccggt gaagccttcgatccggttgaggtggatatgggccgcagtgaggagaataacatcacgcagtccggcggcacggagtggagcaagcgtgacaagtcc acgtatgacccgaccgacgatatcgaagcctacgcgctgaacgccagcggtgtggtgaatatcatcgtgttcgatccgaaaggctgggcgctgttccg ttccttcaaagccgtcaaggagaagctggatacccgtcgtggctctaattccgagctggagacagcggtgaaagacctgggcaaagcggtgtcctata aggggatgtatggcgatgtggccatcgtcgtgtattccggacagtacgtggaaaacggcgtcaaaaagaacttcctgccggacaacacgatggtgct ggggaacactcaggcacgcggtctgcgcacctatggctgcattcaggatgcggacgcacagcgcgaaggcattaacgcctctgcccgttacccgaaa aactgggtgaccaccggcgatccggcgcgtgagttcaccatgattcagtcagcaccgctgatgctgctggctgaccctgatgagttcgtgtccgtacaa ctggcgtaatcatggcccttcggggccattgtttctctgtggaggagtccatgacgaaagatgaactgattgcccgtctccgctcgctgggtgaacaact gaaccgtgatgtcagcctgacggggacgaaagaagaactggcgctccgtgtggcagagctgaaagaggagcttgatgacacggatgaaactgccg gtcaggacacccctctcagccgggaaaatgtgctgaccggacatgaaaatgaggtgggatcagcgcagccggataccgtgattctggatacgtctga actggtcacggtcgtggcactggtgaagctgcatactgatgcacttcacgccacgcgggatgaacctgtggcatttgtgctgccgggaacggcgtttcg tgtctctgccggtgtggcagccgaaatgacagagcgcggcctggccagaatgcaataacgggaggcgctgtggctgatttcgataacctgttcgatgc tgccattgcccgcgccgatgaaacgatacgcgggtacatgggaacgtcagccaccattacatccggtgagcagtcaggtgcggtgatacgtggtgttt ttgatgaccctgaaaatatcagctatgccggacagggcgtgcgcgttgaaggctccagcccgtccctgtttgtccggactgatgaggtgcggcagctgc ggcgtggagacacgctgaccatcggtgaggaaaatttctgggtagatcgggtttcgccggatgatggcggaagttgtcatctctggcttggacggggc gtaccgcctgccgttaaccgtcgccgctgaaagggggatgtatggccataaaaggtcttgagcaggccgttgaaaacctcagccgtatcagcaaaac ggcggtgcctggtgccgccgcaatggccattaaccgcgttgcttcatccgcgatatcgcagtcggcgtcacaggttgcccgtgagacaaaggtacgcc ggaaactggtaaaggaaagggccaggctgaaaagggccacggtcaaaaatccgcaggccagaatcaaagttaaccggggggatttgcccgtaatc aagctgggtaatgcgcgggttgtcctttcgcgccgcaggcgtcgtaaaaaggggcagcgttcatccctgaaaggtggcggcagcgtgcttgtggtggg taaccgtcgtattcccggcgcgtttattcagcaactgaaaaatggccggtggcatgtcatgcagcgtgtggctgggaaaaaccgttaccccattgatgt ggtgaaaatcccgatggcggtgccgctgaccacggcgtttaaacaaaatattgagcggatacggcgtgaacgtcttccgaaagagctgggctatgcg ctgcagcatcaactgaggatggtaataaagcgatgaaacatactgaactccgtgcagccgtactggatgcactggagaagcatgacaccggggcga cgttttttgatggtcgccccgctgtttttgatgaggcggattttccggcagttgccgtttatctcaccggcgctgaatacacgggcgaagagctggacag cgatacctggcaggcggagctgcatatcgaagttttcctgcctgctcaggtgccggattcagagctggatgcgtggatggagtcccggatttatccggt gatgagcgatatcccggcactgtcagatttgatcaccagtatggtggccagcggctatgactaccggcgcgacgatgatgcgggcttgtggagttcag ccgatctgacttatgtcattacctatgaaatgtgaggacgctatgcctgtaccaaatcctacaatgccggtgaaaggtgccgggaccaccctgtgggtt tataaggggagcggtgacccttacgcgaatccgctttcagacgttgactggtcgcgtctggcaaaagttaaagacctgacgcccggcgaactgaccgc tgagtcctatgacgacagctatctcgatgatgaagatgcagactggactgcgaccgggcaggggcagaaatctgccggagataccagcttcacgctg gcgtggatgcccggagagcaggggcagcaggcgctgctggcgtggtttaatgaaggcgatacccgtgcctataaaatccgcttcccgaacggcacgg tcgatgtgttccgtggctgggtcagcagtatcggtaaggcggtgacggcgaaggaagtgatcacccgcacggtgaaagtcaccaatgtgggacgtcc gtcgatggcagaagatcgcagcacggtaacagcggcaaccggcatgaccgtgacgcctgccagcacctcggtggtgaaagggcagagcaccacgct gaccgtggccttccagccggagggcgtaaccgacaagagctttcgtgcggtgtctgcggataaaacaaaagccaccgtgtcggtcagtggtatgacc atcaccgtgaacggcgttgctgcaggcaaggtcaacattccggttgtatccggtaatggtgagtttgctgcggttgcagaaattaccgtcaccgccagt taatccggagagtcagcgatgttcctgaaaaccgaatcatttgaacataacggtgtgaccgtcacgctttctgaactgtcagccctgcagcgcattgag catctcgccctgatgaaacggcaggcagaacaggcggagtcagacagcaaccggaagtttactgtggaagacgccatcagaaccggcgcgtttctg gtggcgatgtccctgtggcataaccatccgcagaagacgcagatgccgtccatgaatgaagccgttaaacagattgagcaggaagtgcttaccacct ggcccacggaggcaatttctcatgctgaaaacgtggtgtaccggctgtctggtatgtatgagtttgtggtgaataatgcccctgaacagacagaggac gccgggcccgcagagcctgtttctgcgggaaagtgttcgacggtgagctgagttttgccctgaaactggcgcgtgagatggggcgacccgactggcgt gccatgcttgccgggatgtcatccacggagtatgccgactggcaccgcttttacagtacccattattttcatgatgttctgctggatatgcacttttccggg ctgacgtacaccgtgctcagcctgtttttcagcgatccggatatgcatccgctggatttcagtctgctgaaccggcgcgaggctgacgaagagcctgaa gatgatgtgctgatgcagaaagcggcagggcttgccggaggtgtccgctttggcccggacgggaatgaagttatccccgcttccccggatgtggcgga catgacggaggatgacgtaatgctgatgacagtatcagaagggatcgcaggaggagtccggtatggctgaaccggtaggcgatctggtcgttgattt gagtctggatgcggccagatttgacgagcagatggccagagtcaggcgtcatttttctggtacggaaagtgatgcgaaaaaaacagcggcagtcgtt gaacagtcgctgagccgacaggcgctggctgcacagaaagcggggatttccgtcgggcagtataaagccgccatgcgtatgctgcctgcacagttca ccgacgtggccacgcagcttgcaggcgggcaaagtccgtggctgatcctgctgcaacagggggggcaggtgaaggactccttcggcgggatgatccc catgttcagggggcttgccggtgcgatcaccctgccgatggtgggggccacctcgctggcggtggcgaccggtgcgctggcgtatgcctggtatcagg gcaactcaaccctgtccgatttcaacaaaacgctggtcctttccggcaatcaggcgggactgacggcagatcgtatgctggtcctgtccagagccggg caggcggcagggctgacgtttaaccagaccagcgagtcactcagcgcactggttaaggcgggggtaagcggtgaggctcagattgcgtccatcagcc agagtgtggcgcgtttctcctctgcatccggcgtggaggtggacaaggtcgctgaagccttcgggaagctgaccacagacccgacgtcggggctgac ggcgatggctcgccagttccataacgtgtcggcggagcagattgcgtatgttgctcagttgcagcgttccggcgatgaagccggggcattgcaggcgg cgaacgaggccgcaacgaaagggtttgatgaccagacccgccgcctgaaagagaacatgggcacgctggaaacctgggcagacaggactgcgcgg gcattcaaatccatgtgggatgcggtgctggatattggtcgtcctgataccgcgcaggagatgctgattaaggcagaggctgcgtataagaaagcaga cgacatctggaatctgcgcaaggatgattattttgttaacgatgaagcgcgggcgcgttactgggatgatcgtgaaaaggcccgtcttgcgcttgaagc cgcccgaaagaaggctgagcagcagactcaacaggacaaaaatgcgcagcagcagagcgataccgaagcgtcacggctgaaatataccgaagag gcgcagaaggcttacgaacggctgcagacgccgctggagaaatataccgcccgtcaggaagaactgaacaaggcactgaaagacgggaaaatcct gcaggcggattacaacacgctgatggcggcggcgaaaaaggattatgaagcgacgctgaaaaagccgaaacagtccagcgtgaaggtgtctgcgg gcgatcgtcaggaagacagtgctcatgctgccctgctgacgcttcaggcagaactccggacgctggagaagcatgccggagcaaatgagaaaatca gccagcagcgccgggatttgtggaaggcggagagtcagttcgcggtactggaggaggcggcgcaacgtcgccagctgtctgcacaggagaaatccc tgctggcgcataaagatgagacgctggagtacaaacgccagctggctgcacttggcgacaaggttacgtatcaggagcgcctgaacgcgctggcgca gcaggcggataaattcgcacagcagcaacgggcaaaacgggccgccattgatgcgaaaagccgggggctgactgaccggcaggcagaacgggaa gccacggaacagcgcctgaaggaacagtatggcgataatccgctggcgctgaataacgtcatgtcagagcagaaaaagacctgggcggctgaaga ccagcttcgcgggaactggatggcaggcctgaagtccggctggagtgagtgggaagagagcgccacggacagtatgtcgcaggtaaaaagtgcagc cacgcagacctttgatggtattgcacagaatatggcggcgatgctgaccggcagtgagcagaactggcgcagcttcacccgttccgtgctgtccatgat gacagaaattctgcttaagcaggcaatggtggggattgtcgggagtatcggcagcgccattggcggggctgttggtggcggcgcatccgcgtcaggc ggtacagccattcaggccgctgcggcgaaattccattttgcaaccggaggatttacgggaaccggcggcaaatatgagccagcggggattgttcacc gtggtgagtttgtcttcacgaaggaggcaaccagccggattggcgtggggaatctttaccggctgatgcgcggctatgccaccggcggttatgtcggta caccgggcagcatggcagacagccggtcgcaggcgtccgggacgtttgagcagaataaccatgtggtgattaacaacgacggcacgaacgggcag ataggtccggctgctctgaaggcggtgtatgacatggcccgcaagggtgcccgtgatgaaattcagacacagatgcgtgatggtggcctgttctccgg aggtggacgatgaagaccttccgctggaaagtgaaacccggtatggatgtggcttcggtcccttctgtaagaaaggtgcgctttggtgatggctattct cagcgagcgcctgccgggctgaatgccaacctgaaaacgtacagcgtgacgctttctgtcccccgtgaggaggccacggtactggagtcgtttctgga agagcacgggggctggaaatcctttctgtggacgccgccttatgagtggcggcagataaaggtgacctgcgcaaaatggtcgtcgcgggtcagtatg ctgcgtgttgagttcagcgcagagtttgaacaggtggtgaactgatgcaggatatccggcaggaaacactgaatgaatgcacccgtgcggagcagtc ggccagcgtggtgctctgggaaatcgacctgacagaggtcggtggagaacgttattttttctgtaatgagcagaacgaaaaaggtgagccggtcacct ggcaggggcgacagtatcagccgtatcccattcaggggagcggttttgaactgaatggcaaaggcaccagtacgcgccccacgctgacggtttctaa cctgtacggtatggtcaccgggatggcggaagatatgcagagtctggtcggcggaacggtggtccggcgtaaggtttacgcccgttttctggatgcggt gaacttcgtcaacggaaacagttacgccgatccggagcaggaggtgatcagccgctggcgcattgagcagtgcagcgaactgagcgcggtgagtgc ctcctttgtactgtccacgccgacggaaacggatggcgctgtttttccgggacgtatcatgctggccaacacctgcacctggacctatcgcggtgacga gtgcggttatagcggtccggctgtcgcggatgaatatgaccagccaacgtccgatatcacgaaggataaatgcagcaaatgcctgagcggttgtaagt tccgcaataacgtcggcaactttggcggcttcctttccattaacaaactttcgcagtaaatcccatgacacagacagaatcagcgattctggcgcacgc ccggcgatgtgcgccagcggagtcgtgcggcttcgtggtaagcacgccggagggggaaagatatttcccctgcgtgaatatctccggtgagccggag gctatttccgtatgtcgccggaagactggctgcaggcagaaatgcagggtgagattgtggcgctggtccacagccaccccggtggtctgccctggctg agtgaggccgaccggcggctgcaggtgcagagtgatttgccgtggtggctggtctgccgggggacgattcataagttccgctgtgtgccgcatctcacc gggcggcgctttgagcacggtgtgacggactgttacacactgttccgggatgcttatcatctggcggggattgagatgccggactttcatcgtgaggat gactggtggcgtaacggccagaatctctatctggataatctggaggcgacggggctgtatcaggtgccgttgtcagcggcacagccgggcgatgtgct gctgtgctgttttggttcatcagtgccgaatcacgccgcaatttactgcggcgacggcgagctgctgcaccatattcctgaacaactgagcaaacgaga gaggtacaccgacaaatggcagcgacgcacacactccctctggcgtcaccgggcatggcgcgcatctgcctttacggggatttacaacgatttggtcg ccgcatcgaccttcgtgtgaaaacgggggctgaagccatccgggcactggccacacagctcccggcgtttcgtcagaaactgagcgacggctggtatc aggtacggattgccgggcgggacgtcagcacgtccgggttaacggcgcagttacatgagactctgcctgatggcgctgtaattcatattgttcccagag tcgccggggccaagtcaggtggcgtattccagattgtcctgggggctgccgccattgccggatcattctttaccgccggagccacccttgcagcatggg gggcagccattggggccggtggtatgaccggcatcctgttttctctcggtgccagtatggtgctcggtggtgtggcgcagatgctggcaccgaaagcca gaactccccgtatacagacaacggataacggtaagcagaacacctatttctcctcactggataacatggttgcccagggcaatgttctgcctgttctgt acggggaaatgcgcgtggggtcacgcgtggtttctcaggagatcagcacggcagacgaaggggacggtggtcaggttgtggtgattggtcgctgatg caaaatgttttatgtgaaaccgcctgcgggcggttttgtcatttatggagcgtgaggaatgggtaaaggaagcagtaaggggcataccccgcgcgaa gcgaaggacaacctgaagtccacgcagttgctgagtgtgatcgatgccatcagcgaagggccgattgaaggtccggtggatggcttaaaaagcgtgc tgctgaacagtacgccggtgctggacactgaggggaataccaacatatccggtgtcacggtggtgttccgggctggtgagcaggagcagactccgcc ggagggatttgaatcctccggctccgagacggtgctgggtacggaagtgaaatatgacacgccgatcacccgcaccattacgtctgcaaacatcgac cgtctgcgctttaccttcggtgtacaggcactggtggaaaccacctcaaagggtgacaggaatccgtcggaagtccgcctgctggttcagatacaacgt aacggtggctgggtgacggaaaaagacatcaccattaagggcaaaaccacctcgcagtatctggcctcggtggtgatgggtaacctgccgccgcgcc cgtttaatatccggatgcgcaggatgacgccggacagcaccacagaccagctgcagaacaaaacgctctggtcgtcatacactgaaatcatcgatgt gaaacagtgctacccgaacacggcactggtcggcgtgcaggtggactcggagcagttcggcagccagcaggtgagccgtaattatcatctgcgcggg cgtattctgcaggtgccgtcgaactataacccgcagacgcggcaatacagcggtatctgggacggaacgtttaaaccggcatacagcaacaacatgg cctggtgtctgtgggatatgctgacccatccgcgctacggcatggggaaacgtcttggtgcggcggatgtggataaatgggcgctgtatgtcatcggcc agtactgcgaccagtcagtgccggacggctttggcggcacggagccgcgcatcacctgtaatgcgtacctgaccacacagcgtaaggcgtgggatgt gctcagcgatttctgctcggcgatgcgctgtatgccggtatggaacgggcagacgctgacgttcgtgcaggaccgaccgtcggataagacgtggacct ataaccgcagtaatgtggtgatgccggatgatggcgcgccgttccgctacagcttcagcgccctgaaggaccgccataatgccgttgaggtgaactgg attgacccgaacaacggctgggagacggcgacagagcttgttgaagatacgcaggccattgcccgttacggtcgtaatgttacgaagatggatgcctt tggctgtaccagccgggggcaggcacaccgcgccgggctgtggctgattaaaacagaactgctggaaacgcagaccgtggatttcagcgtcggcgca gaagggcttcgccatgtaccgggcgatgttattgaaatctgcgatgatgactatgccggtatcagcaccggtggtcgtgtgctggcggtgaacagcca gacccggacgctgacgctcgaccgtgaaatcacgctgccatcctccggtaccgcgctgataagcctggttgacggaagtggcaatccggtcagcgtgg aggttcagtccgtcaccgacggcgtgaaggtaaaagtgagccgtgttcctgacggtgttgctgaatacagcgtatgggagctgaagctgccgacgctg cgccagcgactgttccgctgcgtgagtatccgtgagaacgacgacggcacgtatgccatcaccgccgtgcagcatgtgccggaaaaagaggccatcg tggataacggggcgcactttgacggcgaacagagtggcacggtgaatggtgtcacgccgccagcggtgcagcacctgaccgcagaagtcactgcag acagcggggaatatcaggtgctggcgcgatgggacacaccgaaggtggtgaagggcgtgagtttcctgctccgtctgaccgtaacagcggacgacg gcagtgagcggctggtcagcacggcccggacgacggaaaccacataccgcttcacgcaactggcgctggggaactacaggctgacagtccgggcgg taaatgcgtgggggcagcagggcgatccggcgtcggtatcgttccggattgccgcaccggcagcaccgtcgaggattgagctgacgccgggctatttt cagataaccgccacgccgcatcttgccgtttatgacccgacggtacagtttgagttctggttctcggaaaagcagattgcggatatcagacaggttgaa accagcacgcgttatcttggtacggcgctgtactggatagccgccagtatcaatatcaaaccgggccatgattattacttttatatccgcagtgtgaaca ccgttggcaaatcggcattcgtggaggccgtcggtcgggcgagcgatgatgcggaaggttacctggattttttcaaaggcaagataaccgaatcccat ctcggcaaggagctgctggaaaaagtcgagctgacggaggataacgccagcagactggaggagttttcgaaagagtggaaggatgccagtgataa gtggaatgccatgtgggctgtcaaaattgagcagaccaaagacggcaaacattatgtcgcgggtattggcctcagcatggaggacacggaggaagg caaactgagccagtttctggttgccgccaatcgtatcgcatttattgacccggcaaacgggaatgaaacgccgatgtttgtggcgcagggcaaccaga tattcatgaacgacgtgttcctgaagcgcctgacggcccccaccattaccagcggcggcaatcctccggccttttccctgacaccggacggaaagctga ccgctaaaaatgcggatatcagtggcagtgtgaatgcgaactccgggacgctcagtaatgtgacgatagctgaaaactgtacgataaacggtacgct gagggcggaaaaaatcgtcggggacattgtaaaggcggcgagcgcggcttttccgcgccagcgtgaaagcagtgtggactggccgtcaggtacccg tactgtcaccgtgaccgatgaccatccttttgatcgccagatagtggtgcttccgctgacgtttcgcggaagtaagcgtactgtcagcggcaggacaac gtattcgatgtgttatctgaaagtactgatgaacggtgcggtgatttatgatggcgcggcgaacgaggcggtacaggtgttctcccgtattgttgacatg ccagcgggtcggggaaacgtgatcctgacgttcacgcttacgtccacacggcattcggcagatattccgccgtatacgtttgccagcgatgtgcaggtt atggtgattaagaaacaggcgctgggcatcagcgtggtctgagtgtgttacagaggttcgtccgggaacgggcgttttattataaaacagtgagaggt gaacgatgcgtaatgtgtgtattgccgttgctgtctttgccgcacttgcggtgacagtcactccggcccgtgcggaaggtggacatggtacgtttacggt gggctattttcaagtgaaaccgggtacattgccgtcgttgtcgggcggggataccggtgtgagtcatctgaaagggattaacgtgaagtaccgttatga gctgacggacagtgtgggggtgatggcttccctggggttcgccgcgtcgaaaaagagcagcacagtgatgaccggggaggatacgtttcactatgag agcctgcgtggacgttatgtgagcgtgatggccggaccggttttacaaatcagtaagcaggtcagtgcgtacgccatggccggagtggctcacagtcg gtggtccggcagtacaatggattaccgtaagacggaaatcactcccgggtatatgaaagagacgaccactgccagggacgaaagtgcaatgcggca tacctcagtggcgtggagtgcaggtatacagattaatccggcagcgtccgtcgttgttgatattgcttatgaaggctccggcagtggcgactggcgtact gacggattcatcgttggggtcggttataaattctgattagccaggtaacacagtgttatgacagcccgccggaaccggtgggcttttttgtggggtgaat atggcagtaaagatttcaggagtcctgaaagacggcacaggaaaaccggtacagaactgcaccattcagctgaaagccagacgtaacagcaccac ggtggtggtgaacacggtgggctcagagaatccggatgaagccgggcgttacagcatggatgtggagtacggtcagtacagtgtcatcctgcaggtt gacggttttccaccatcgcacgccgggaccatcaccgtgtatgaagattcacaaccggggacgctgaatgattttctctgtgccatgacggaggatgat gcccggccggaggtgctgcgtcgtcttgaactgatggtggaagaggtggcgcgtaacgcgtccgtggtggcacagagtacggcagacgcgaagaaa tcagccggcgatgccagtgcatcagctgctcaggtcgcggcccttgtgactgatgcaactgactcagcacgcgccgccagcacgtccgccggacaggc tgcatcgtcagctcaggaagcgtcctccggcgcagaagcggcatcagcaaaggccactgaagcggaaaaaagtgccgcagccgcagagtcctcaa aaaacgcggcggccaccagtgccggtgcggcgaaaacgtcagaaacgaatgctgcagcgtcacaacaatcagccgccacgtctgcctccaccgcgg ccacgaaagcgtcagaggccgccacttcagcacgagatgcggtggcctcaaaagaggcagcaaaatcatcagaaacgaacgcatcatcaagtgcc ggtcgtgcagcttcctcggcaacggcggcagaaaattctgccagggcggcaaaaacgtccgagacgaatgccaggtcatctgaaacagcagcggaa cggagcgcctctgccgcggcagacgcaaaaacagcggcggcggggagtgcgtcaacggcatccacgaaggcgacagaggctgcgggaagtgcggt atcagcatcgcagagcaaaagtgcggcagaagcggcggcaatacgtgcaaaaaattcggcaaaacgtgcagaagatatagcttcagctgtcgcgct tgaggatgcggacacaacgagaaaggggatagtgcagctcagcagtgcaaccaacagcacgtctgaaacgcttgctgcaacgccaaaggcggtta aggtggtaatggatgaaacgaacagaaaagcccactggacagtccggcactgaccggaacgccaacagcaccaaccgcgctcaggggaacaaac aatacccagattgcgaacaccgcttttgtactggccgcgattgcagatgttatcgacgcgtcacctgacgcactgaatacgctgaatgaactggccgca gcgctcgggaatgatccagattttgctaccaccatgactaacgcgcttgcgggtaaacaaccgaagaatgcgacactgacggcgctggcagggctttc cacggcgaaaaataaattaccgtattttgcggaaaatgatgccgccagcctgactgaactgactcaggttggcagggatattctggcaaaaaattccg ttgcagatgttcttgaataccttggggccggtgagaattcggcctttccggcaggtgcgccgatcccgtggccatcagatatcgttccgtctggctacgtc ctgatgcaggggcaggcgtttgacaaatcagcctacccaaaacttgctgtcgcgtatccatcgggtgtgcttcctgatatgcgaggctggacaatcaag gggaaacccgccagcggtcgtgctgtattgtctcaggaacaggatggaattaagtcgcacacccacagtgccagtgcatccggtacggatttgggga cgaaaaccacatcgtcgtttgattacgggacgaaaacaacaggcagtttcgattacggcaccaaatcgacgaataacacgggggctcatgctcacag tctgagcggttcaacaggggccgcgggtgctcatgcccacacaagtggtttaaggatgaacagttctggctggagtcagtatggaacagcaaccatta caggaagtttatccacagttaaaggaaccagcacacagggtattgcttatttatcgaaaacggacagtcagggcagccacagtcactcattgtccggt acagccgtgagtgccggtgcacatgcgcatacagttggtattggtgcgcaccagcatccggttgttatcggtgctcatgcccattctttcagtattggttc acacggacacaccatcaccgttaacgctgcgggtaacgcggaaaacaccgtcaaaaacattgcatttaactatattgtgaggcttgcataatggcatt cagaatgagtgaacaaccacggaccataaaaatttataatctgctggccggaactaatgaatttattggtgaaggtgacgcatatattccgcctcata ccggtctgcctgcaaacagtaccgatattgcaccgccagatattccggctggctttgtggctgttttcaacagtgatgaggcatcgtggcatctcgttga agaccatcggggtaaaaccgtctatgacgtggcttccggcgacgcgttatttatttctgaactcggtccgttaccggaaaattttacctggttatcgccg ggaggggaatatcagaagtggaacggcacagcctgggtgaaggatacggaagcagaaaaactgttccggatccgggaggcggaagaaacaaaaa aaagcctgatgcaggtagccagtgagcatattgcgccgcttcaggatgctgcagatctggaaattgcaacgaaggaagaaacctcgttgctggaagc ctggaagaagtatcgggtgttgctgaaccgtgttgatacatcaactgcacctgatattgagtggcctgctgtccctgttatggagtaatcgttttgtgata tgccgcagaaacgttgtatgaaataacgttctgcggttagttagtatattgtaaagctgagtattggtttatttggcgattattatcttcaggagaataat ggaagttctatgactcaattgttcatagtgtttacatcaccgccaattgcttttaagactgaacgcatgaaatatggtttttcgtcatgttttgagtctgct gttgatatttctaaagtcggttttttttcttcgttttctctaactattttccatgaaatacatttttgattattatttgaatcaattccaattacctgaagtcttt catctataattggcattgtatgtattggtttattggagtagatgcttgcttttctgagccatagctctgatatccaaatgaagccataggcatttgttatttt ggctctgtcagctgcataacgccaaaaaatatatttatctgcttgatcttcaaatgttgtattgattaaatcaattggatggaattgtttatcataaaaaa ttaatgtttgaatgtgataaccgtcctttaaaaaagtcgtttctgcaagcttggctgtatagtcaactaactcttctgtcgaagtgatatttttaggcttatc taccagttttagacgctctttaatatcttcaggaattattttattgtcatattgtatcatgctaaatgacaatttgcttatggagtaatcttttaattttaaat aagttattctcctggcttcatcaaataaagagtcgaatgatgttggcgaaatcacatcgtcacccattggattgtttatttgtatgccaagagagttaca gcagttatacattctgccatagattatagctaaggcatgtaataattcgtaatcttttagcgtattagcgacccatcgtctttctgatttaataatagatga ttcagttaaatatgaaggtaatttcttttgtgcaagtctgactaacttttttataccaatgtttaacatactttcatttgtaataaactcaatgtcattttctt caatgtaagatgaaataagagtagcctttgcctcgctatacatttctaaatcgccttgtttttctatcgtattgcgagaatttttagcccaagccattaatg gatcatttttccatttttcaataacattattgttataccaaatgtcatatcctataatctggtttttgtttttttgaataataaatgttactgttcttgcggtttg gaggaattgattcaaattcaagcgaaataattcagggtcaaaatatgtatcaatgcagcatttgagcaagtgcgataaatctttaagtcttctttcccat ggttttttagtcataaaactctccattttgataggttgcatgctagatgctgatatattttagaggtgataaaattaactgcttaactgtcaatgtaataca agttgtttgatctttgcaatgattcttatcagaaaccatatagtaaattagttacacaggaaatttttaatattattattatcattcattatgtattaaaatt agagttgtggcttggctctgctaacacgttgctcataggagatatggtagagccgcagacacgtcgtatgcaggaacgtgctgcggctggctggtgaa cttccgatagtgcgggtgttgaatgatttccagttgctaccgattttacatattttttgcatgagagaatttgtaccacctcccaccgaccatctatgactg tacgccactgtccctaggactgctatgtgccggagcggacattacaaacgtccttctcggtgcatgccactgttgccaatgacctgcctaggaattggtt agcaagttactaccggattttgtaaaaacagccctcctcatataaaaagtattcgttcacttccgataagcgtcgtaattttctatctttcatcatattcta gatccctctgaaaaaatcttccgagtttgctaggcactgatacataactcttttccaataattggggaagtcattcaaatctataataggtttcagatttg cttcaataaattctgactgtagctgctgaaacgttgcggttgaactatatttccttataacttttacgaaagagtttctttgagtaatcacttcactcaagt gcttccctgcctccaaacgatacctgttagcaatatttaatagcttgaaatgatgaagagctctgtgtttgtcttcctgcctccagttcgccgggcattca acataaaaactgatagcacccggagttccggaaacgaaatttgcatatacccattgctcacgaaaaaaaatgtccttgtcgatatagggatgaatcgc ttggtgtacctcatctactgcgaaaacttgacctttctctcccatattgcagtcgcggcacgatggaactaaattaataggcatcaccgaaaattcagga taatgtgcaataggaagaaaatgatctatattttttgtctgtcctatatcaccacaaaatggacatttttcacctgatgaaacaagcatgtcatcgtaat atgttctagcgggtttgtttttatctcggagattattttcataaagcttttctaatttaacctttgtcaggttaccaactactaaggttgtaggctcaagagg gtgtgtcctgtcgtaggtaaataactgacctgtcgagcttaatattctatattgttgttctttctgcaaaaaagtggggaagtgagtaatgaaattatttct aacatttatctgcatcataccttccgagcatttattaagcatttcgctataagttctcgctggaagaggtagttttttcattgtactttaccttcatctctgtt cattatcatcgcttttaaaacggttcgaccttctaatcctatctgaccattataattttttagaatggtttcataagaaagctctgaatcaacggactgcga taataagtggtggtatccagaatttgtcacttcaagtaaaaacacctcacgagttaaaacacctaagttctcaccgaatgtctcaatatccggacggat aatatttattgcttctcttgaccgtaggactttccacatgcaggattttggaacctcttgcagtactactggggaatgagttgcaattattgcta caeca tt gcgtgcatcgagtaagtcgcttaatgttcgtaaaaaagcagagagcaaaggtggatgcagatgaacctctggttcatcgaataaaactaatgactttt cgccaacgacatctactaatcttgtgatagtaaataaaacaattgcatgtccagagctcattcgaagcagatatttctggatattgtcataaaacaattt agtgaatttatcatcgtccacttgaatctgtggttcattacgtcttaactcttcatatttagaaatgaggctgatgagttccatatttgaaaagttttcatca ctacttagttttttgatagcttcaagccagagttgtctttttctatctactctcatacaaccaataaatgctgaaatgaattctaagcggagatcgcctagt gattttaaactattgctggcagcattcttgagtccaatataaaagtattgtgtaccttttgctgggtcaggttgttctttaggaggagtaaaaggatcaaa tgcactaaacgaaactgaaacaagcgatcgaaaatatccctttgggattcttgactcgataagtctattattttcagagaaaaaatattcattgttttct gggttggtgattgcaccaatcattccattcaaaattgttgttttaccacacccattccgcccgataaaagcatgaatgttcgtgctgggcatagaattaa ccgtcacctcaaaaggtatagttaaatcactgaatccgggagcactttttctattaaatgaaaagtggaaatctgacaattctggcaaaccatttaaca cacgtgcgaactgtccatgaatttctgaaagagttacccctctaagtaatgaggtgttaaggacgctttcattttcaatgtcggctaatcgatttggccat actactaaatcctgaatagctttaagaaggttatgtttaaaaccatcgcttaatttgctgagattaacatagtagtcaatgctttcacctaaggaaaaaa acatttcagggagttgactgaattttttatctattaatgaataagtgcttacttcttctttttgacctacaaaaccaattttaacatttccgatatcgcatttt tcaccatgctcatcaaagacagtaagataaaacattgtaacaaaggaatagtcattccaaccatctgctcgtaggaatgccttatttttttctactgcag gaatatacccgcctctttcaataacactaaactccaacatatagtaacccttaattttattaaaataaccgcaatttatttggcggcaacacaggatctc tcttttaagttactctctattacatacgttttccatctaaaaattagtagtattgaacttaacggggcatcgtattgtagttttccatatttagctttctgcttc cttttggataacccactgttattcatgttgcatggtgcactgtttataccaacgatatagtctattaatgcatatatagtatcgccgaacgattagctcttc aggcttctgaagaagcgtttcaagtactaataagccgatagatagccacggacttcgtagccatttttcataagtgttaacttccgctcctcgctcataa cagacattcactacagttatggcggaaaggtatgcatgctgggtgtggggaagtcgtgaaagaaaagaagtcagctgcgtcgtttgacatcactgcta tcttcttactggttatgcaggtcgtagtgggtggcacacaaagctttgcactggattgcgaggctttgtgcttctctggagtgcgacaggtttgatgacaa aaaattagcgcaagaagacaaaaatcaccttgcgctaatgctctgttacaggtcactaataccatctaagtagttgattcatagtgactgcatatgttgt gttttacagtattatgtagtctgttttttatgcaaaatctaatttaatatattgatatttatatcattttacgtttctcgttcagcttttttatactaagttggca ttataaaaaagcattgcttatcaatttgttgcaacgaacaggtcactatcagtcaaaataaaatcattatttgatttcaattttgtcccactccctgcctct gtcatcacgatactgtgatgccatggtgtccgacttatgcccgagaagatgttgagcaaacttatcgcttatctgcttctcatagagtcttgcagacaaa ctgcgcaactcgtgaaaggtaggcggatccccttcgaaggaaagacctgatgcttttcgtgcgcgcataaaataccttgatactgtgccggatgaaag cggttcgcgacgagtagatgcaattatggtttctccgccaagaatctctttgcatttatcaagtgtttccttcattgatattccgagagcatcaatatgca atgctgttgggatggcaatttttacgcctgttttgctttgctcgacataaagatatccatctacgatatcagaccacttcatttcgcataaatcaccaactc gttgcccggtaacaacagccagttccattgcaagtctgagccaacatggtgatgattctgctgcttgataaattttcaggtattcgtcagccgtaagtctt gatctccttacctctgattttgctgcgcgagtggcagcgacatggtttgttgttatatggccttcagctattgcctctcggaatgcatcgctcagtgttgat ctgattaacttggctgacgccgccttgccctcgtctatgtatccattgagcattgccgcaatttcttttgtggtgatgtcttcaagtggagcatcaggcaga cccctccttattgctttaattttgctcatgtaatttatgagtgtcttctgcttgattcctctgctggccaggattttttcgtagcgatcaagccatgaatgtaa cgtaacggaattatcactgttgattctcgctgtcagaggcttgtgtttgtgtcctgaaaataactcaatgttggcctgtatagcttcagtgattgcgattcg cctgtctctgcctaatccaaactctttacccgtccttgggtccctgtagcagtaatatccattgtttcttatataaaggttagggggtaaatcccggcgctc atgacttcgccttcttcccatttctgatcctcttcaaaaggccacctgttactggtcgatttaagtcaacctttaccgctgattcgtggaacagatactctct tccatccttaaccggaggtgggaatatcctgcattcccgaacccatcgacgaactgtttcaaggcttcttggacgtcgctggcgtgcgttccactcctga agtgtcaagtacatcgcaaagtctccgcaattacacgcaagaaaaaaccgccatcaggcggcttggtgttctttcagttcttcaattcgaatattggtta cgtctgcatgtgctatctgcgcccatatcatccagtggtcgtagcagtcgttgatgttctccgcttcgataactctgttgaatggctctccattccattctcc tgtgactcggaagtgcatttatcatctccataaaacaaaacccgccgtagcgagttcagataaaataaatccccgcgagtgcgaggattgttatgtaat attgggtttaatcatctatatgttttgtacagagagggcaagtatcgtttccaccgtactcgtgataataattttgcacggtatcagtcatttctcgcacat tgcagaatggggatttgtcttcattagacttataaaccttcatggaatatttgtatgccgactctatatctataccttcatctacataaacaccttcgtgat gtctgcatggagacaagacaccggatctgcacaacattgataacgcccaatctttttgctcagactctaactcattgatactcatttataaactccttgc aatgtatgtcgtttcagctaaacggtatcagcaatgtttatgtaaagaaacagtaagataatactcaacccgatgtttgagtacggtcatcatctgaca ctacagactctggcatcgctgtgaagacgacgcgaaattcagcattttcacaagcgttatcttttacaaaaccgatctcactctcctttgatgcgaatgc cagcgtcagacatcatatgcagatactcacctgcatcctgaacccattgacctccaaccccgtaatagcgatgcgtaatgatgtcgatagttactaacg ggtcttgttcgattaactgccgcagaaactcttccaggtcaccagtgcagtgcttgataacaggagtcttcccaggatggcgaacaacaagaaactgg tttccgtcttcacggacttcgttgctttccagtttagcaatacgcttactcccatccgagataacaccttcgtaatactcacgctgctcgttgagttttgatt ttgctgtttcaagctcaacacgcagtttccctactgttagcgcaatatcctcgttctcctggtcgcggcgtttgatgtattgctggtttctttcccgttcatcc agcagttccagcacaatcgatggtgttaccaattcatggaaaaggtctgcgtcaaatccccagtcgtcatgcattgcctgctctgccgcttcacgcagtg cctgagagttaatttcgctcacttcgaacctctctgtttactgataagttccagatcctcctggcaacttgcacaagtccgacaaccctgaacgaccagg cgtcttcgttcatctatcggatcgccacactcacaacaatgagtggcagatatagcctggtggttcaggcggcgcatttttattgctgtgttgcgctgtaa ttcttctatttctgatgctgaatcaatgatgtctgccatctttcattaatccctgaactgttggttaatacgcttgagggtgaatgcgaataataaaaaag gagcctgtagctccctgatgattttgcttttcatgttcatcgttccttaaagacgccgtttaacatgccgattgccaggcttaaatgagtcggtgtgaatcc catcagcgttaccgtttcgcggtgcttcttcagtacgctacggcaaatgtcatcgacgtttttatccggaaactgctgtctggctttttttgatttcagaatt agcctgacgggcaatgctgcgaagggcgttttcctgctgaggtgtcattgaacaagtcccatgtcggcaagcataagcacacagaatatgaagcccg ctgccagaaaaatgcattccgtggttgtcatacctggtttctctcatctgcttctgctttcgccaccatcatttccagcttttgtgaaagggatgcggctaa cgtatgaaattcttcgtctgtttctactggtattggcacaaacctgattccaatttgagcaaggctatgtgccatctcgatactcgttcttaactcaacaga agatgctttgtgcatacagcccctcgtttattatttatctcctcagccagccgctgtgctttcagtggatttcggataacagaaaggccgggaaataccc agcctcgctttgtaacggagtagacgaaagtgattgcgcctacccggatattatcgtgaggatgcgtcatcgccattgctccccaaatacaaaaccaat ttcagccagtgcctcgtccattttttcgatgaactccggcacgatctcgtcaaaactcgccatgtacttttcatcccgctcaatcacgacataatgcaggc cttcacgcttcatacgcgggtcatagttggcaaagtaccaggcattttttcgcgtcacccacatgctgtactgcacctgggccatgtaagctgactttatg gcctcgaaaccaccgagccggaacttcatgaaatcccgggaggtaaacgggcatttcagttcaaggccgttgccgtcactgcataaaccatcgggag agcaggcggtacgcatactttcgtcgcgatagatgatcggggattcagtaacattcacgccggaagtgaattcaaacagggttctggcgtcgttctcgt actgttttccccaggccagtgctttagcgttaacttccggagccacaccggtgcaaacctcagcaagcagggtgtggaagtaggacattttcatgtcag gccacttctttccggagcggggttttgctatcacgttgtgaacttctgaagcggtgatgacgccgagccgtaatttgtgccacgcatcatccccctgttcg acagctctcacatcgatcccggtacgctgcaggataatgtccggtgtcatgctgccaccttctgctctgcggctttctgtttcaggaatccaagagctttt actgcttcggcctgtgtcagttctgacgatgcacgaatgtcgcggcgaaatatctgggaacagagcggcaataagtcgtcatcccatgttttatccagg gcgatcagcagagtgttaatctcctgcatggtttcatcgttaaccggagtgatgtcgcgttccggctgacgttctgcagtgtatgcagtattttcgacaat gcgctcggcttcatccttgtcatagataccagcaaatccgaaggccagacgggcacactgaatcatggctttatgacgtaacatccgtttgggatgcga ctgccacggccccgtgatttctctgccttcgcgagttttgaatggttcgcggcggcattcatccatccattcggtaacgcagatcggatgattacggtcct tgcggtaaatccggcatgtacaggattcattgtcctgctcaaagtccatgccatcaaactgctggttttcattgatgatgcgggaccagccatcaacgcc caccaccggaacgatgccattctgcttatcaggaaaggcgtaaatttctttcgtccacggattaaggccgtactggttggcaacgatcagtaatgcgat gaactgcgcatcgctggcatcacctttaaatgccgtctggcgaagagtggtgatcagttcctgtgggtcgacagaatccatgccgacacgttcagccag cttcccagccagcgttgcgagtgcagtactcattcgttttatacctctgaatcaatatcaacctggtggtgagcaatggtttcaaccatgtaccggatgtg ttctgccatgcgctcctgaaactcaacatcgtcatcaaacgcacgggtaatggattttttgctggccccgtggcgttgcaaatgatcgatgcatagcgat tcaaacaggtgctggggcaggcctttttccatgtcgtctgccagttctgcctctttctcttcacgggcgagctgctggtagtgacgcgcccagctctgagc ctcaagacgatcctgaatgtaataagcgttcatggctgaactcctgaaatagctgtgaaaatatcgcccgcgaaatgccgggctgattaggaaaaca ggaaagggggttagtgaatgcttttgcttgatctcagtttcagtattaatatccattttttataagcgtcgacggcttcacgaaacatcttttcatcgccaa taaaagtggcgatagtgaatttagtctggatagccataagtgtttgatccattctttgggactcctggctgattaagtatgtcgataaggcgtttccatcc gtcacgtaatttacgggtgattcgttcaagtaaagattcggaagggcagccagcaacaggccaccctgcaatggcatattgcatggtgtgctccttatt tatacataacgaaaaacgcctcgagtgaagcgttattggtatgcggtaaaaccgcactcaggcggccttgatagtcatatcatctgaatcaaatattcc tgatgtatcgatatcggtaattcttattccttcgctaccatccattggaggccatccttcctgaccatttccatcattccagtcgaactcacacacaacacc atatgcatttaagtcgcttgaaattgctataagcagagcatgttgcgccagcatgattaatacagcatttaatacagagccgtgtttattgagtcggtat tcagagtctgaccagaaattattaatctggtgaagtttttcctctgtcattacgtcatggtcgatttcaatttctattgatgctttccagtcgtaatcaatga tgtattttttgatgtttgacatctgttcatatcctcacagataaaaaatcgccctcacactggagggcaaagaagatttccaataatcagaacaagtcgg ctcctgtttagttacgagcgacattgctccgtgtattcactcgttggaatgaatacacagtgcagtgtttattctgttatttatgccaaaaataaaggcca ctatcaggcagctttgttgttctgtttaccaagttctctggcaatcattgccgtcgttcgtattgcccatttatcgacatatttcccatcttccattacagga aacatttcttcaggcttaaccatgcattccgattgcagcttgcatccattgcatcgcttgaattgtccacaccattgatttttatcaatagtcgtagtcata cggatagtcctggtattgttccatcacatcctgaggatgctcttcgaactcttcaaattcttcttccatatatcaccttaaatagtggattgcggtagtaaa gattgtgcctgtcttttaaccacatcaggctcggtggttctcgtgtacccctacagcgagaaatcggataaactattacaacccctacagtttgatgagt atagaaatggatccactcgttattctcggacgagtgttcagtaatgaacctctggagagaaccatgtatatgatcgttatctgggttggacttctgctttt aagcccagataactggcctgaatatgttaatgagagaatcggtattcctcatgtgtggcatgttttcgtctttgctcttgcattttcgctagcaattaatgt gcatcgattatcagctattgccagcgccagatataagcgatttaagctaagaaaacgcattaagatgcaaaacgataaagtgcgatcagtaattcaa aaccttacagaagagcaatctatggttttgtgcgcagcccttaatgaaggcaggaagtatgtggttacatcaaaacaattcccatacattagtgagttg attgagcttggtgtgttgaacaaaactttttcccgatggaatggaaagcatatattattccctattgaggatatttactggactgaattagttgccagcta tgatccatataatattgagataaagccaaggccaatatctaagtaactagataagaggaatcgattttcccttaattttctggcgtccactgcatgttat gccgcgttcgccaggcttgctgtaccatgtgcgctgattcttgcgctcaatacgttgcaggttgctttcaatctgtttgtggtattcagccagcactgtaag gtctatcggatttagtgcgctttctactcgtgatttcggtttgcgattcagcgagagaatagggcggttaactggttttgcgcttaccccaaccaacaggg gatttgctgctttccattgagcctgtttctctgcgcgacgttcgcggcggcgtgtttgtgcatccatctggattctcctgtcagttagctttggtggtgtgtg gcagttgtagtcctgaacgaaaaccccccgcgattggcacattggcagctaatccggaatcgcacttacggccaatgcttcgtttcgtatcacacaccc caaagccttctgctttgaatgctgcccttcttcagggcttaatttttaagagcgtcaccttcatggtggtcagtgcgtcctgctgatgtgctcagtatcacc gccagtggtatttatgtcaacaccgccagagataatttatcaccgcagatggttatctgtatgttttttatatgaatttattttttgcaggggggcattgttt ggtaggtgagagatctgaattgctatgtttagtgagttgtatctatttatttttcaataaatacaattggttatgtgttttgggggcgatcgtgaggcaaa gaaaacccggcgctgaggccgggtcaggactttttacgcgaggcttttttacccccgctggctgcgcgttcagctttgattttctccagcaacgcggcgg cgctgttttcaccgctgattaattccgggttttcggcccgccactgggcggtgagttcaccacggaacgcttttgccaggatggattgcgtcagattgttg acgcgggctaaggcgttgttgacctgtttttctattgtgtcggcgtaggcgaagagttgctcgacgcggcgaacgatttcagcttgttcgggaaggggg ggaacagttattgggtatgcaattaatttctgtgcattaatatttgattgactgaccccatcagatttaactgaatagcaatattgtctccccaaaatgct atttaaatggtagttaagatagtcagggtttaaatcaggtaaacattgaaccctaatcaaataacctgcataaatagctggttgctctgatttatatatg gctgttttcccgacaagttcaggactatttgtacgattgaataatacatcattgtactcaagtttatacttttcaatttctatggtatctgacgtatagacta gatccgtccagtccaactttcctccctgaagattgcccattcttaatacaggaatgagtccttctttattagacttagatgacgttccgtaacttggtttttt tataaactccccaagtttgccttctctccacccagacaggcttgaattatctctccagtcttcagttagttttcctctcaccgctgcacccagaaccgcctg acgaaaacgtttcaggatttgtgggatttgctcaagacgtgctttggtgctgtctacctgtgccagcagcgtatcgagtttttcagcgatgattttttgttc ggcaaacggtacaagaacaaatggtaatgttttcgcagaactaccagaaatttctttaaaagttgttcctgtgcccatttcctctgcaatatgtctgatgt tgcggagaaagtaatatgcataatcaggaaaaatgtctgaaggaaaagcaaaacttttgaaaccctggttagtcgcaatctcatttgcagcaatagc aacatatccaatcggtgcccggctactaaaaagtatagtgcctttgggcattagttttgctgaacaagaggaatagcctagtgtagttaaatctctggc accatgtgagatatatttctctttataaccgctcaaatcggcaggcgtaagccaggcgactccctcacccgaaggtgcaaaattttcagcgactcccga ttttggtgtcccaccagagatgacatcagcaatgtcgcctatttcaatctgttcccacccctccggcaatttcccagcactcattccttcaccccaccaaa tgcttcttccagcaactggcgctgcgcatccgcctcatcgctcgcccccagttcacgcatcagcgcatccagttcagacagcgcctgtaccagttcaccc atcgcttctgccgctaatacatccggctccggcaggttgtcggcatcaatactgtctttatctttcagccaggagatatccagcgaatcggatttcgcggt gcggatccactcgcggctgaacttgcgccagcggctggtagcaagatgctggtcggtgtttttgttctcttcgctgtcggcaatttccgtctcttcggcgtt aaaactccattcaccttcagtgcgcgggcttaagccgtgcgggtcttcgccaaacacgcgctcaaacggttgcaaatgctcgtcggtaaacggcgtgc gtttaccaaagctcggcatattggtccgcaggtcgtacacccacacgtcatcggtgcagttcttatcctgattcgggttcgccaccgtccctttggtaaag aacagcacgttggtcttcacgccctgcgcgtaaaaaataccggtcggcagacgcagaatagtgtgcagatgacacttatccatcaggtcacgacgga tgtccgtacctttgccgccttcaaacagcacgttatccggcaccaccaccgccgcacgaccgccggggtgcagcgtttcgataatatgctgcataaagc acaactgtttgttgctggtcgggtgaacaaaggtgcgggtaatgttggtgcctgcggcgctgccaaacggcgggttagtggcgacaatatgcgccttcg gcaggttttcgccgtcgctgcccagggtgttgcccagacggattgcgccgccgtggtcgaggttgccttcaatatcgtgcagcaggcagttcatcagcg ccagacgacgggtgccgggcaccagttcgaggccgataaacgcgcggtggatctggaaatcctgcgtgtcgccatcaaggtcgtccagatcgttagtc tgcgacttaacgtagcggtcggcttcaatcaaaaagcccgccgtaccggctgccggatcctgtaccacttcacgtggctgcggtttcagcagatgaata atggttttaatcagcggacgcggggtgaagtactggcccgcgccagacttggtttcattggcgttcttctgcagcagcccttcgtacatatcgccgaagt catcgcgcgacttaccgtgcgcaccgttgtaccagtccagtgaatccatattgctgaccagtgcggttatttgtttcggctcggtgatggtggtactaaca ttatgaaaaactgcctgtaccagctttttgtcatcttcgcctaaatgcacgagcatttttcggtagaactgcaactgctcctggccgatgcgggatttcag gtcatcccagcggtaaccttccggcaggtattccgcttcctgcccAgtctctttacacattttcaaaaacagcagcgaggcgagttcattgacgtagtttt gataggaaacgccgccatcgcgcaggttgtcgcacagcttccacagcttcgcgaccagatcgttattgttcatacgttaaatctatcaccgcaagggat aaatatctaacaccgtgcgtgttgactattttacctctggcggtgataatggttgcatgtactaaggaggttgtatggaacaacgcataaccctgaaag attatgcaatgcgctttgggcaaaccaagacagctaaagatctcggcgtatatcaaagcgcgatcaacaaggccattcatgcaggccgaaagattttt ttaactataaacgctgatggaagcgtttatgcggaagaggtaaagcccttcccgagtaacaaaaaaacaacagcataaataaccccgctcttacaca ttccagccctgaaaaagggcatcaaattaaaccacacctatggtgtatgcatttatttgcatacattcaatcaattgttatctaaggaaatacttacata tggttcgtgcaaacaaacgcaacgaggctctacgaatcgagagtgcgttgcttaacaaaatcgcaatgcttggaactgagaagacagcggaagctgt gggcgttgataagtcgcagatcagcaggtggaagagggactggattccaaagttctcaatgctgcttgctgttcttgaatggggggtcgttgacgacg acatggctcgattggcgcgacaagttgctgcgattctcaccaataaaaaacgcccggcggcaaccgagcgttctgaacaaatccagatggagttctg aggtcattactggatctatcaacaggagtcattatgacaaatacagcaaaaatactcaacttcggcagaggtaactttgccggacaggagcgtaatgt ggcagatctcgatgatggttacgccagactatcaaatatgctgcttgaggcttattcgggcgcagatctgaccaagcgacagtttaaagtgctgcttgc cattctgcgtaaaacctatgggtggaataaaccaatggacagaatcaccgattctcaacttagcgagattacaaagttacctgtcaaacggtgcaatg aagccaagttagaactcgtcagaatgaatattatcaagcagcaaggcggcatgtttggaccaaataaaaacatctcagaatggtgcatccctcaaaa cgagggaaaatcccctaaaacgagggataaaacatccctcaaattgggggattgctatccctcaaaacagggggacacaaaagacactattacaaa agaaaaaagaaaagattattcgtcagagaattctggcgaatcctctgaccagccagaaaacgacctttctgtggtgaaaccggatgctgcaattcag agcggcagcaagtgggggacagcagaagacctgaccgccgcagagtggatgtttgacatggtgaagactatcgcaccatcagccagaaaaccgaa ttttgctgggtgggctaacgatatccgcctgatgcgtgaacgtgacggacgtaaccaccgcgacatgtgtgtgctgttccgctgggcatgccaggacaa cttctggtccggtaacgtgctgagcccggccaaactccgcgataagtggacccaactcgaaatcaaccgtaacaagcaacaggcaggcgtgacagcc agcaaaccaaaactcgacctgacaaacacagactggatttacggggtggatctatgaaaaacatcgccgcacagatggttaactttgaccgtgagca gatgcgtcggatcgccaacaacatgccggaacagtacgacgaaaagccgcaggtacagcaggtagcgcagatcatcaacggtgtgttcagccagtt actggcaactttcccggcgagcctggctaaccgtgaccagaacgaagtgaacgaaatccgtcgccagtgggttctggcttttcgggaaaacgggatca ccacgatggaacaggttaacgcaggaatgcgcgtagcccgtcggcagaatcgaccatttctgccatcacccgggcagtttgttgcatggtgccgggaa gaagcatccgttaccgccggactgccaaacgtcagcgagctggttgatatggtttacgagtattgccggaagcgaggcctgtatccggatgcggagtc ttatccgtggaaatcaaacgcgcactactggctggttaccaacctgtatcagaacatgcgggccaatgcgcttactgatgcggaattacgccgtaagg ccgcagatgagcttgtccatatgactgcgagaattaaccgtggtgaggcgatccctgaaccagtaaaacaacttcctgtcatgggcggtagacctcta aatcgtgcacaggctctggcgaagatcgcagaaatcaaagctaagttcggactgaaaggagcaagtgtatgacgggcaaagaggcaattattcatt acctggggacgcataatagcttctgtgcgccggacgttgccgcgctaacaggcgcaacagtaaccagcataaatcaggccgcggctaaaatggcacg ggcaggtcttctggttatcgaaggtaaggtctggcgaacggtgtattaccggtttgctaccagggaagaacgggaaggaaagatgagcacgaacctg gtttttaaggagtgtcgccagagtgccgcgatgaaacgggtattggcggtatatggagttaaaagatgaccatctacattactgagctaataacaggc ctgctggtaatcgcaggcctttttatttgggggagagggaagtcatgaaaaaactaacctttgaaattcgatctccagcacatcagcaaaacgctattc acgcagtacagcaaatccttccagacccaaccaaaccaatcgtagtaaccattcaggaacgcaaccgcagcttagaccaaaacaggaagctatggg cctgcttaggtgacgtctctcgtcaggttgaatggcatggtcgctggctggatgcagaaagctggaagtgtgtgtttaccgcagcattaaagcagcagg atgttgttcctaaccttgccgggaatggctttgtggtaataggccagtcaaccagcaggatgcgtgtaggcgaatttgcggagctattagagcttatac aggcattcggtacagagcgtggcgttaagtggtcagacgaagcgagactggctctggagtggaaagcgagatggggagacagggctgcatgataaa tgtcgttagtttctccggtggcaggacgtcagcatatttgctctggctaatggagcaaaagcgacgggcaggtaaagacgtgcattacgttttcatgga tacaggttgtgaacatccaatgacatatcggtttgtcagggaagttgtgaagttctgggatataccgctcaccgtattgcaggttgatatcaacccgga gcttggacagccaaatggttatacggtatgggaaccaaaggatattcagacgcgaatgcctgttctgaagccatttatcgatatggtaaagaaatatg gcactccatacgtcggcggcgcgttctgcactgacagattaaaactcgttcccttcaccaaatactgtgatgaccatttcgggcgagggaattacacca cgtggattggcatcagagctgatgaaccgaagcggctaaagccaaagcctggaatcagatatcttgctgaactgtcagactttgagaaggaagatat cctcgcatggtggaagcaacaaccattcgatttgcaaataccggaacatctcggtaactgcatattctgcattaaaaaatcaacgcaaaaaatcggac ttgcctgcaaagatgaggagggattgcagcgtgtttttaatgaggtcatcacgggatcccatgtgcgtgacggacatcgggaaacgccaaaggagatt atgtaccgaggaagaatgtcgctggacggtatcgcgaaaatgtattcagaaaatgattatcaagccctgtatcaggacatggtacgagctaaaagatt cgataccggctcttgttctgagtcatgcgaaatatttggagggcagcttgatttcgacttcgggagggaagctgcatgatgcgatgttatcggtgcggtg aatgcaaagaagataaccgcttccgaccaaatcaaccttactggaatcgatggtgtctccggtgtgaaagaacaccaacaggggtgttaccactacc gcaggaaaaggaggacgtgtggcgagacagcgacgaagtatcaccgacataatctgcgaaaactgcaaataccttccaacgaaacgcaccagaaa taaacccaagccaatcccaaaagaatctgacgtaaaaaccttcaactacacggctcacctgtgggatatccggtggctaagacgtcgtgcgaggaaa acaaggtgattgaccaaaatcgaagttacgaacaagaaagcgtcgagcgagctttaacgtgcgctaactgcggtcagaagctgcatgtgctggaagt tcacgtgtgtgagcactgctgcgcagaactgatgagcgatccgaatagctcgatgcacgaggaagaagatgatggctaaaccagcgcgaagacgat gtaaaaacgatgaatgccgggaatggtttcaccctgcattcgctaatcagtggtggtgctctccagagtgtggaaccaagatagcactcgaacgacga agtaaagaacgcgaaaaagcggaaaaagcagcagagaagaaacgacgacgagaggagcagaaacagaaagataaacttaagattcgaaaact cgccttaaagccccgcagttactggattaaacaagcccaacaagccgtaaacgccttcatcagagaaagggaccgcgacttaccatgtatctcgtgcg gaacgctcacgtctgctcagtgggatgccggacattaccggacaactgctgcggcacctcaactccgatttaatgaacgcaatattcacaagcaatgc gtggtgtgcaaccagcacaaaagcggaaatctcgttccgtatcgcgtcgaactgattagccgcatcgggcaggaagcagtagacgaaatcgaatcaa accataaccgccatcgctggactatcgaagagtgcaaggcgatcaaggcagagtaccaacagaaactcaaagacctgcgaaatagcagaagtgag gccgcatgacgttctcagtaaaaaccattccagacatgctcgttgaagcatacggaaatcagacagaagtagcacgcagactgaaatgtagtcgcgg tacggtcagaaaatacgttgatgataaagacgggaaaatgcacgccatcgtcaacgacgttctcatggttcatcgcggatggagtgaaagagatgcg ctattacgaaaaaattgatggcagcaaataccgaaatatttgggtagttggcgatctgcacggatgctacacgaacctgatgaacaaactggatacg attggattcgacaacaaaaaagacctgcttatctcggtgggcgatttggttgatcgtggtgcagagaacgttgaatgcctggaattaatcacattcccc tggttcagagctgtacgtggaaaccatgagcaaatgatgattgatggcttatcagagcgtggaaacgttaatcactggctgcttaatggcggtggctgg ttctttaatctcgattacgacaaagaaattctggctaaagctcttgcccataaagcagatgaacttccgttaatcatcgaactggtgagcaaagataaa aaatatgttatctgccacgccgattatccctttgacgaatacgagtttggaaagccagttgatcatcagcaggtaatctggaaccgcgaacgaatcagc aactcacaaaacgggatcgtgaaagaaatcaaaggcgcggacacgttcatctttggtcatacgccagcagtgaaaccactcaagtttgccaaccaaa tgtatatcgataccggcgcagtgttctgcggaaacctaacattgattcaggtacagggagaaggcgcatgagactcgaaagcgtagctaaatttcatt cgccaaaaagcccgatgatgagcgactcaccacgggccacggcttctgactctctttccggtactgatgtgatggctgctatggggatggcgcaatca caagccggattcggtatggctgcattctgcggtaagcacgaactcagccagaacgacaaacaaaaggctatcaactatctgatgcaatttgcacaca aggtatcggggaaataccgtggtgtggcaaagcttgaaggaaatactaaggcaaaggtactgcaagtgctcgcaacattcgcttatgcggattattgc cgtagtgccgcgacgccgggggcaagatgcagagattgccatggtacaggccgtgcggttgatattgccaaaacagagctgtgggggagagttgtcg agaaagagtgcggaagatgcaaaggcgtcggctattcaaggatgccagcaagcgcagcatatcgcgctgtgacgatgctaatcccaaaccttaccc aacccacctggtcacgcactgttaagccgctgtatgacgctctggtggtgcaatgccacaaagaagagtcaatcgcagacaacattttgaatgcggtc acacgttagcagcatgattgccacggatggcaacatattaacggcatgatattgacttattgaataaaattgggtaaatttgactcaacgatgggttaa ttcgctcgttgtggtagtgagatgaaaagaggcggcgcttactaccgattccgcctagttggtcacttcgacgtatcgtctggaactccaaccatcgcag gcagagaggtctgcaaaatgcaatcccgaaacagttcgcaggtaatagttagagcctgcataacggtttcgggattttttatatctgcacaacaggta agagcattgagtcgataatcgtgaagagtcggcgagcctggttagccagtgctctttccgttgtgctgaattaagcgaataccggaagcagaaccgga tcaccaaatgcgtacaggcgtcatcgccgcccagcaacagcacaacccaaactgagccgtagccactgtctgtcctgaattcattagtaatagttacg ctgcggccttttacacatgaccttcgtgaaagcgggtggcaggaggtcgcgctaacaacctcctgccgttttgcccgtgcatatcggtcacgaacaaat ctgattactaaacacagtagcctggatttgttctatcagtaatcgaccttattcctaattaaatagagcaaatccccttattgggggtaagacatgaaga tgccagaaaaacatgacctgttggccgccattctcgcggcaaaggaacaaggcatcggggcaatccttgcgtttgcaatggcgtaccttcgcggcaga tataatggcggtgcgtttacaaaaacagtaatcgacgcaacgatgtgcgccattatcgcctggttcattcgtgaccttctcgacttcgccggactaagta gcaatctcgcttatataacgagcgtgtttatcggctacatcggtactgactcgattggttcgcttatcaaacgcttcgctgctaaaaaagccggagtaga agatggtagaaatcaataatcaacgtaaggcgttcctcgatatgctggcgtggtcggagggaactgataacggacgtcagaaaaccagaaatcatg gttatgacgtcattgtaggcggagagctatttactgattactccgatcaccctcgcaaacttgtcacgctaaacccaaaactcaaatcaacaggcgccg gacgctaccagcttctttcccgttggtgggatgcctaccgcaagcagcttggcctgaaagacttctctccgaaaagtcaggacgctgtggcattgcagc agattaaggagcgtggcgctttacctatgattgatcgtggtgatatccgtcaggcaatcgaccgttgcagcaatatctgggcttcactgccgggcgctg gttatggtcagttcgagcataaggctgacagcctgattgcaaaattcaaagaagcgggcggaacggtcagagagattgatgtatgagcagagtcacc gcgattatctccgctctggttatctgcatcatcgtctgcctgtcatgggctgttaatcattaccgtgataacgccattacctacaaagcccagcgcgacaa aaatgccagagaactgaagctggcgaacgcggcaattactgacatgcagatgcgtcagcgtgatgttgctgcgctcgatgcaaaatacacgaagga gttagctgatgctaaagctgaaaatgatgctctgcgtgatgatgttgccgctggtcgtcgtcggttgcacatcaaagcagtctgtcagtcagtgcgtgaa gccaccaccgcctccggcgtggataatgcagcctccccccgactggcagacaccgctgaacgggattatttcaccctcagagagaggctgatcactat gcaaaaacaactggaaggaacccagaagtatattaatgagcagtgcagatagagttgcccatatcgatgggcaactcatgcaattattgtgagcaat acacacgcgcttccagcggagtataaatgcctaaagtaataaaaccgagcaatccatttacgaatgtttgctgggtttctgttttaacaacattttctgc gccgccacaaattttggctgcatcgacagttttcttctgcccaattccagaaacgaagaaatgatgggtgatggtttcctttggtgctactgctgccggtt tgttttgaacagtaaacgtctgttgagcacatcctgtaataagcagggccagcgcagtagcgagtagcatttttttcatggtgttattcccgatgcttttt gaagttcgcagaatcgtatgtgtagaaaattaaacaaaccctaaacaatgagttgaaatttcatattgttaatatttattaatgtatgtcaggtgcgatg aatcgtcattgtattcccggattaactatgtccacagccctgacggggaacttctctgcgggagtgtccgggaataattaaaacgatgcacacagggtt tagcgcgtacacgtattgcattatgccaacgccccggtgctgacacggaagaaaccggacgttatgatttagcgtggaaagatttgtgtagtgttctga atgctctcagtaaatagtaatgaattatcaaaggtatagtaatatcttttatgttcatggatatttgtaacccatcggaaaactcctgctttagcaagatt ttccctgtattgctgaaatgtgatttctcttgatttcaacctatcataggacgtttctataagatgcgtgtttcttgagaatttaacatttacaacctttttaa gtccttttattaacacggtgttatcgttttctaacacgatgtgaatattatctgtggctagatagtaaatataatgtgagacgttgtgacgttttagttcag aataaaacaattcacagtctaaatcttttcgcacttgatcgaatatttctttaaaaatggcaacctgagccattggtaaaaccttccatgtgatacgagg gcgcgtagtttgcattatcgtttttatcgtttcaatctggtctgacctccttgtgttttgttgatgatttatgtcaaatattaggaatgttttcacttaatagta ttggttgcgtaacaaagtgcggtcctgctggcattctggagggaaatacaaccgacagatgtatgtaaggccaacgtgctcaaatcttcatacagaaa gatttgaagtaatattttaaccgctagatgaagagcaagcgcatggagcgacaaaatgaataaagaacaatctgctgatgatccctccgtggatctga ttcgtgtaaaaaatatgcttaatagcaccatttctatgagttaccctgatgttgtaattgcatgtatagaacataaggtgtctctggaagcattcagagc aattgaggcagcgttggtgaagcacgataataatatgaaggattattccctggtggttgactgatcaccataactgctaatcattcaaactatttagtct gtgacagagccaacacgcagtctgtcactgtcaggaaagtggtaaaactgcaactcaattactgcaatgccctcgtaattaagtgaatttacaatatc gtcctgttcggagggaagaacgcgggatgttcattcttcatcacttttaattgatgtatatgctctcttttctgacgttagtctccgacggcaggcttcaat gacccaggctgagaaattcccggaccctttttgctcaagagcgatgttaatttgttcaatcatttggttaggaaagcggatgttgcgggttgttgttctgc gggttctgttcttcgttgacatgaggttgccccgtattcagtgtcgctgatttgtattgtctgaagttgtttttacgttaagttgatgcagatcaattaatac gatacctgcgtcataattgattatttgacgtggtttgatggcctccacgcacgttgtgatatgtagatgataatcattatcactttacgggtcctttccggt gatccgacaggttacg

Appendix IV GenBank DNA sequence files of orfs encoding RM subunits

LOCUS M.EcoKI 2981 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 5)

DEFINITION .

FEATURES Location/Qualifiers misc_feature 1..1590

/label="M"

CDS 1..1590

/label="Translation 1-1590" misc_feature 1587..2981

/label="S"

CDS 1587..2981

/label="Translation 1587-2981"

ORIGIN

1 atgaacaata acgatctggt cgcgaagctg tggaagctgt gcgacaacct gcgcgatggc

61 ggcgtttcct atcaaaacta cgtcaatgaa ctcgcctcgc tgctgttttt gaaaatgtgt

121 aaagagacgg gtcaggaagc ggaatacctg ccggaaggtt accgctggga tgacctgaaa

181 tcccgcatcg gccaggagca gttgcagttc taccgaaaaa tgctcgtgca tttaggcgaa

241 gatgacaaaa agctggtaca ggcagttttt cataatgtta gtaccaccat caccgagccg

301 aaacaaataa ccgcactggt cagcaatatg gattcgctgg actggtacaa cggcgcgcac

361 ggtaagtcgc gcgatgactt cggcgatatg tacgaagggc tgttgcagaa gaacgcgaat 421 gaaaccaagt ctggtgcagg ccagtacttc accccgcgtc cgctgattaa aaccattatt

481 catctgctga aaccgcagcc gcgtgaagtg gtgcaggacc cggcggcagg tacggcgggc

541 tttttgattg aagccgaccg ctatgttaag tcgcaaacca atgatctgga cgaccttgat

601 ggcgacacgc aggatttcca gatccaccgc gcgtttatcg gcctcgaact ggtgcccggc

661 acccgtcgtc tggcactgat gaactgcctg ctgcacgata ttgaaggcaa cctcgaccac

721 ggcggcgcaa tccgtctggg caacacTctg ggTagcgacg gtgaaaacct gccgaaggcg

781 catattgtcg ccactaaccc gccgtttggc agcgccgcag gcaccaacat tacccgcacc

841 tttgttcacc cgaccagcaa caaacagTtg tgctttatgc agcatattat cgaaacgctg

901 catcccggcg gtcgtgcggc ggtggtggtg ccGgataacg tgCtgtttga aggcggcaaa

961 ggcaccgaca tTcgtcgtga cctgatggat aagtgtcatc tgcacaccat tctgcgtctg

1021 ccgaccggta ttttttacgc Tcagggcgtg aagaccaacg tgctgttctt taccaaaggG

1081 acGgtggcga acccgAatca ggataagaac tgTaccgaTg aTgtgtgggt gtatgacctg

1141 cgtaccaata tgccgagttt cggcaagcgc acaccgttta ccgacgagca tttgcagccg

1201 tttgagcgcg tgtatggcga agacccgcac ggtttaagcc cgcgcactga aggtgaatgg

1261 agttttaacg ccgaagagac ggaagttgcc gacagcgaag agaacaaaaa caccgaccag

1321 catcttgcta ccagccgctg gcgcaagttc agccgtgagt ggatccgcac cgcaaaatcc

1381 gattcgctgg atatctcctg gctgaaagat aaagacagta ttgatgccga cagcctgccg

1441 gagccggatg tattagcggc agaagcgatg ggcgaactgg tacaggcgct gtctgaactg

1501 gatgcgctga tgcgtgaact gggggcgagc gatgaggccg atttgcagcg tcagttgctg

1561 gaagaagcgt ttggtggggt gaaggaatga gtgcggggaa attgccggag gggtgggtta

1621 tcgccccagt atctacggtc acaactctaa tccgaggagt aacgtataaa aaagagcagg

1681 caataaatta tctaaaagat gattatttgc ctcttatccg tgcgaacaat attcagaatg

1741 gcaagtttga tactacggac ttggtttttg ttcctaaaaa tcttgttaaa gaaaGtcaaa

1801 aaatatctcc tgaagatatt gttattgcaa tgtcatcagg gagcaaatcc gtagttggta

1861 aatccgcaca tcagcatcta ccatttgaat gtagtttcgg cgcattttgc ggtgtattac

1921 gtcctgaaaa acttatattt tctggtttta ttgctcattt cacaaaatct tctctttatc

1981 gaaacaaaat ttcatcactt tctgctggtg caaatattaa taatattaag ccggcaagct

2041 ttgatttgat aaatatacca atcccaccac ttgccgaaca aaaaatcatc gctgaaaaac

2101 tcgatacgct gctggcgcag gtagacagca ccaaagcacg ttttgagcaa atcccacaaa

2161 tcctgaaacg ttttcgtcaa gcggtattgg ggggcgcagt taatggaaaa ttgacagaaa

2221 aatggcgtaa ttttgagccg caacattctg tatttaagaa gttaaatttt gaatctatct

2281 taactgaatt acgtaatggt ctttcatcaa agccaaatga aagtggtgtt ggtcatccaa

2341 tactacgcat tagttctgta cgtgctggcc atgtagatca aaacgatatt cggtttctag

2401 aatgttcaga aagtgaacta aaccgccaca aattacaaga tggagatctt ttatttactc

2461 gctataacgg aagtttagaa tttgttggtg tttgtgggtt attgaaaaaa ttacaacatc

2521 aaaatttgct atatcctgat aaacttattc gagctcgatt aaccaaagat gctttaccag 2581 aatatatcga aatatttttt tcatccccct cagcacgaaa tgcaatgatg aactgcgtga

2641 aaacaacttc tggtcaaaaa ggtatttcag gaaaagatat caaatcccaa gttgttttat

2701 tacctccagt aaaagaacaa gccgaaatcg ttcgccgcgt cgagcaactc ttcgcctacg

2761 ccgacaccat agaaaaacag gtcaacaacg ccttagcccg cgtcaacaac ctgacgcaat

2821 ccatcctggc aaaagcgttc cgtggtgaac ttaccgccca gtggcgggcc gaaaacccgg

2881 atttgatcag cggagaaaac agcgccgccg cgttgctgga aaaaatcaaa gctgaacgcg

2941 cagctagcgg gggtaaaaaa gcctcacgta aaaaatcctg a

//

LOCUS mutant JVLEcoKI 2981 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 6)

DEFINITION .

FEATURES Location/Qualifiers misc_feature 1..1590

/label="M"

CDS 1..1590

/label="Translation 1-1590" misc_feature 1587..2981

/label="S"

CDS 1587..2981

/label="Translation 1587-2981"

ORIGIN

1 atgaacaata acTAActggt cgcgaagctg tggaagctgt gcgacaacct gcgcgatggc

61 ggcgtttcct atcaaaacta cgtcaatgaa ctcgcctcgc tgctgttttt gaaaatgtgt

121 aaagagacgg gtcaggaagc ggaatacctg ccggaaggtt accgctggga tgacctgaaa

181 tcccgcatcg gccaggagca gttgcagttc taccgaaaaa tgctcgtgca tttaggcgaa

241 gatgacaaaa agctggtaca ggcagttttt cataatgtta gtaccaccat caccgagccg

301 aaacaaataa ccgcactggt cagcaatatg gattcgctgg actggtacaa cggcgcgcac

361 ggtaagtcgc gcgatgactt cggcgatatg tacgaagggc tgttgcagaa gaacgcgaat

421 gaaaccaagt ctggtgcagg ccagtacttc accccgcgtc cgctgattaa aaccattatt

481 catctgctga aaccgcagcc gcgtgaagtg gtgcaggacc cggcggcagg tacggcgggc

541 tttttgattg aagccgaccg ctatgttaag tcgcaaacca atgatctgga cgaccttgat

601 ggcgacacgc aggatttcca gatccaccgc gcgtttatcg gcctcgaact ggtgcccggc

661 acccgtcgtc tggcactgat gaactgcctg ctgcacgata ttgaaggcaa cctcgaccac

721 ggcggcgcaa tccgtctggg caacacTctg ggTagcgacg gtgaaaacct gccgaaggcg

781 catattgtcg ccactaaccc gccgtttggc agcgccgcag gcaccaacat tacccgcacc 841 tttgttcacc cgaccagcaa caaacagTtg tgctttatgc agcatattat cgaaacgctg

901 catcccggcg gtcgtgcggc ggtggtggtg ccGgataacg tgCtgtttga aggcggcaaa

961 ggcaccgaca tTcgtcgtga cctgatggat aagtgtcatc tgcacaccat tctgcgtctg

1021 ccgaccggta ttttttacgc Tcagggcgtg aagaccaacg tgctgttctt taccaaaggG

1081 acGgtggcga acccgAatca ggataagaac tgTaccgaTg aTgtgtgggt gtatgacctg

1141 cgtaccaata tgccgagttt cggcaagcgc acaccgttta ccgacgagca tttgcagccg

1201 tttgagcgcg tgtatggcga agacccgcac ggtttaagcc cgcgcactga aggtgaatgg

1261 agttttaacg ccgaagagac ggaagttgcc gacagcgaag agaacaaaaa caccgaccag

1321 catcttgcta ccagccgctg gcgcaagttc agccgtgagt ggatccgcac cgcaaaatcc

1381 gattcgctgg atatctcctg gctgaaagat aaagacagta ttgatgccga cagcctgccg

1441 gagccggatg tattagcggc agaagcgatg ggcgaactgg tacaggcgct gtctgaactg

1501 gatgcgctga tgcgtgaact gggggcgagc gatgaggccg atttgcagcg tcagttgctg

1561 gaagaagcgt ttggtggggt gaaggaatga gtgcgTAAaa attgccggag gggtgggtta

1621 tcgccccagt atctacggtc acaactctaa tccgaggagt aacgtataaa aaagagcagg

1681 caataaatta tctaaaagat gattatttgc ctcttatccg tgcgaacaat attcagaatg

1741 gcaagtttga tactacggac ttggtttttg ttcctaaaaa tcttgttaaa gaaaGtcaaa

1801 aaatatctcc tgaagatatt gttattgcaa tgtcatcagg gagcaaatcc gtagttggta

1861 aatccgcaca tcagcatcta ccatttgaat gtagtttcgg cgcattttgc ggtgtattac

1921 gtcctgaaaa acttatattt tctggtttta ttgctcattt cacaaaatct tctctttatc

1981 gaaacaaaat ttcatcactt tctgctggtg caaatattaa taatattaag ccggcaagct

2041 ttgatttgat aaatatacca atcccaccac ttgccgaaca aaaaatcatc gctgaaaaac

2101 tcgatacgct gctggcgcag gtagacagca ccaaagcacg ttttgagcaa atcccacaaa

2161 tcctgaaacg ttttcgtcaa gcggtattgg ggggcgcagt taatggaaaa ttgacagaaa

2221 aatggcgtaa ttttgagccg caacattctg tatttaagaa gttaaatttt gaatctatct

2281 taactgaatt acgtaatggt ctttcatcaa agccaaatga aagtggtgtt ggtcatccaa

2341 tactacgcat tagttctgta cgtgctggcc atgtagatca aaacgatatt cggtttctag

2401 aatgttcaga aagtgaacta aaccgccaca aattacaaga tggagatctt ttatttactc

2461 gctataacgg aagtttagaa tttgttggtg tttgtgggtt attgaaaaaa ttacaacatc

2521 aaaatttgct atatcctgat aaacttattc gagctcgatt aaccaaagat gctttaccag

2581 aatatatcga aatatttttt tcatccccct cagcacgaaa tgcaatgatg aactgcgtga

2641 aaacaacttc tggtcaaaaa ggtatttcag gaaaagatat caaatcccaa gttgttttat

2701 tacctccagt aaaagaacaa gccgaaatcg ttcgccgcgt cgagcaactc ttcgcctacg

2761 ccgacaccat agaaaaacag gtcaacaacg ccttagcccg cgtcaacaac ctgacgcaat

2821 ccatcctggc aaaagcgttc cgtggtgaac ttaccgccca gtggcgggcc gaaaacccgg

2881 atttgatcag cggagaaaac agcgccgccg cgttgctgga aaaaatcaaa gctgaacgcg

2941 cagctagcgg gggtaaaaaa gcctcacgta aaaaatcctg a //

LOCUS M.Eco0015 2954 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 7)

DEFINITION .

FEATURES Location/Qualifiers misc_feature 1..1590

/label="M"

CDS 1..1590

/label="Translation 1-1590" misc_feature 1587..2954

/label="S"

CDS 1587..2954

/label="Translation 1587-2954"

ORIGIN

1 atgaacaata acgatctggt cgcgaagctg tggaagctgt gcgacaacct gcgcgatggc

61 ggcgtttcct atcaaaacta cgtcaatgaa ctcgcctcgc tgctgttttt gaaaatgtgt

121 aaagagacTg ggcaggaagc ggaatacctg ccggaaggtt accgctggga tgacctgaaa

181 tcccgcatcg gccaggagca gttgcagttc taccgaaaaa tgctcgtgca tttaggcgaa

241 gatgacaaaa agctggtaca ggcagttttt cataatgtta gtaccaccat caccgagccg

301 aaacaaataa ccgcactggt cagcaatatg gattcactgg actggtacaa cggtgcgcac

361 ggtaagtcgc gcgatgactt cggcgatatg tacgaagggc tgctgcagaa gaacgccaat

421 gaaaccaagt ctggcgcggg ccagtacttc accccgcgtc cgctgattaa aaccattatt

481 catctgctga aaccgcagcc acgtgaagtg gtacaggatc cggcagccgg tacggcgggc

541 tttttgattg aagccgaccg ctacgttaag tcgcagacta acgatctgga cgaccttgat

601 ggcgacacgc aggatttcca gatccaccgc gcgtttatcg gcctcgaact ggtgcccggc

661 acccgtcgtc tggcgctgat gaactgcctg ctgcacgata ttgaaggcaa cctcgaccac

721 ggcggcgcaa tccgtctggg caacaccctg ggcagcgacg gcgaaaacct gccgaaggcg

781 catattgtcg ccactaaccc gccgtttggc agcgccgcag gcaccaacat tacccgcacc

841 tttgttcacc cgaccagcaa caaacagttg tgctttatgc agcatattat cgaaacgctg

901 caccccggcg gtcgtgcggc ggtggtggtg ccggataacg tgctgtttga aggcggcaaa

961 ggtacggaca tccgtcgtga cctgatggat aagtgtcatc tgcacactat tctgcgtctg

1021 ccgaccggta ttttttacgc gcagggcgtg aagaccaacg tgctgttctt taccaaaggg

1081 acggtggcga acccgaatca ggataagaac tgcaccgatg acgtgtgggt gtacgacctg

1141 cggaccaata tgccgagctt tggtaaacgc acgccgttta ccgacgagca tttgcaaccg

1201 tttgagcgcg tgtttggcga agacccgcac ggcttaagcc cgcgcactga aggtgaatgg 1261 agttttaacg ccgaagagac ggaaattgcc gacagcgaag agaacaaaaa caccgaccag

1321 catcttgcta ccagccgctg gcgcaagttc agccgcgagt ggatccgcac cgcgaaatcc

1381 gattcgctgg atatctcctg gctgaaagat aaagacagta ttgatgccga caacctgccg

1441 gagccggatg tattagcggc agaagcgatg ggtgaactgg tacaggcgct gtctgaactg

1501 gatgcgctga tgcgtgaact gggggcgagc gatgaggcgg atgcgcagcg ccagttgctg

1561 gaagaagcat ttggtggggt gaaggaatga gtgctgggaa attgccggag gggtgggaac

1621 agattgaaat aggcgacatt gctgatgtca tctctggtgg gacaccaaaa tcgggagtcg

1681 ctgaaaattt tgcaccttcg ggtgagggag tcgcctggct tacgcctgcc gatttgagcg

1741 gttataaaga gaaatatatc tcacatggtg ccagagattt aactacacta ggctattcct

1801 cttgttcagc aaaactaatg cccaaaggca ctatactttt tagtagccgg gcaccgattg

1861 gatatgttgc tattgctgca aatgagattg cgactaacca gggtttcaaa agttttgctt

1921 ttccttcaga catttttcct gattatgcat attactttct ccgcaacatc agacatattg

1981 cagaggaaat gggcacagga acaactttta aagaaatttc tggtagttct gcgaaaacat

2041 taccatttgt tcttgtaccg tttgccgaac aaaaaatcat cgctgaaaaa ctcgatacgc

2101 tgctggcaca ggtagacagc accaaagcac gtcttgagca aatcccacaa atcctgaaac

2161 gttttcgtca ggcggttctg ggtgcagcgg tgagaggaaa actaactgaa gactggagag

2221 ataattcaag cctgtctggg tggagagaag gcaaacttgg ggagtttata aaaaaaccaa

2281 gttacggaac gtcatctaag tctaataaag aaggactcat tcctgtatta agaatgggca

2341 atcttcaggg aggaaagttg gactggacgg atctagtcta tacgtcagat accatagaaa

2401 ttgaaaagta taaacttgag tacaatgatg tattattcaa tcgtacaaat agtcctgaac

2461 ttgtcgggaa aacagccata tataaatcag agcaaccagc tatttatgca ggttatttga

2521 ttagggttca atgtttacct gatttaaacc ctgactatct taactaccat ttaaatagca

2581 ttttggggag acaatattgc tattcagtta aatctgatgg ggtcagtcaa tcaaatatta

2641 atgcacagaa attaattgca tacccaataa ctgttccccc ccttcccgaa caagctgaaa

2701 tcgttcgccg cgtcgagcaa ctcttcgcct acgccgacac aatagaaaaa caggtcaaca

2761 acgccttagc ccgcgtcaac aatctgacgc aatccatcct ggcaaaagcg ttccgtggtg

2821 aactcaccgc ccagtggcgg gccgaaaacc cggaattaat cagcggtgaa aacagcgccg

2881 ccgcgttgct ggagaaaatc aaagctgaac gcgcagccag cgggggtaaa aaagcctcgc

2941 gtaaaaagtc ctga

//

LOCUS mutant_M.Eco0015 2954 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 8)

DEFINITION .

FEATURES Location/Qualifiers misc_feature 1..1590

/label="M" CDS 1..1590

/label="Translation 1-1590" misc_feature 1587..2954

/label="S"

CDS 1587..2954

/label="Translation 1587-2954"

ORIGIN

1 atgaacaata acTAActggt cgcgaagctg tggaagctgt gcgacaacct gcgcgatggc

61 ggcgtttcct atcaaaacta cgtcaatgaa ctcgcctcgc tgctgttttt gaaaatgtgt

121 aaagagacTg ggcaggaagc ggaatacctg ccggaaggtt accgctggga tgacctgaaa

181 tcccgcatcg gccaggagca gttgcagttc taccgaaaaa tgctcgtgca tttaggcgaa

241 gatgacaaaa agctggtaca ggcagttttt cataatgtta gtaccaccat caccgagccg

301 aaacaaataa ccgcactggt cagcaatatg gattcactgg actggtacaa cggtgcgcac

361 ggtaagtcgc gcgatgactt cggcgatatg tacgaagggc tgctgcagaa gaacgccaat

421 gaaaccaagt ctggcgcggg ccagtacttc accccgcgtc cgctgattaa aaccattatt

481 catctgctga aaccgcagcc acgtgaagtg gtacaggatc cggcagccgg tacggcgggc

541 tttttgattg aagccgaccg ctacgttaag tcgcagacta acgatctgga cgaccttgat

601 ggcgacacgc aggatttcca gatccaccgc gcgtttatcg gcctcgaact ggtgcccggc

661 acccgtcgtc tggcgctgat gaactgcctg ctgcacgata ttgaaggcaa cctcgaccac

721 ggcggcgcaa tccgtctggg caacaccctg ggcagcgacg gcgaaaacct gccgaaggcg

781 catattgtcg ccactaaccc gccgtttggc agcgccgcag gcaccaacat tacccgcacc

841 tttgttcacc cgaccagcaa caaacagttg tgctttatgc agcatattat cgaaacgctg

901 caccccggcg gtcgtgcggc ggtggtggtg ccggataacg tgctgtttga aggcggcaaa

961 ggtacggaca tccgtcgtga cctgatggat aagtgtcatc tgcacactat tctgcgtctg

1021 ccgaccggta ttttttacgc gcagggcgtg aagaccaacg tgctgttctt taccaaaggg

1081 acggtggcga acccgaatca ggataagaac tgcaccgatg acgtgtgggt gtacgacctg

1141 cggaccaata tgccgagctt tggtaaacgc acgccgttta ccgacgagca tttgcaaccg

1201 tttgagcgcg tgtttggcga agacccgcac ggcttaagcc cgcgcactga aggtgaatgg

1261 agttttaacg ccgaagagac ggaaattgcc gacagcgaag agaacaaaaa caccgaccag

1321 catcttgcta ccagccgctg gcgcaagttc agccgcgagt ggatccgcac cgcgaaatcc

1381 gattcgctgg atatctcctg gctgaaagat aaagacagta ttgatgccga caacctgccg

1441 gagccggatg tattagcggc agaagcgatg ggtgaactgg tacaggcgct gtctgaactg

1501 gatgcgctga tgcgtgaact gggggcgagc gatgaggcgg atgcgcagcg ccagttgctg

1561 gaagaagcat ttggtggggt gaaggaatga gtgctgggTA Attgccggag gggtgggaac

1621 agattgaaat aggcgacatt gctgatgtca tctctggtgg gacaccaaaa tcgggagtcg

1681 ctgaaaattt tgcaccttcg ggtgagggag tcgcctggct tacgcctgcc gatttgagcg 1741 gttataaaga gaaatatatc tcacatggtg ccagagattt aactacacta ggctattcct

1801 cttgttcagc aaaactaatg cccaaaggca ctatactttt tagtagccgg gcaccgattg

1861 gatatgttgc tattgctgca aatgagattg cgactaacca gggtttcaaa agttttgctt

1921 ttccttcaga catttttcct gattatgcat attactttct ccgcaacatc agacatattg

1981 cagaggaaat gggcacagga acaactttta aagaaatttc tggtagttct gcgaaaacat

2041 taccatttgt tcttgtaccg tttgccgaac aaaaaatcat cgctgaaaaa ctcgatacgc

2101 tgctggcaca ggtagacagc accaaagcac gtcttgagca aatcccacaa atcctgaaac

2161 gttttcgtca ggcggttctg ggtgcagcgg tgagaggaaa actaactgaa gactggagag

2221 ataattcaag cctgtctggg tggagagaag gcaaacttgg ggagtttata aaaaaaccaa

2281 gttacggaac gtcatctaag tctaataaag aaggactcat tcctgtatta agaatgggca

2341 atcttcaggg aggaaagttg gactggacgg atctagtcta tacgtcagat accatagaaa

2401 ttgaaaagta taaacttgag tacaatgatg tattattcaa tcgtacaaat agtcctgaac

2461 ttgtcgggaa aacagccata tataaatcag agcaaccagc tatttatgca ggttatttga

2521 ttagggttca atgtttacct gatttaaacc ctgactatct taactaccat ttaaatagca

2581 ttttggggag acaatattgc tattcagtta aatctgatgg ggtcagtcaa tcaaatatta

2641 atgcacagaa attaattgca tacccaataa ctgttccccc ccttcccgaa caagctgaaa

2701 tcgttcgccg cgtcgagcaa ctcttcgcct acgccgacac aatagaaaaa caggtcaaca

2761 acgccttagc ccgcgtcaac aatctgacgc aatccatcct ggcaaaagcg ttccgtggtg

2821 aactcaccgc ccagtggcgg gccgaaaacc cggaattaat cagcggtgaa aacagcgccg

2881 ccgcgttgct ggagaaaatc aaagctgaac gcgcagccag cgggggtaaa aaagcctcgc

2941 gtaaaaagtc ctga //

LOCUS pM0-Eco0015_R+M+S 6708 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 9) DEFINITION .

FEATURES Location/Qualifiers misc_feature 1..36

/label="pM0" misc_feature 41..3553

/label="R"

CDS 41..3553

/label="Translation 41-3553" misc_feature 3755..5344

/label="M"

CDS 3755..5344 /label="Translation 3755-5344" misc_feature 5341..6708

/label="S"

CDS 5341..6708

/label="Translation 5341-6708"

ORIGIN

1 ggattaacaa tataagctga ccttcaagta ttgaatTGAC atgatgaata aatccaactt

61 cgaattcctg aagggcgtca acgacttcac ttatgccatc gcctgtgcgg cggaaaataa

121 ctacccggat gatcccaaca cgacgctgat taaaatgcgt atgtttggcg aagctacagc

181 gaaacatctt ggtctgttac tcaacatccc cccttgtgag aatcaacacg atctcctgcg

241 cgaactcggc aaaatcgcct tcgttgatga caacattctc tctgtattcc acaaattacg

301 ccgcattggt aaccaggcgg tgcacgaata ccataacgat ctcgacgatg cccagatgtg

361 cctgcgactc gggttccgcc tggctgtctg gtactaccgt ctggtcacca aagattatga

421 cttcccggtg ccggtatttg tgttgccgga acgtggcgaa aacctctatc accaggaagt

481 gctgacgcta aaacaacagc ttgaacagca ggcgcgagaa aaagcgcaga ctcaggcaga

541 agtcgaagcg caacagcaga agctggttgc cctgaacggc tatatcgcca ttctggaagg

601 caaacagcag gaaacagaag cgcaaaccca ggctcgcctt gcggcgctgg aagcacagct

661 cgccgagaag aacgcggagc tggccaaaca gaccgaacag gaacgtaagg cttaccacaa

721 agaaattacc gatcaggcca tcaagcgcac actcaacctt agcgaagaag agagtcgctt

781 cctgattgac gcgcaactgc gtaaagcagg ctggcaggcc gatagcaaaa ccctgcgctt

841 ctccaaaggc gcacgcccgg aacccggcgt caataaagcc attgccgaat ggccgaccgg

901 gaaagatgaa acgggtaatc agggctttgc ggattatgtg ctgtttgtcg gcctcaagcc

961 catcgcggtg gtagaggcga aacgtaaaaa tatcgacgtt cccggcaagc tcaatgagtc

1021 gtatcgctac agcaaatgct tcgataatgg cttcctgcgg gaaaccttgc ttgagcatta

1081 ctcaccggat gaagtgcatg aagctgtgcc ggagtatgaa actagctggc aggacaccag

1141 cggccaacaa cggtttaaaa tccccttctg ctactccacc aacgggcgcg aatatcgcgc

1201 agcaatgaag actaaaagtg gcatctggta tcgcgacgtg cgtgataccc gcaatatgtc

1261 gaaagcctta cccgagtggc atcgcccgga agagctgctg gaaatgctcg gcagcgaacc

1321 gcaaaaacag aatcagtggt ttgccgataa cccgggcatg agcgagctgg gcctgcgtta

1381 ttaccaggaa gatgccgtcc gcgcggtgga aaaggcaatc gtcaagggtc aacaagagat

1441 cctgctggcg atggcgaccg gtaccggtaa aacccgcacc gcaattgcca tgatgttccg

1501 cctgatccag tcccagcgtt ttaaacgcat tctcttcctt gtcgatcgcc gttctcttgg

1561 cgagcaggca ctgggggcgt ttgaagatac gcgtattaac ggcgacacct tcaacagcat

1621 tttcgacatt aaagggctga cggataaatt ccccgaagac agcaccaaaa ttcacgttgc

1681 caccgtacag tcgctggtga aacgcaccct acaatcagat gaaccgatgc cggtggcccg

1741 ttacgactgt atcgtcgtcg atgaagcgca tcgcggctat attctcgata aagagcagac 1801 cgaaggcgag ctgcagttcc gcagccagct ggattacgtt tctgcctacc gtcgcattct

1861 cgatcacttc gatgcggtaa aaatcgccct caccgccacc ccggcgctgc atacggtgca

1921 gattttcggc gagccggttt accgttatac ctaccgtacc gcggttatcg acggttttct

1981 gatcgaccag gatccgccga ttcagatcat cacccgcaac gcccaggagg gggtttatct

2041 ctccaagggc gaacaggtag agcgcatcag cccgcaggga gaagtgatca acgataccct

2101 ggaagacgat caggattttg aggtcgccga ctttaaccgt ggcctggtga tcccggcgtt

2161 taaccgcgcc gtctgtaacg agctcaccaa ctaccttgac ccgaccgggt cgcaaaaaac

2221 gctggtcttc tgcgttacca atgcccatgc cgatatggtg gtggaagagt tgcgtaccgc

2281 gttcaagaaa aagtatccgc aactggagca cgacgcgatc atcaagatca ccggtgatgc

2341 cgataaagac gcgcgcaaag tgcagaccat gatcacccgc ttcaataaag agcggctgcc

2401 caatatcgtg gtaaccgtcg acctgctgac aaccggcgtc gatattccgt cgatctgtaa

2461 tatcgtgttc ctgcgtaaag tacgcagccg tattctgtac gagcagatga aaggccgcgc

2521 cacgcgctta tgcccggacg tgaataaaac cagctttaag attttcgact gtgtcgatat

2581 ctacagcacg ctggagagcg tcgacaccat gcgtccggtg gtggtgcgtc cgaaggtgga

2641 actgcaaacg ctggtcaatg aaattaccga ttcagaaacc tataaaatca ccgaagcgga

2701 tggccgtagc tttgccgagc acagccatga acaattggtg gcgaagctcc agcgcatcat

2761 cggcctggcc acgtttaacc gtgaccgcag cgaaacgata gataaacagg tgcgtcgtct

2821 ggatgagcta tgccaggacg cggcgggcgt gaactttaac ggcttcgcct cgcgcctgcg

2881 ggaaaaaggg ccgcactgga gcgccgaagt ctttaacaaa ctgcctggct ttatcgcccg

2941 tctggaaaag ctgaaaacgg acatcaacaa cctgaatgat gcgccgatct tcctcgatat

3001 cgacgatgaa gtggtgagtg taaaatcgct gtacggtgat tacgacacgc cgcaggattt

3061 cctcgaagcc tttgactcgc tggtgcaacg ttccccgaac gcgcagccgg cattgcaggc

3121 ggtgatcaat cgcccgcgcg atctcacccg taaagggctg gtcgagctcc aggagtggtt

3181 tgaccgccag cattttgagg aatcttccct gcgcaaagca tggaaagaga cgcgcaatga

3241 agatatcgcc gcccggctga ttggtcatat tcgccgcgct gcggtgggcg atgcgctgaa

3301 accgtttgag gaacgtgtcg atcacgcgct gacgcgcatt aagggtgaaa acgactggag

3361 cagcgagcaa ttaagctggc tcgatcgttt agcgcaggcg ctgaaagaga aagtggtgct

3421 cgacgacgat gtcttcaaaa ccggcaactt ccaccgccgc ggcggtaagg cgatgctaca

3481 aagaaccttt gacgataatc tcgacaacct gcttgataaa ttcagcgatt atatctggga

3541 cgagctggcc tgacacgtat acacttcatc cttcaggctg cctctgcgtt ggctgcgctc

3601 gttcaccccg gtcacgtact tctgtacgct cccggggatt cactcacttg ccgccttgat

3661 gcaacctgaa tgattttgtg tatattaccc tcggcaattt cttcttctgc ggccccaaac

3721 aatgcgggcc gttgtttaat ttacggaact cacaatgaac aataacgatc tggtcgcgaa

3781 gctgtggaag ctgtgcgaca acctgcgcga tggcggcgtt tcctatcaaa actacgtcaa

3841 tgaactcgcc tcgctgctgt ttttgaaaat gtgtaaagag acTgggcagg aagcggaata

3901 cctgccggaa ggttaccgct gggatgacct gaaatcccgc atcggccagg agcagttgca 3961 gttctaccga aaaatgctcg tgcatttagg cgaagatgac aaaaagctgg tacaggcagt

4021 ttttcataat gttagtacca ccatcaccga gccgaaacaa ataaccgcac tggtcagcaa

4081 tatggattca ctggactggt acaacggtgc gcacggtaag tcgcgcgatg acttcggcga

4141 tatgtacgaa gggctgctgc agaagaacgc caatgaaacc aagtctggcg cgggccagta

4201 cttcaccccg cgtccgctga ttaaaaccat tattcatctg ctgaaaccgc agccacgtga

4261 agtggtacag gatccggcag ccggtacggc gggctttttg attgaagccg accgctacgt

4321 taagtcgcag actaacgatc tggacgacct tgatggcgac acgcaggatt tccagatcca

4381 ccgcgcgttt atcggcctcg aactggtgcc cggcacccgt cgtctggcgc tgatgaactg

4441 cctgctgcac gatattgaag gcaacctcga ccacggcggc gcaatccgtc tgggcaacac

4501 cctgggcagc gacggcgaaa acctgccgaa ggcgcatatt gtcgccacta acccgccgtt

4561 tggcagcgcc gcaggcacca acattacccg cacctttgtt cacccgacca gcaacaaaca

4621 gttgtgcttt atgcagcata ttatcgaaac gctgcacccc ggcggtcgtg cggcggtggt

4681 ggtgccggat aacgtgctgt ttgaaggcgg caaaggtacg gacatccgtc gtgacctgat

4741 ggataagtgt catctgcaca ctattctgcg tctgccgacc ggtatttttt acgcgcaggg

4801 cgtgaagacc aacgtgctgt tctttaccaa agggacggtg gcgaacccga atcaggataa

4861 gaactgcacc gatgacgtgt gggtgtacga cctgcggacc aatatgccga gctttggtaa

4921 acgcacgccg tttaccgacg agcatttgca accgtttgag cgcgtgtttg gcgaagaccc

4981 gcacggctta agcccgcgca ctgaaggtga atggagtttt aacgccgaag agacggaaat

5041 tgccgacagc gaagagaaca aaaacaccga ccagcatctt gctaccagcc gctggcgcaa

5101 gttcagccgc gagtggatcc gcaccgcgaa atccgattcg ctggatatct cctggctgaa

5161 agataaagac agtattgatg ccgacaacct gccggagccg gatgtattag cggcagaagc

5221 gatgggtgaa ctggtacagg cgctgtctga actggatgcg ctgatgcgtg aactgggggc

5281 gagcgatgag gcggatgcgc agcgccagtt gctggaagaa gcatttggtg gggtgaagga

5341 atgagtgctg ggaaattgcc ggaggggtgg gaacagattg aaataggcga cattgctgat

5401 gtcatctctg gtgggacacc aaaatcggga gtcgctgaaa attttgcacc ttcgggtgag

5461 ggagtcgcct ggcttacgcc tgccgatttg agcggttata aagagaaata tatctcacat

5521 ggtgccagag atttaactac actaggctat tcctcttgtt cagcaaaact aatgcccaaa

5581 ggcactatac tttttagtag ccgggcaccg attggatatg ttgctattgc tgcaaatgag

5641 attgcgacta accagggttt caaaagtttt gcttttcctt cagacatttt tcctgattat

5701 gcatattact ttctccgcaa catcagacat attgcagagg aaatgggcac aggaacaact

5761 tttaaagaaa tttctggtag ttctgcgaaa acattaccat ttgttcttgt accgtttgcc

5821 gaacaaaaaa tcatcgctga aaaactcgat acgctgctgg cacaggtaga cagcaccaaa

5881 gcacgtcttg agcaaatccc acaaatcctg aaacgttttc gtcaggcggt tctgggtgca

5941 gcggtgagag gaaaactaac tgaagactgg agagataatt caagcctgtc tgggtggaga

6001 gaaggcaaac ttggggagtt tataaaaaaa ccaagttacg gaacgtcatc taagtctaat

6061 aaagaaggac tcattcctgt attaagaatg ggcaatcttc agggaggaaa gttggactgg 6121 acggatctag tctatacgtc agataccata gaaattgaaa agtataaact tgagtacaat

6181 gatgtattat tcaatcgtac aaatagtcct gaacttgtcg ggaaaacagc catatataaa

6241 tcagagcaac cagctattta tgcaggttat ttgattaggg ttcaatgttt acctgattta

6301 aaccctgact atcttaacta ccatttaaat agcattttgg ggagacaata ttgctattca

6361 gttaaatctg atggggtcag tcaatcaaat attaatgcac agaaattaat tgcataccca

6421 ataactgttc ccccccttcc cgaacaagct gaaatcgttc gccgcgtcga gcaactcttc

6481 gcctacgccg acacaataga aaaacaggtc aacaacgcct tagcccgcgt caacaatctg

6541 acgcaatcca tcctggcaaa agcgttccgt ggtgaactca ccgcccagtg gcgggccgaa

6601 aacccggaat taatcagcgg tgaaaacagc gccgccgcgt tgctggagaa aatcaaagct

6661 gaacgcgcag ccagcggggg taaaaaagcc tcgcgtaaaa agtcctga

//

LOCUS M.EcoMII 2914 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 10)

DEFINITION .

FEATURES Location/Qualifiers misc_feature 1..1620

/label="M"

CDS 1..1620

/label="Translation 1-1620" misc_feature 1610..2914

/label="S"

CDS 1610..2914

/label="Translation 1610-2914"

ORIGIN

1 atggccaaag caccaactaa aaaagccaaa gcaaaaaaag gctttgaaga aacattatgg

61 gataccgcaa accaacttcg cggtagcgtt gagtcctccg aatacaagca cgtggtgtta

121 agcctcgtgt tcctgaaatt catcagcgat aagtttgaag cacgccgcaa aaaaatgatt

181 gccgatgggc aagctgactt ccttgagatg gaagtgttct accagcagga caacattttc

241 tacctgccgg aagaggcgcg ctggtcgttt atcaaacaaa atgccaaaca ggacgatatt

301 gcggttcgta ttgacaccgc cctctcgaca attgagaaac gtaacccaac cctgaaaggc

361 gcgctgccag acaactactt cagccgtcag aatctggaaa ccaaaaaact ggcgtcactg

421 attgatacca tcgacaacat cgaaacgctg gcacacgaaa ctgacgttga aacgttgtcg

481 aaagaagacc tggtcggacg cgtttacgaa tacttcctcg gtaagtttgc cgcaaccgaa

541 ggcaaaggcg gtggagagtt ctacacgccc aaatgtgtgg tcacgctgtt aactgaaatg

601 ctcgaaccct tccagggcaa aatttatgac ccgtgctgcg gctcggcagg gatgttcgtg 661 cagtcggtga agttcgtcga gagccatcag ggtaaaagcc gtgatatcgc gctgtatggt

721 caggaactga cagccacgac gtataaactg gcaaaaatga acctcgccat tcgcggtctt

781 tcagctaacc tcggcgaacg cccggcaaac actttcttta gcgaccagca cccggacctg

841 aaagccgact acattctggc gaacccgccg ttcaacctga aagactggcg caatgaagcc

901 gaattaaccg aagatccacg ctttgccggt tatcgtatgc cgccaaccgg taacgccaac

961 tacggctgga ttttgcatat gctctccaag ctgtcggcta acggcacagc aggttttgtg

1021 ctggcaaacg gttcgatgag ttctaacacc agcggtgaag gcgagatccg tgcacagatg

1081 atcgaaaatg atctgattga ctgcatgatc gccctacctg gacagttgtt ctacaccacg

1141 cagatcccgg tgtgtttatg gtttatgacc aaatcgaagg ctgccgaccc ggccaaaggt

1201 tatcgtgatc gtcagggcga gacgctgttt attgatgcgc gtaacctcgg caccatgatt

1261 agccgcacaa ctaaagagtt aacggcggaa gatattgcca caatcgccga tacctaccat

1321 gcctggcgca gcacgccgga agaactggct gcacggattg cgcgtggtga cagcaagctg

1381 gaaaaatatg aagaccaggc aggcttctgc aaagttgcga ccctgcaaga tattaaagat

1441 aacgactacg ttctgacacc gggccgctat gtgggtgcag ccgagcagga agaagacggc

1501 gtggcatttg agacgaaaat gcgtgaattg tcgaagacgt tgtttgagca gatgaagcag

1561 gcggaagaac tggatcgtgc gattcgccag aatctggagg cgctgggtta tggggagtaa

1621 gtggtgtata tatcgcttag gtgatatagc tgaattttca tacggaaaaa tgcctaaaaa

1681 ggagtttgtt ggtacaggtg actatgtcat tttttcagga tataaataca cagaaagcta

1741 ccctgaaaaa aatacttcgg cCggtgagtt aattgttgtt gcccgaggtg ttggtggaac

1801 tggcgatgtt aaaataacga ctagagattg ttatttaaca aatttgtcca ttaaaataaa

1861 tttggatgaa acaaaagtaa gaaaagaata tttttattat ctgtttctac agagtaattt

1921 acgatatctt gatagcggtt ctgctcaaag ccaaataacc atcaatgatc tagcgaatgt

1981 agaaatatct gctccttcgc taaatgttca agatttaata gtcaagagtc tcaaagcttt

2041 tgatgacaaa atcacaacac tttcatccat gaaccaaacc ttggaacaaa tgtcccaaac

2101 cctgttcaaa tcctggtttg tggattttga tccagtgatt gataacgctc tggatgcagg

2161 aaatccaatc ccggaagccc tgcaaactcg cgccaaattg cgtcaaaaag tacgtaatag

2221 tgcagatttt aaaccgcttc cggcggaaat tcgctcgctt ttcccaagtg aatttgaaga

2281 aacggagttg ggttgggtgc caaaggggtg gaaagaaggg acgcttccag agatagcttt

2341 tataaactcc acatcgtgga ctaataaaaa tcaaccagat tatatcaatt atgtagatct

2401 ctctaatgcg aaagatggca gaatatactc aattgaaaaa ttatcattta atgatgctcc

2461 aagtcgagct cgaagaatat taaaaaaaga tgatctgatt tttggccttg tcagacctgc

2521 taacagatca tttgcatatg tacatgttga tggtctaacg ggtagcacag gctttgccgt

2581 tattagagcc caaaagacga tatataaaaa tttcatttat tactttgtga cctatgataa

2641 aaatatagaa gaactagcga gaattgctga tggtggagca tatcctgcta taaaacctga

2701 tgatatttgt acattacctc taattattcc atcaataaat ataattgaga aatttgatga

2761 attaacaagc aatttcagag agatgatgaa taactcatta attcaaaacg aatatctcac 2821 caacctccga ggcaccttgc tcccgaaact catctccggc gagctatccc tggaagatct

2881 tccggatctc accaccgata cagaagccgc ataa

//

LOCUS M.EcoAl 3251 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 11)

DEFINITION .

FEATURES Location/Qualifiers misc_feature 1..1482

/label="M"

CDS 1..1482

/label="Translation 1-1482" misc_feature 1482..3251

/label="S"

CDS 1482..3251

/label="Translation 1482-3251"

ORIGIN

1 atggaataca acatgtctat cagctcagta atcaaatcat tacaagatat tatgcgcaaa

61 gatgccggtg tggacggaga tgcgcagcgt ctcggtcagc tctcctggct gctgtttttg

121 aaaatcttcg atgcccagga agaggcgctg gaactggagc aggataacta tcaatatccg

181 atcccacagc gttatttatg gcgcagttgg gccgcaaacg cgcagggcat taccggtgat

241 tccctgctgg aattcgttaa tgatgatctg ttcccggcgt tgaaaaacct cactgcgcct

301 atcgataaaa acccacgcgg ctacgtggtg aagcaggcgt tcagcgatgc ctataactat

361 atgaaaaacg gtacgctact gcgtcaggtg atcaacaagc tgaacgaaat tgactttacc

421 agcgccagcg aacgccatct gtttggtgat atttacgaac agatccttaa agatctgcaa

481 tctgcgggca atgctggcga attctatact ccacgcgccg tcactcgctt tatggtggat

541 cgcgttgatc cgaaactcgg cgaatccatt atggacccgg cctgcggtac gggcggtttt

601 cttgcctgcg catttgatca tgtaaagaac aaatacgtga agagcgtcgc cgatcatcag

661 acgctgcaac aacagatcca cggtgttgag aaaaaacagc ttccgcacct gctggcgacc

721 accaatatgc tgctgcacgg cattgaagta ccagtacaaa ttcgtcacga caacaccctg

781 aacaaaccgc tttcctcctg ggatgagcaa ctggatgtca ttgttaccaa cccgccgttt

841 ggtggcacgg aagaagacgg tattgagaag aactttccgg cagagatgca aacccgcgaa

901 acggcggatc tgttcctgca actgattgtg gaagtactgg cgaaaaacgg tcgtgcggcg

961 gtggtattgc cggatggcac actatttggc gaaggcgtta aaaccaaaat caaaaagcta

1021 cttaccgaag agtgcaacct gcataccatc gtgcgtttac cgaatggtgt gtttaacccc

1081 tataccggca ttaaaaccaa cctgttgttc tttaccaaag gtcagccaac caaagagatt 1141 tggttctatg agcatccgta tccggcgggc gtgaaaaact acagcaaaac caagccgatg

1201 aagtttgaag agtttcaggc ggagatcgac tggtggggta acgaggccga tggttttgcc

1261 agccgcgtcg agaatgagca agcgtggaaa gtcagcattg atgacgtgat tgcgcgtaac

1321 ttcaatctgg atattaaaaa cccacatcag gcggaaaccg tcagccatga tccggacgaa

1381 ctgttagcgc agtatgcaaa gcaacaggcg gagatccaga cgctgcgtaa tcaactgcgc

1441 gatattctcg gcgctgcgct gtctgtcaag gaggttaact gatgagtgtg gaaaagctga

1501 tcgttgatca tatggaaacc tggacctcgg cgttgcaaac ccgttccacc gccgggcgcg

1561 gcagttccgg taaaattgat ttgtatggca ttaagaaatt acgtgagctg attctggaac

1621 tggcagtgcg cggtaaactg gtgccgcagg atccgaacga tgaaccggcg tcggagctgc

1681 tgaagcgtat tgcggcggaa aaagcagagc tggtgaaaca ggggaaaatt aaaaagcaaa

1741 aaccactgcc ggaaattagc gaggaagaga agccgtttga attgccggat ggatgggagt

1801 ggacaacgct aactagaatt gcggaaataa atcctaaaat tgatgtcagt gatgatgagc

1861 aagaaatatc atttattcca atgccactca tatcaactaa atttgatggc tcgcatgaat

1921 ttgaaataaa aaaatggaaa gatgttaaaa aaggttatac acactttgct aatggtgata

1981 tcgctattgc aaaaataaca ccttgctttg aaaatagtaa agcagctatt ttttctggct

2041 taaaaaacgg cattggtgtt ggaacaacag aattacatgt tgcacgccct tttagcgata

2101 taattaatcg aaaatatctt cttttaaact ttaaatcacc taattttctt aagtctggtg

2161 aatcacaaat gactggttct gctggtcaga aacgtgttcc aaggtttttt tttgaaaata

2221 atcctattcc cttccctcca ttgcaagaac aagagcgcat tattattagg tttactcaat

2281 taatgtctct ctgcgaccaa ctggaacagc aatccctaac cagtctggac gcacatcagc

2341 aactggttga aaccctgttg ggaacactta cagacagcca aaacgtcgag gaactggctg

2401 aaaactgggc gcgtattagc gagcatttcg acacactatt taccactgaa gccagcgtgg

2461 atgcgttaaa acagaccatt ctgcaactgg ccgtaatggg taaacttgtg ccgcaggatc

2521 cgaatgacga acccgcctct gaactgctca aacgtattgc gcagaaaaaa gcgcaattgg

2581 tgaaagaagg gaaaataaaa aaacaaaaac cgttgccgcc aattagtgat gaggaaaaac

2641 cgtttgaact gccggaaggg tgggagtggt gtcgattggg ttctatttat aattttttaa

2701 atggatatgc ctttaaaagt gagtggttta cctctgtcgg tttacgttta ttgcggaatg

2761 ctaacattgc tcatggagtt accaattgga aagatgttgt acatatacca aatgacatga

2821 tatccgattt tgaaaattat attttgtcag aaaatgatat tgttatttca ttagacagac

2881 caattattaa taccgggtta aaatatgcta tcattagcaa atcagattta ccctgtttac

2941 tactccaacg agtagcaaaa tttaaaaatt atgcaaacac tgtgtctaac tccttcttga

3001 caatatggtt gcaatcttat ttcttcataa attcaattga tcctggaaga agtaatggag

3061 ttccacatat atctacgaaa cagttagaga tgacactatt tcctcttcta ccgcaaagtg

3121 aacaagatcg cattatttca aaaacggatg aattaataca aacctgtaat aaactgaaat

3181 atattatcaa aaccgccaaa caaacccaac tgcaccttgc agacgcactc actgacgcgg

3241 cgataaacta a ill //

LOCUS M.EcoAO83l 3176 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 12) DEFINITION .

FEATURES Location/Qualifiers misc_feature 1..1470

/label="M"

CDS 1..1470

/label="Translation 1-1470" misc_feature 1470..3176

/label="S"

CDS 1470..3176

/label="Translation 1470-3176"

ORIGIN

1 atgtctatca gttcagtaat caaatcatta caagatatta tgcgcaaaga tgccggtgtg

61 gacggcgatg cgcagcgtct cggtcagctc tcctggctgc tgtttttgaa aatcttcgat

121 gcccaggaag aggcgctgga actggagcag gataactatc aatatccgat cccacagcgt

181 tatttatggc gcagttgggc cgcaaacgct cagggcatta ccggtgattc cctgctggaa

241 ttcgttaatg atgatctgtt cccggcgttg aaaaacctca ctgcgcctat cgataaaaac

301 ccacgcggct acgtggtaaa gcaggcgttc agcgatgcct ataactacat gaaaaacggt

361 acgctactgc gccaggtgat caacaagctg aacgaaattg actttaccag cgccagcgaa

421 cgccatctgt ttggtgatat ttacgaacag atccttaaag atctgcaatc tgcgggcaat

481 gcgggcgaat tctatactcc acgcgccgtc actcgcttta tggtggatcg cattgatccg

541 aaactcggcg aatccattat ggacccggcc tgcggtacgg gcggttttct tgcctgcgcg

601 ttcgatcatg taaagaacaa atacgtgaag agcgtcgccg atcatcagac gctgcaacaa

661 cagatccacg gtgtcgagaa aaaacagctt ccgcacctgc tggcgaccac caatatgctg

721 ctgcacggca ttgaagtgcc tgtacaaatt cgtcacgaca acaccctgaa caaaccgctt

781 tcctcctggg atgagcaact ggatgtcatt gttaccaacc cgccgtttgg tggcacggaa

841 gaagacggta ttgagaagaa ctttccggca gagatgcaaa cccgcgaaac ggcggatctg

901 tttctgcaac tgattgtgga agtactggcg aaaaacggtc gcgcggcggt ggtattgccg

961 gatggcacgc tatttggcga aggcgttaaa accaaaatca aaaagctgct taccgaagag

1021 tgcaatctgc ataccatcgt gcgtttaccg aatggtgtgt ttaaccccta taccggcatt

1081 aaaaccaacc tgttgttttt taccaaaggt cagccaacca aagagatttg gttctatgag

1141 catccgtatc cggcgggcgt gaaaaattac agtaaaacca agccgatgaa gttcgaagag

1201 tttcaggcgg agattgactg gtggggtaac gaggccgatg gttttgccag ccgcatcgag 1261 aatgagcaag cgtggaaagt cagcattgat gacgtgattg cgcgtaactt caatctggat

1321 attaaaaacc cacatcaggc ggaaaccgtc agccatgacc cggacgaact gttagcgcag

1381 tatgcaaaac agcaggcgga gatccagacg ctgcgtaatc aactgcgcga tattcttggc

1441 gctgcgctgt ctggcaagga ggttaactaa tgaatgtgga aaagctgatc gttgatcata

1501 tggaaacctg gacctcggcg ttgcaaaccc gttccaccgc cgggcgcggc agttccggaa

1561 aaattgattt atatggcatt aagaaattac gtgagctgat tctggaactg gctgtgcgcg

1621 gtaaactggt gccgcaggat ccgaatgatg aaccggcgtc ggagctgctg aagcgtattg

1681 cggcggaaaa agccgagctg gtgaaacagg ggaaaattaa aaagcaaaaa ccactgccgg

1741 aaattagcga ggaagagaag ctatttgaat tgccggaggg atgggagtgg gtgcgatttg

1801 gtaatatcta tgaaatggag tacggtaata atttaccaca agaaaaaaga tctaattctg

1861 gtgaatataa tgtttatggc tccaatggag ttgtaggtac acacaatgaa gcatgtatta

1921 aatcaccatg tattattatt ggcagaaaag gttccgcagg agcattaaat ctttcaaatc

1981 agccagcctg ttgggtaaca gacgtagctt atagcactat acccccaatt gctatggttc

2041 ttgaatttgt ctttatacaa tttcatacat tgggcctcga taaattaggg aagggaatta

2101 agccaggctt gaatcgtaac gatgcttata gtttagtaat tgctattcca cctcgaagcg

2161 aacagaaagc catagtgtcg aaagtaaatg aattaatgtc cctatgcgac caactggaac

2221 agcaatcctt gaccagtctg gacgcacatc agcaactggt tgaaaccctg ttgggaacac

2281 ttgcagacag ccagaacgcc gaggaactgg ctgaaaactg ggcgcgtatt agcgagcatt

2341 tcgacacact ttttaccacc gaagccagcg tggatgcgtt aaaacagacc attctgcaac

2401 tggccgtaat gggtaaactt gtgccgcagg atccgaatga cgaacccgcc tctgaactgc

2461 tcaaacgaat tgcgcaggaa aaagctcaac tggtgaaaga agggaaaata aaaaaacaaa

2521 aaccgttgct gccaattagc gatgaggaaa aaccgtttga actgcctaat ggatgggaat

2581 ggtgcaggct cggagagctt attgattcca ttgatgctgg gtggagtcct gcatgttctt

2641 ctgaaccagc cgctccagga gaatgggggg tgttaaaaac gaccgcagtt caatctcttg

2701 aatatagaga gtacgaaaac aaggcgctcc ctaaaaataa agccccaagg ccacaactag

2761 aagtaaaagc aggagatatt cttataacaa gagctggtcc taaaaatcgg gttggtattt

2821 catgccttgt tgaaaacaca agagaaaatt taatgatttc agataaaata atcagatttc

2881 atttgatatc agaggatatc tccgagaaat acatttcctt atgtctaaac tatgggttta

2941 cttcaacata tcttgaaaac tccaaatctg gcatggcaga aagtcagatg aatatttcac

3001 aagatatttt aaaaatggca cctatagcta tcccgactac acatgaacaa ttaaaaataa

3061 cagataaaat aaatgaaatg atggattatt ttataactct aaagagtcaa attcaatctg

3121 cccaacaaac ccaactgcac cttgcagacg cactgactaa cgcggcgata aactaa LOCUS M.EcoMIII 2875 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 13)

DEFINITION .

FEATURES Location/Qualifiers misc_feature 1..1638

/label="M"

CDS 1..1638

/label="Translation 1-1638" misc_feature 1628..2875

/label="S"

CDS 1628..2875

/label="Translation 1628-2875"

ORIGIN

1 atggtagaac tggaatttag agacaaaacc aaagccctga tcgacagcct gaaaagcatc

61 tgtgcgaact acggcctggg caacgacggt aacgaattca aaattatcac tcaggcattc

121 ctgtacaaat tcctgaacga caaattcgct tttgaggcca aacagaaaga taaaagtatc

181 gccagcgccg aaagctggga agatgcgctc agcgcaatga gcgaagatca actgaaaaag

241 ctgcaacagc ggatggcccc ggataccgcc cgcctgaagc cgcaccattt tattcgttac

301 ttgtataacc gacaaaacgc agcggacttt gccagaactt ttgacgacac cctgatggac

361 atcgccgcca ccaacaacga cgtcttcgcg gtcaaaaccg acggcggcgc gaaagtggtg

421 ctgtttgagc gcctcagcca gtacatcgcc gacgaatcaa agcgcgatga tttctgccgc

481 gccatcatca acaagctggc cgatttcagc ttcgaacgca tcttcacgca gaaattcgac

541 ttctacgcca ccatctttga atacctgatc aaagactaca acagcaactc cggcggcaaa

601 tacgcggaat actacacgcc ccacgccgtg gcgcgcatca tggcggaaat cctggtgccg

661 aaagcgcagc agggcgtggt gcgcaacgta agctgttacg acccgtccgc cggttccggc

721 acgctgctga tgaacgtggc gcacgccatt ggggaagatc gttgcagcat ctttgcccag

781 gatatctcgc agaaatcctc cagcctgctg cgtctgaacc tgatcctcaa caacctggtg

841 cattcgatcc ctaacgttat ccagggcaac actattctgc atccgttcca caaggacggc

901 ggcgcgctga aacgctttga ctacatcgtc tccaacccgc cgtttaagat ggatttcagc

961 gactttcgtg acgcgctgga cagcaaggaa aaccagcaac gtttctttgc cgggataccg

1021 aaaatcaaag ccaaagccag ggacaaaatg gagatttacc agttgttcct gcaacacatc

1081 attttctcgc tgaaaccggg tggcaaggcg gcggtcgtcg tgccgaccgg gtttatcacc

1141 gcgcaatcgg gcattgataa aggcattcgc gaacatctgg tgcaaaacaa gatgctggcg

1201 ggcgtggttt ccatgccgtc gaatatcttc gccaccaccg gtaccaacgt gtcgatcctg

1261 tttattgacg ccagcaacaa agagaaagtg gtgctgatcg acgcctcgaa tttaggtgaa

1321 aaggtcaaag acggcaaaaa ccagaaaacg gtgctgacgg agtgcgaaga aaaacggatc

1381 tgcgaagcgt ttaacaacaa gtggagcgaa gaggatttct cggtggtggt cagctatgac 1441 gacatcgccg cgaaaaatta ctcgttcagc gccggacagt attttgatgt gaaaatcgaa

1501 tacaccgaca tgacgccgga acagtttgcc gcgaagatga aaggatttac ggagaatctg

1561 aataatctct ttgaacagtc gcgcgagtta gaagtgcaaa taaaaatgca gatggcagat

1621 atcaaatatg aaatttaatt atctgaaaat taaagatgta tgtgattttg tcggaggaag

1681 tcaacctcca aaatctcaat ttatttatgt atctaaacct ggatatgtca ggttgatcca

1741 gacgcgtgat tacaaaacgg atgcatttcc cacgtatata ccaatatcgt cgacgaagaa

1801 attttgtgat gaattcgata ttatgattgg taggtatggt cccccaattt ttcaaatatg

1861 tcgcggattg aaaggagcat ataacgttgc cttgctgaag gttataccta aagaaggtgt

1921 aagtcgtgat tttttatact attttttgaa gcaagactct gttttccaat atgttgataa

1981 attatctgcc agaaccggag gacaaactgg agttgatcta gtttctttaa aagaatatcc

2041 agtcaggata cctgaagaaa ttgaatgtca agaaaaattg gtgactattc ttagtgttat

2101 tgataaaaaa atcgccctca acaaccgcat caacacggaa ctggaagcga tggcgaaaac

2161 actgtatgac tactggtttg tacagtttga tttccctgat gccaacggaa aaccgtataa

2221 aacctccggc gggaagatgg agtataacgc cacgctgaaa cgggaaattc cggcaggttg

2281 gaatgactcc attcttggta agtttattga gttagatcgt ggcgttactt atagcaaaga

2341 ggatgttcga actcaggatg ataaagatac gataggcatt cttcgcgcta caaatgtgac

2401 gggaaataat gttgatattg atgatttagt cttcattcct tctagtagag taaacgttaa

2461 tcaaatgcta aataaatttg acatattgat cgtgatgtca agtggtagta aggagcatgt

2521 agggaagaat ggcgtgtatt atttcgaaaa aaaacatgca tttggcgcat tttgttcaaa

2581 aattacaccc gtcagaaaat acagatattt tataaataca ttcctgcaat cgaaatggtt

2641 taagtcttat ataaacaatc agtgcttggg aactaatatt aataacctga ccaatacgca

2701 tatcactaat tgtgaaatca tatgccccac tcctgatgtt gtggcgttat tcgagaataa

2761 aatgatgcca atttataata aactggcatc aaacactcaa gagaatagtc atttaatcca

2821 actccgcgac tggctcctcc ctctacttat gaacggacag gtcacggtca aataa

//

LOCUS M.ECO1167 3794 bp ds-DNA linear 09-NOV-2021 (SEQ ID NO: 14)

DEFINITION .

FEATURES Location/Qualifiers misc_feature 1..2439

/label="M"

CDS 1..2439

/label="Translation 1-2439" misc_feature 2451..3794

/label="S" CDS 2451..3794

/label="Translation 2451-3794"

ORIGIN

1 atggctatca aaaaaaccga actctattct tccctgtggg caagctgtga cgaactgcgt

61 ggcgggatgg atgccagcca gtacaaagac tatgtactga ccctgctgtt tatgaagtac

121 gtttccgata agtataaagg cgatccttac gggatgatcg tgatacccaa aggagccagc

181 tttgacgata tggttgcgct gaaaaatgac aaagagatcg gcgacaaaat caacaaaatc

241 atccacaagc tggcggaaga gaacggcctg aagggcgtca tcgacgaagc cgactttaac

301 gacgaagata agctcggcaa aggcaaggaa atgattgacc gcctgagcaa gcttgtcggt

361 atctttgaag ggttaaatct ctccagcaac cgggccgaag gcgacgatct gctgggcgat

421 gcctatgaat atctgatgcg tcacttcgca accgaatccg gtaagagtaa aggacagttt

481 tacactccag cggaggtgtc acgtattctg gcgaaggtga ttggtattac tcccgatact

541 ccgcaagatg caacggttta tgacccaacc tgtggctctg gttcgctgct gttgaaagtg

601 aatgatgaag cccggcgcgg actgtcgatt tttggccagg agatggataa cgctaccagt

661 gcgctggcgc ggatgaacat gattctgcat aacaatgcca ccgcgaaaat ctggcagggc

721 aatacgctga gcgatccgca gtggaaagag gcgaacggca aactgaaagc cttcgatttt

781 gccgtggcga acccgccttt ttccaataaa aactggacca acgggctgac gccgaaaaaa

841 gatccgtttg aacgcttcgg ctggggtatt ccgccggaaa aaaacggcga ttatgctttt

901 ttgttgcata tcattaaaag cctgaaaagt accggcaaag gcgcggtgat cctgccccac

961 ggcgtactgt ttcgcggtaa cgccgaagcg aatatccgtg aaaacctaat caagcagggc

1021 tatatcaaag gcgttattgg tttgccagcc aacctgtttt acggcaccgg catcccggcg

1081 tgcatcattg tgattgataa agagcacgcc cacagccgca aaggcatttt tatgatcgac

1141 gccagccggg gctttatcaa agatggcaac aagaaccgcc tgcgtagccg agatattcac

1201 cgcattgtgg acgtgttcaa ccatcagcga acggtacctg gctacagccg gatggtgccc

1261 tccagcgaga tcgccggaaa tgattacaac ctgaatattc ctcgctatat cgagagcggc

1321 gagccggaag atttacacga tctgaccgcg cacttacagg gcggtattcc ggcacgcgat

1381 gtggatgctt tgcaagatta ctggcgggta ttccctgccc tgcgcaacgt gctgtttgct

1441 gacgaccgtc ccggttactg tcgggcacag gttaacgcgc aacaggtgaa accgatcatt

1501 ctggcgcatc aggagtttaa ggattttgcc acccgcagcc tgctaccgtt taaagcgtgg

1561 gtgaaagagg caagtctgga agagattcgc aaaggtgata aacccaaagc gctaatccac

1621 gatatcagcg aaatgctatt ggcgcagtac gccaacagtg agttgctgaa caaatacagc

1681 gtgtaccaga tcctgatgga ttactggact gatgtcatgc aagacgatgt gtatgcgatt

1741 atgcaggatg gctggcaagc cgccgcgcaa atccgcgagc ttcagccggt gaaaggcaaa

1801 gatggcaaaa acatctggaa agaaacgcac gatttcgagt ttaccaaacg gcgctacaag

1861 gccgatgtgc tgccccgttc gctagtggaa attcgctgtt tcccggaact gctggatgcg

1921 ctgaaccgcg cgcaggagcg cagtgaagaa gccagtcgtc agctagtgga attcatcgag 1981 gagcaggcag gggaagagga tctgctggct gaggcgaaaa acgataaaga taaggtgacg

2041 caaaagctgg tgaatgcccg tctggcacag ttgaagaaaa cccctgccga cccggatgag

2101 ctggcagtgc tgaaccgctg tctggcgctg attaaggccg aggccagtgc gaaaaaagcg

2161 ctgaaagccg cacaggaagc gttggatcga gcggtattta aacattatcc cacgctggat

2221 gaagcagcga ttaaaacgct ggtagtgcag gataagtggc tggcaacgtt gcaggcaggg

2281 atcaaagcgg agatagagcg tattacccag caactggcgt cgcgcgtaaa ggagctggaa

2341 gagcgttatg ctgaaccgtt gccagcactt gaagccgccg ttgaggcgtt gagcgagaaa

2401 gtggccgggc atttgcgggc gatggggctg gagtggtgat ggtggattta atggtgaatg

2461 agcaacggtc gggtacggtg gggaaaggtg tgcctgcggg gtataagctg actgagatgg

2521 agatgattcc tgaggattgg gttgtatcca cgatattaaa cctgacaacc aacataattg

2581 attatcgcgg cagaactccc aaaaaacttg gaatggattg gggggatggg gatatcgttg

2641 ccctttctgc tgctaatgtt aaaaaaggat atatagattt atctactgaa tgctactttg

2701 gttccgaaga actatataag cgctggatga catccggtca tccccaaaaa ggggacatag

2761 ctttcacaat ggaagcccct ctggggaatg cagcatctat tcctgataac agaaaatata

2821 ttcttagcca gagaacaatt cttttacaaa tagatagaga gaatttttct ccatcactaa

2881 tactccaaat cttattatca gagcgttttc aatcatatat ttctgaaagc gcgactggtt

2941 caacggccca agggattaaa cgttcagttt tagagaagtt atacatatct ataccgaaaa

3001 gtatcgtaga gcaaaaggct attgctaatg tattaaccaa tgttgatagc ttgattctat

3061 ccctagaaaa attactctca aaaaaacagt ccatcaaaac tgccacaatg cagcaactac

3121 tgactggtaa aacccgtttg ccacagtttg cgttgcgcaa agatggctct gctaagggat

3181 ataaaaagag cgagttgggg gagattcctg aagattggaa ttgtattaat cttggtgagc

3241 taggtaattg cattattggg ttaacttaca aacctgaaga tacatctgat tatggtacat

3301 tggtattacg ctcatccaac attcaaaata atcacttagc ttatgatgat aacgtcttcg

3361 tcagcataga accacctgag cgcacaattg tgaaagaagg tgatatcttg gtatgtgtaa

3421 gaaatggcag tcgccaactt attggcaaat gcgcattaat tgacaaaaat gcgcagggct

3481 cagcattcgg tgcatttatg tcagtattcc gaactgatat atatggattt acatttcacc

3541 aattccagtc tgatattatt caaaagcaaa tcgctgaagt tatgggtgcc actatcaatc

3601 agataactaa caaagatatg ttgggcttta aaatcccatt acctgaaaat acagaagaac

3661 aagccgccat cgccgccatc ctctccgata tggacaagga aatccaaatc ctgcaacagc

3721 gtctggacaa aacccgccag cttaagcaag ggatgatgca ggaactgtta accgggaaaa

3781 tccgcttaat atag Appendix V : GenBank DNA sequence file of pl065

LOCUS pl065 9705 bp ds-DNA circular 01-NOV-2021 (SEQ ID NO: 15)

DEFINITION .

KEYWORDS "Plasmid_plO65:P2 test construct for methylation protection" "Resistance:Spc"

FEATURES Location/Qualifiers misc_feature 1..92

/label="Promoter"

CDS 93..881

/label="aadA" terminator 1066..1153

/label="rrnBT2 term" misc_feature 1164..1197

/label="J23100" misc_feature 1205..1218

/label="rbsJ6110" misc_feature 1225..1941

/label="sGFP" misc_feature 1946..1997

/label="terminator BBa_K864600" misc_feature 2936..2949

/label="Eco0015" misc_feature 2936..4322

/label="Test Block" misc_feature 2971..2984

/label="Ecoll67" misc_feature 3010..3020

/label="EcoMIH" misc_feature 3053..3063

/label="EcoMH" misc_feature 3103..3115

/label="EcoKI" misc_feature 3149..3162 /label="EcoAI" misc_feature 3179..3193 /label="EcoAO83l" misc_feature 4038..4052 /label="EcoAO83l" misc_feature 4083..4096 /label="EcoAI" misc_feature 4141..4153 /label="EcoKI" misc_feature 4186..4196 /label- 'EcoMH" misc_feature 4226..4236 /label- 'EcoMIII" misc_feature 4265..4278 /label="Ecoll67" misc_feature 4309..4322 /label="Eco0015" terminator 5716..5787

/label="rrnB T1 terminator"

CDS complement(5814..6692) /label="araC" promoter 6719..7003

/label="araBAD promoter" misc_feature 7004..8559 /label="P4" cds 7039..7535

/label="delta (P2 activation)" cds 7609..8181

/label="psu" misc_feature 8246..8472

/label="Essential P4 packaging signal" misc_feature 8920..9658

/label="CloDF13 ori" ORIGIN

1 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat 61 aaatgcttca ataatattga aaaaggaaga gtatgaggga agcggtgatc gccgaagtat

121 cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg

181 ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt

241 tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc

301 ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca

361 ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg

421 gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg

481 atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg

541 cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct

601 taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt

661 tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg

721 actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg

781 cttatcttgg acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg

841 tccactacgt gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta acaattcgtt

901 caagccgagg ggccgcaaga tccggccacg atgacccggt cgtcggttca gggcagggta

961 ccaggcacgc ctaaccgtca gtgagattgg atgagtgaac gatattgatc gagaagagcc

1021 ctgcgcagcc gctgccgtgc ctgcaggaag caacggcccg gagggtggcg ggcaggacgc

1081 ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt

1141 gcgtttctac aaactctgct agcttgacgg ctagctcagt cctaggtaca gtgctagctc

1201 tagagaaaga ggcgaattac tagtatgcgt aaaggcgaag aactgttcac gggcgtagtt

1261 ccgattctgg tcgagctgga cggcgatgtg aacggtcata agtttagcgt tcgcggtgaa

1321 ggtgagggcg acgcgaccaa cggcaaactg accctgaagt tcatctgcac caccggtaaa

1381 ctgccggtgc cttggccgac cttggtgacg acgttgacgt atggcgtgca gtgttttgcg

1441 cgttatccgg accacatgaa acaacacgat ttcttcaaat ctgcgatgcc ggagggttac

1501 gtccaggagc gtaccatttc cttcaaggat gatggctact acaaaactcg cgcagaggtt

1561 aagtttgaag gtgacacgct ggtcaatcgt atcgaattga agggtatcga ctttaaagag

1621 gatggtaaca ttctgggcca taaactggag tataacttca acagccataa tgtttacatt

1681 acggcagaca agcaaaagaa cggcatcaag gccaatttca agattcgcca caatgttgag

1741 gacggtagcg tccaactggc cgaccattac cagcagaaca ccccaattgg tgacggtccg

1801 gttttgctgc cggataatca ctatctgagc acccaaagcg tgctgagcaa agatccgaac

1861 gaaaaacgtg atcacatggt cctgctggaa tttgtgaccg ctgcgggcat cacccacggt

1921 atggacgagc tgtataagta atgagttgtt cagaacgctc ggtcttgcac accgggcgtt

1981 ttttctttgt gagtccaaat tacagctttc aaaagatact tacgatgatg atttagataa

2041 tttattggcg caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc

2101 agatgctatt ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct

2161 atcagcttca atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc 2221 tttagttcga caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa

2281 cggatatgca ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa

2341 accaatttta gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga

2401 tttgctgcgc aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg

2461 tgagctgcat gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg

2521 tgagaagatt gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg

2581 tggcaatagt cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa

2641 ttttgaagaa gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa

2701 ctttgataaa aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta

2761 ttttacggtt tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc

2821 agcatttctt tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg

2881 aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttccatc

2941 gattgcttct taccgcgtgc gagatagcca ttcatgaatc gttcagcctg cagtcaggtc

3001 tattagttta aaggtcagtt gtaacgttga tgcccctaag aacctctcgg tggtaccatt

3061 gtgcgacgca agcgattaca ctcctgtcac atcataatcg ttaacacgtc agtgcgtttg

3121 ctattcaggg cttgaccaac actggattga gataactagt catggtaaac actccacagg

3181 aattcttaca tgcttgatgc tacggcgatt cttggagagc cagcagcgac tgcaaatgtg

3241 agatcagagt aatattagca agcgataagt ccctaactgg ttgtggcctt ttgtagagtg

3301 aacttcataa catatgctgt ctcaggcacg tggatggttt ggacaaatca gattcaagtc

3361 tgatcaacct tcatacagat ctagagtcta aagcagtgat ctcaaaatac taggtaacta

3421 gagggactgc gacgttctaa acgttggtcc gtcagaagcg ccatccagga tcacgttacc

3481 ccgaaaaaaa gatatcagga gctctcctcc tcagaaacta caggactaac cttcctggca

3541 accgggaggt gggaatccgt cacatatgag aaggtatttg cccgataatc aatactgcct

3601 taagccggct tgccctttct gcctgtagat ccattggact ggtgccaacg cgcaggcata

3661 gttcgaggag aattatccgg gggcaatgac aaccagcatc tcgggtcttg cccaacccgt

3721 ctacacgctc cttaactctg gcaggcaatt aaagggaacg tatatataac gcaaagaagc

3781 tggaaaattg gcgagagaat cttctctctg tctatcgaag aatggccacg cggtggcaac

3841 cgtcatgcta gcgtgcgggg tacacttgct aaccatttgg gacacgggac actcgctgtt

3901 ttcgaaatta ccctttatgc gcgggtattg aaccacgctt atgcccagca tcgttacaag

3961 cagactcata ctagatgtat tatgcccgcc atgcagacga aaccagtcgg agattaccga

4021 gcattctatc acgtcgggga attcttacat gccgaccata agtgagctac tggagccgag

4081 gggagataac tagtcagctt ttcacttaaa gtattatgca cgacagggtg cgtgtacgtt

4141 aacacgtcag tgccatgtaa acctgttata acttacctca gttaggtacc attgtgttgg

4201 aagtgtggct agatcttagc tttttaaagg tcagttttcc ggtgccacgc gatggaacga

4261 gccattcatg aatcgttcta ttgttatagc gaatcagcgg gaacgcaacc atcgattgct

4321 tctttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc 4381 aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa

4441 tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg

4501 ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat

4561 tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag

4621 aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt

4681 tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc

4741 gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt

4801 tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt

4861 aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct

4921 ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa

4981 ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca

5041 aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag

5101 tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata

5161 taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac

5221 gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa

5281 acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg

5341 tctttatgaa acacgcattg atttgagtca gctaggaggt gactgaccgg ctgataaatt

5401 tctttgaatt tctccttgat tatttgttat aaaagttata aaataatctt gttggaacca

5461 ttcaaaacag catagcaagt taaaataagg ctagtccgtt atcaacttga aaaagtggca

5521 ccgagtcggt gctttttttg atacttctat tctactctga ctgcaaacca aaaaaacaag

5581 cgctttcaaa acgcttgttt tatcattttt agggaaatta atctcttaat ccttttatgc

5641 gatcgcgggc catcgccctg atagactata aacgcagaaa ggcccacccg aaggtgagcc

5701 agtgtgactc tagtagagag cgttcaccga caaacaacag ataaaacgaa aggcccagtc

5761 tttcgactga gcctttcgtt ttatttgatg cctggagatc cttactcgag ttattatgac

5821 aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg

5881 gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg

5941 cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata

6001 cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac

6061 agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct

6121 gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg

6181 agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc

6241 agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg

6301 ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac

6361 ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac

6421 cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa

6481 atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga 6541 atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga

6601 taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc

6661 ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa

6721 gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc

6781 tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa

6841 gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat

6901 tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag attagcggat

6961 cctacctgac gctttttatc gcaactctct actgtttctc cataattttc ttaacctgaa

7021 gtgacgagga gccggaaatt tactgtccgt cgtgtggaca tgttgctcac acccgtcgcg

7081 cacatttcat ggacgatggc accaagataa tgattgcaca gtgccggaat atttattgct

7141 ctgcgacatt tgaagcgagt gaaagctttt tctctgacag taaagattca ggaatggaat

7201 acatttcagg caaacagaga taccgcgatt cactgacgtc agcctcctgc ggtatgaaac

7261 gcccgaaaag aatgcttgtt accggatatt gttgtcggag atgtaaaggc cttgcactgt

7321 caagaacatc gcggcgtctg tctcaggaag tcaccgagcg tttttatgtg tgcacggatc

7381 cgggctgtgg tctggtgttt aaaacgcttc agaccatcaa ccgcttcatt gtccgcccgg

7441 tcacgccgga cgaactggca gaacgcctgc atgaaaaaca ggaactgccg ccagtacggt

7501 taaaaacaca atcatattcg ctgcgtctgg aatgagggct gccggttaac accggccgtc

7561 gccgcacacc gtatttttat tcttcagcat gatgagaaag agataacgat ggaaagcaca

7621 gccttacagc aggcctttga cacctgtcag aataacaaag cagcatggct gcaacgcaaa

7681 aatgagctgg cagcggccga acaggaatat ctgcggcttc tgtcaggaga aggcagaaac

7741 gtcagtcgcc tggacgaatt acgcaatatt atcgaagtca gaaaatggca ggtgaatcag

7801 gccgccggtc gttatattcg ttcgcatgaa gccgttcagc acatcagcat ccgcgaccgg

7861 ctgaatgatt ttatgcagca gcacggcaca gcactggcgg ccgcactggc accggagctg

7921 atgggctaca gtgagctgac ggccattgcc cgaaactgtg ccatacagcg tgccacagat

7981 gccctgcgtg aagcccttct gtcctggctt gcgaagggtg aaaaaattaa ttattccgca

8041 caggatagcg acattttaac gaccatcgga ttcaggcctg acgtggcttc ggtggatgac

8101 agccgtgaaa aattcacccc tgcgcagaac atgatttttt cgcgtaaaag tgcgcaactg

8161 gcatcacgtc agtcagtgta aaattccccg aaaatccgcc cgtttttact gaaaaaagcc

8221 atgcatcgat aaggtgcatg gctttgcatg cgttttcctg cctcattttc tgcaaaccgc

8281 gccattcccg gcgcggtctg agcgtgtcag tgcaactgca ttaaaaccgc cccgcaaagc

8341 gggcgggcga ggcggggaaa gcaccgcgcg caaaccgaca agttagttaa ttatttgtgt

8401 agtcaaagtg ccttcagtac atacctcgtt aatacattgg agcataatga agaaaatcta

8461 tggcctatgg tccaaaactg tcttttttga tggcactatc ctgaaaaata tgcaaaaaat

8521 agattgatgt aaggtggttc ttgtcagtgt cgcaagatcg ggccccattc tacattttta

8581 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcagcgc

8641 gggtgtggtt gtgcttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt 8701 atcaacttga aaaagtggca ccgagtcggt gctttttttg gcgctgccat cttggcggcc

8761 taaggcgatg ccccctcgac ctcgatcagg gaggcgttca ggacgactca caaagaaagc

8821 cgggcaatgc ccggcttttt ccacgcctcc tgggctgact tcaggtgcta catttgaaga

8881 gataaattgc actgaaatct agagcggttc agtagaaaag atcaaaggat cttcttgaga

8941 tccttttttt ctgcgcgtaa tcttttgccc tgtaaacgaa aaaaccacct ggggaggtgg

9001 tttgatcgaa ggttaagtca gttggggaac tgcttaaccg tggtaactgg atttcgcaga

9061 gcacagcaac caaatctgtc cttccagtgt agccggactt tggcgcacac ttcaagagca

9121 accgcgtgtt tagctaaaca aatcctctgc gaactcccag ttaccaatgg ctgctgccag

9181 tggcgtttta ccgtgctttt ccgggttgga ctcaagtgaa cagttaccgg ataaggcgca

9241 gcagtcgggc tgaacgggga gttcttgctt acagcccagc ttggagcgaa cgacctacac

9301 cgagccgaga taccagtgtg tgagctatga gaaagcgcca cacttcccgt aagggagaaa

9361 ggcggaacag gtatccggta aacggcaggg tcggaacagg agagcgcaag agggagcgac

9421 ccgccggaaa cggtggggat ctttaagtcc tgtcgggttt cgcccgtact gtcagattca

9481 tggttgagcc tcacggctcc cacagatgca ccggaaaagc gtctgtttat gtgaactctg

9541 gcaggagggc ggagcctatg gaaaaacgcc accggcgcgg ccctgctgtt ttgcctcaca

9601 tgttagtccc ctgcttatcc acggaatctg tgggtaactt tgtatgtgtc cgcagcgccc

9661 gccgcagtct cacgcccgga gcgtagcgac cgagtgagct agcta

//

References

1. Antoine, R and Locht, C (1992) Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from Gram-positive organisms. Molecular Microbiology 6(13): 1785-1799.

2. Kroger, C., Dillon, S.C., Cameron, A.D.S., Papenfort, K. et al. (2012) The transcriptional landscape and small RNAs of Salmonella enterica serovar Typhimurium. Proceedings National Academy of Sciences USA 109(2): E1277-E1286.

3. Uribe, R.V., van der Helm, E., Misiakou, M., Lee, S., Koi, S. and Sommer M.O.A. (2019) Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla. Cell Host and Microbe 25: 233-241.

4. Harms, A., Liesch, M., Kbmer, J., Quebatte, M., Engel, P. amd Dehio, C. (2017) A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effects of Bartonella. PLOS Genetics 13(10): el007077.

Claims

1 A carrier bacterial cell comprising a conjugative plasmid, the plasmid encoding a methyltransferase enzyme.

2 A carrier bacterial cell according to claim Iwherein the carrier bacterial cell is selected from a Bacteriodes, Escherichia, Clostridium, Caulobacter, Lactobacillus, Pseudomonas, Bifidobacterium, Salmonella, Listeria, Proteus or Streptococcus cell.

3 A carrier bacterial call according to claim 1 or 2, wherein the plasmid comprises an OriT and optionally one or more or all of the tra genes necessary for bacterial conjugation.

4 A method for delivery of DNA from a carrier bacterial cell to a target bacteria or an archeal cell by conjugation, the method comprising contacting the target bacterial species with the carrier bacterial cell according to any one of claims 1-3, wherein the target bacteria comprises a restriction modification system that cleaves DNA which is unmethylated at a target site of methylation of the methyltransferase enzyme.

5 A bacteriophage encoding a methyltransferase enzyme, optionally wherein the methyltransferase enzyme protects against a restriction modification system found in a target bacterial which is pathogenic in humans or animals, and/or in a target bacteria which is associated with biofouling or microbial corrosion.

6 A mixture of at least 2 bacteriophage of claim 5, each bacteriophage encoding a different methyltransferase enzyme.

7 A bacteriophage or mixture according to claim 5 or 6 respectively wherein the bacteriophage is from a genus selected from Dhakavirus, Gaprivervirus, Gelderlandvirus, Jiaodavirus, Karamvirus, Krischvirus, Moonvirus, Mosigvirus, Schizotequatrovirus, Slopekvirus and Tequatrovirus. Each phage herein may be an enterobacteria phage, E coli phage, Myoviridae phage, Tevenvirinae phage, Tequatrovirus phage, Caudovirales phage, adeno- associated viruses (AAV), herpes simplex viruses, retroviruses or lentiviruses, or may be a Klebsiella phage (e.g., Klebsiella phage PMBT1, Klebsiella phage PKO111, Klebsiella phage phi KpNIH-6, Klebsiella phage Miro, Klebsiella phage vB_KpnM_KpV477, Klebsiella phage KPV15, Klebsiella phage vB_Kpn_F48, Klebsiella phage KPN5, Klebsiella phage KP27, Klebsiella phage KPI 5, Klebsiella phage KPI or Klebsiella phage JD18), Acinetobacter phage (e.g., Acinetobacter virus 133), Aeromonas phage (e.g., Aeromonas virus 65 or Aeromonas virus Aehl), Escherichia phage (e.g., Escherichia virus RB16, Escherichia virus RB32 or Escherichia virus RB43) or Pseudomonas phage (e.g., Pseudomonas virus 42) or may be a Tevenvirinae phage, e.g., a phage selected from Table D.

8 A method for delivery of bacteriophage DNA to a target bacteria, the method comprising contacting the target bacterial species with a bacteriophage encoding a methyltransferase enzyme, wherein the target bacteria comprises a restriction modification system that cleaves DNA which is unmethylated at a target site of methylation of the methyltransferase enzyme.

9 A method for methylation of a bacteriophage DNA or plasmid or phagemid DNA, the method comprising: introducing into a production bacterium (i) a polynucleotide encoding a plurality of methyltransferases, or (ii) polynucleotides which together encode a plurality of methyltransferases, thereby producing a modified production bacterium expressing at least 2 introduced methyltransferases; infecting the modified production bacterium with a bacteriophage particle comprising bacteriophage DNA or phagemid DNA or plasmid DNA , thereby methylating said bacteriophage DNA or phagemid or plasmid DNA; and producing a bacteriophage particle comprising bacteriophage DNA or phagemid DNA or plasmid DNA having a modified methylation pattern, optionally purifying the bacteriophage particle comprising bacteriophage DNA or phagemid DNA or plasmid DNA.

10 A method according to claim 9 wherein the bacteriophage DNA or plasmid DNA or phagemid DNA produced encodes a methyltransferase.

11 A method, carrier bacterial cell, bacteriophage, mixture, plasmid or phagemid according to or disclosed in any one of claims 1-10 wherein

(i) the MTase is a type I, II or III MTase; or

(ii) The MTase is selected from Eco0015, EcoKl, EcoAI, M.EcoAO83I, M.EcoMII, M.EcoMIII, Ml M2 Eco3 II, and M.Ecol 167; or

(iii) The MTase methylates within a recognition sequence selected from

GAGNNNNNNNGTCA

GGANNNNNNNNATGC

AACNNNNNNGTGC

RTACNNNNGTG

AAAGNNNNGTT

GGTCTC

CCANNNNNNNCTTC

YTCANNNNNNGTTY; or

(iv) the phage, phagemid, or plasmid e.g. carrier bacterial cell conjugative plasmid DNA encodes multiple MTases, for example including a type I, and a type II MTase, or a type I and a type III MTase, or a type II and III MTase, or all of type I, II and III MTases, or wherein the production bacteria encodes multiple MTases which are not native to the production bacteria, such as a type I, and a type II MTase, or a type I and a type III MTase, or a type II and III MTase, or all of type I, II and III MTase. 12 A method, carrier bacterial cell mixture, bacteriophage, phagemid or plasmid according to or as disclosed in any one of claims 1-11, wherein the carrier bacterial cell plasmid, the bacteriophage or phagemid comprises a target bacteria-modifying CRISPR array to modify a target sequence in the target bacteria.

13 A method, carrier bacterial cell, mixture, bacteriophage, phagemid or plasmid according to claim 12 wherein the plasmid, bacteriophage or phagemid additionally encodes:

(i) a cas endonuclease, and/ or

(ii) a tracrRNA sequence or a DNA sequence expressing a tracrRNA sequence.

14 A pharmaceutical composition comprising a carrier bacterial cell or bacteriophage, mixture, phagemid or plasmid according to or disclosed in any one of claims 1-13 in combination with a pharmaceutically acceptable carrier or excipient.

15 A method, carrier bacterial cell bacteriophage, mixture, phagemid or plasmid according to or disclosed in any one of claims 4-8 and 11-14 when dependent on claims 4-8, wherein the target bacteria is pathogenic in a human or animal.

16 A method of prevention or treatment of disease caused by a pathogenic target bacteria in a human or animal, the method comprising delivery of a bacteriophage, mixture, phagemid, plasmid or carrier bacterial cell according to or disclosed in any one of claims 1-13 or a pharmaceutical composition according to claim 14 to a human or animal in need thereof.

17 A bacteriophage, mixture, phagemid, plasmid or carrier bacterial cell according to or disclosed in claims 1-13 or a pharmaceutical composition according to claim 14, for use in a method of prevention or treatment of disease caused by a pathogenic target bacteria in a human or animal, the method comprising delivery of the bacteriophage, phagemid or carrier bacterial cell or pharmaceutical composition to the human or animal in need thereof.

18 A method according to claim 16 or a bacteriophage, phagemid, mixture, plasmid or carrier bacterial cell for use according to claim 17 wherein the pathogenic bacteria is selected from any bacteria listed in Table 1 of WO 2017/211753A1, which list is specifically herein incorporated by reference, or may be Salmonella, Clostridium, Bifidobacterium, Lactobacillus, Escherichia, Pseudomonas, Caulobacter, Listeria, Proteus and Streptococcus.

19 A method of killing of a target bacteria or the targeted modification of the genome of a target bacteria, the method comprising delivery of a bacteriophage, phagemid, plasmid or carrier bacterial cell according to or disclosed in claims 1-13, or a pharmaceutical composition according to claim 14, to the target bacteria, wherein the target bacteria is comprised within in a naturally occurring population and associated with microbial corrosion or biofouling of a substrate or fluid in an industrial or domestic system. 20 A method of killing of a target bacteria or targeted modification of the genome of a target bacteria according to claim 19 wherein the target bacteria is selected from a Bacillus (e.g., thuringiensis), an E. coli, a Salmonella, a Listeria, a Clostridium (e.g., botulinum), a sulphate- reducing bacterium, e.g., a Desulfovibrio, a Lactobacillus, a Legionella, Streptococcus, a Lactococcus or a Listeria.

21 A method for targeted modification of a target bacterial species or strain, the method comprising contacting a target bacteria with a carrier bacterial cell, plasmid, bacteriophage, mixture or phagemid of claim 12 , such that the target sequence of the target bacterial species is modified by CRISPR mediated DNA modification.

22 A method or method for use according to any one of claims 16-18 and 21 wherein the target bacteria is comprised within the microbiome of a human or animal.

129