CA3137804A1 - Phage and transduction particles - Google Patents

Abstract

The invention relates to the production of phage and transduction particles using DNAs (eg, plasmids and helper phage, mobile genetic elements (MGEs) or plasmids with chromosomally integrated helper phage genes), as well as the phage, helper phage, kits, compositions and methods involving these. The particles are particularly useful for delivering toxic payloads into target bacteria for antibacterial action. Embodiments enable production of highly pure compositions of such particles for medical or environmental use and for containment of the particles, which may be useful for containing antibacterial action.

Description

PHAGE AND TRANSDUCTION PARTICLES
TECHNICAL FIELD
[0001] The invention relates to the production of phage and transduction particles comprising phage proteins using DNAs (eg, plasmids and helper phage, or plasmids with chromosomally integrated helper phage genes), as well as the phage, helper phage, kits, compositions and methods involving these. The particles are particularly useful for delivering toxic payloads into target bacteria for antibacterial action. Embodiments enable production of highly pure compositions of such particles for medical or environmental use and for containment of the particles, which may be useful for containing antibacterial action, controlling dosing or reducing the risk of acquisition of undesirable foreign genes.
BACKGROUND

[0002] The use of helper phage to package phagemid DNA into phage virus particles is known. An example is the M13K07 helper phage, a derivative of M13, used in E coli host cells. Other examples are R408 and CM13.
SUMMARY OF THE INVENTION

[0003] The invention relates to tbe production of phage and transduction particles and provides:-

[0004] In a First Configuration A kit comprising a) A first DNA; and b) One or more second DNAs;
Wherein (i) the DNAs together comprise all phage structural protein genes required to produce a packaged phage particle comprising a copy of the first DNA;
(ii) the first DNA comprises none or at least one, but not all, of the genes; and wherein the one or more second DNAs comprise the remainder of the genes;
(iii) the first DNA comprises a phage packaging signal for producing the packaged phage particle;
and (iv) the second DNA is devoid of a nucleotide sequence (eg, a packaging signal) required for packaging the second DNA into phage particles;
wherein the DNAs are operable when co-existing in a host bacterium for producing packaged phage that comprise the first DNA, wherein the phage require the second DNA for replicaton thereof to produce further phage particles.

There is also provided A method of producing phage, the method comprising expressing in a cell comprising the DNAs the phage protein genes, wherein packaged phage are produced that comprise the first DNA, wherein the phage require the second DNA for replicaton thereof to produce further phage particles.

[0005] In a Seond Configuration A population of helper phage, wherein the helper phage are capable of packaging first phage, wherein the first phage are different from the helper phage and the helper phage are incapable of self-replication.

[0006] In a third Configuration A composition comprising a population of first phage, wherein the first phage require helper phage according to the First Configuration for replication; and wherein less than [20%] of total phage comprised by the composition are such helper phage.

[0007] In a Fourth Configuration

[0008] A method of producing first phage, wherein the first phage require helper phage to replicate, the method comprising (a) Providing DNA comprising a packaging signal;
(b) Introducing the DNA into a host bacterial cell;
(c) Wherein the host bacterial cell comprises helper phage or wherein helper phage are introduced into the bacterial cell simultaneously or sequentially with step (b);
(d) Wherein the helper phage are according to the invention;
(e) Causing or allowing the helper phage to produce phage proteins, wherein the packaging signal is recognised in the host cell, whereby first phage are produced using the proteins, the first phage packaging the DNA;
(f) Wherein helper phage replication in the host cell is inhibited or reduced, thereby limiting the availability of helper phage;
(g) Optionally lysing the host cell and obtaining the first phage;
(h) Thereby producing a composition comprising first phage which require the helper phage for replication, wherein propagation of first phage is prevented or reduced by the limitation of helper phage availability.

[0009] In a Fifth Configuration A phage production system, for producing phage (eg, the first phage of any preceding claim) comprising a nucleotide sequence of interest (NSI-phage), the system comprising components (i) to (iii):-(i) A first DNA;

(ii) A second DNA; and (iii) a NSI-phage production factor (NPF) or an expressible nucleotide sequence that encodes a NPF;
Wherein a) The first DNA encodes a helper phage (eg, said first helper phage recited in any preceding claim);
b) The second DNA comprises the nucleotide sequence of interest (NSI);
c) When the system is comprised by a bacterial host cell, helper phage proteins are expressed from the first DNA to form phage that package the second DNA in the presence of the NPF, thereby producing NSI-phage;
d) The system is devoid of a helper phage production factor (HPF) that is required for forming phage that package the first DNA, or is devoid of an expressible nucleotide sequence that encodes a functional HPF; or the system comprises a nucleotide sequence that comprises or encodes a functional HPF, the system further comprising means for targeted inactivation in the host cell of the HPF
sequence to eliminate or minimise production of helper phage comprising the first DNA; and Whereby the system is capable of producing a product comprising a population of NSI-phage, wherein each NSI-phage requires a said helper phage for propagation, wherein the NSI-phage in the product are not mixed with helper phage or less than [20%] of total phage comprised by the product are said helper phage.
The invention also provides:_

[0010] A composition for use in antibacterial treatment of bacteria, the composition comprising an engineered mobile genetic element (MGE) that is capable of being mobilised in a first bacterial host cell of a first species or strain, the cell comprising a first phage genome, wherein in the cell the MGE
is mobilised using proteins encoded by the phage and replication of first is inhibited, wherein the MGE encodes an antibacterial agent or encodes a component of such an agent.

[0011] A nucleic acid vector comprising the MGE integrated therein, wherein the vector is capable of transferring the MGE or a copy thereof into a host bacterial cell.

[0012] A non-self replicative transduction particle comprising said MGE or vector of the invention.

[0013] A composition comprising a plurality of transduction particles, wherein each particle comprises a MGE or vector according to the invention, wherein the transduction particles are capable of transferring the MGEs, or nucleic acid encoding the agent or component, or copies thereof into target bacterial cells, wherein (i)target cells are killed by the antibacterial agent;
(ii) growth or proliferation of target cells is reduced; or (iii)target cells are sensitised to an antibiotic, whereby the antibiotic is toxic to the cells.

[0014] A composition comprising a plurality of non-self replicative transduction particles, wherein each particle comprises a MGE or plasmid according to the invention, wherein the transduction particles are capable of transferring the MGEs, or nucleic acid encoding the agent or component, or copies thereof into target bacterial cells, wherein the agent is a CRISPR/Cas system and the component comprises a nucleic acid encoding a crRNA or a guide RNA that is operable with a Cas in a target bacterial cell to guide the Cas to a target nucleic acid sequence of the cell to modify the sequence, whereby (i)target cells are killed by the antibacterial agent;
(ii)growth or proliferation of target cells is reduced; or (iii)target cells are sensitised to an antibiotic, whereby the antibiotic is toxic to the cells.

[0015] A method of producing a plurality of transduction particles, the method comprising combining the composition of the invention with host bacterial cells of said first species, wherein the cells comprise the first phage, allowing a plurality of said MGEs to be introduced into host cells and culturing the host cells under conditions in which first phage-encoded proteins are expressed and MGE copies are packaged by first phage proteins to produce a plurality of transduction particles, and optionally separating the transduction particles from cells and obtaining a plurality of transduction particles separated from cells.

[0016] A bacterial host cell comprising a first phage and a MGE, vector or particle of the invention, wherein the agent is toxic to cells of the same species as the host cell, and wherein the host cell has been engineered so that the agent is not toxic to the host cell.

[0017] A bacterial host cell comprising a first phage, wherein the cell is comprised by a kit, the kit further comprising a composition of the invention, wherein the agent is toxic to cells of the same species as the host cell, and wherein the host cell has been engineered so that the agent is not toxic to the host cell.

[0018] A bacterial host cell comprising a first phage and a MGE, vector or particle of the invention, wherein the agent is not toxic to the host cell, but the agent is toxic to second cells of a species or strain that is different from the species or strain of the host cell, wherein the MGE is mobilizable in transduction particles producible by the host cell that are capable of transferring the MGE or a copy thereof into a said second cell, whereby the second cell is exposed to the antibacterial agent.

[0019] A bacterial host cell comprising a first phage, wherein the cell is comprised by a kit, the kit further comprising a composition of the invention, wherein the agent is not toxic to the host cell, but the agent is toxic to second cells of a species or strain that is different from the species or strain of the host cell, wherein the MGE is mobilizable in transduction particles producible by the host cell that are capable of transferring the MGE or a copy thereof into a said second cell, whereby the second cell is exposed to the antibacterial agent.

[0020] A bacterial host cell comprising a MGE, vector or particle of the invention and nucleic acid under the control of one or more inducible promoters, wherein the nucleic acid encodes all structural proteins necessary to produce a transduction particle that packages a copy of the MGE or plasmid, wherein the agent is not toxic to the host cell, but the agent is toxic to second cells of a species or strain that is different from the species or strain of the host cell, wherein the MGE is mobilizable in transduction particles producible by the host cell that are capable of transferring the MGE or a copy thereof into a said second cell, whereby the second cell is exposed to the antibacterial agent.

[0021] A plasmid comprising (a) A nucleotide sequence encoding an antibacterial agent or component thereof for expression in target bacterial cells;
(b) A constitutive promoter for controlling the expression of the agent or component;
(c) An optional terS nucleotide sequence;
(d) An origin of replication (on); and (e) A phage packaging sequence (optionally pac, cos or a homologue thereof); and the plasmid being devoid of (f) All nucleotide sequences encoding phage structural proteins necessary for the production of a transduction particle (optionally a phage), or the plasmid being devoid of at least one of such sequences; and (g) Optionally terL.

[0022] A bacterial host cell comprising the genome of a helper phage that is incapable of self-replication, optionally wherein the genome is present as a prophage, and a plasmid according to the invention, wherein the helper phage is operable to package copies of the plasmid in transduction particles, wherein the particles are capable of infecting bacterial target cells to which the antibacterial agent is toxic.

[0023] A method of making a plurality of transduction particles, the method comprising culturing a plurality of host cells according to the invention, optionally inducing a lytic cycle of the helper phage, and incubating the cells under conditions wherein transducing particles comprising packaged copies of the plasmid are created, and optionally separating the particles from the cells to obtain a plurality of transduction particles.

[0024] A plurality of transduction particles obtainable by the method of the invention for use in medicine, eg, for treating or preventing an infection of a human or animal subject by target bacterial .. cells, wherein transducing particles are administered to the subject for infecting target cells and killing the cells using the antibacterial agent.

[0025] A method of making a plurality of transduction particles, the method comprising (a) Producing host cells whose genomes comprise nucleic acid encoding structural proteins necessary to produce transduction particles that can package first DNA, wherein the genomes are devoid of a phage packaging signal, wherein the expression of the proteins is under the control of inducible promoter(s);
(b) Producing first DNA encoding an antibacterial agent or a component thereof, wherein the DNA comprises a phage packaging signal;
(c) Introducing the DNA into the host cells;
(d) Inducing production of the structural proteins in host cells, whereby transduction particles are produced that package the DNA;
(e) Optionally isolating a plurality of the transduction particles; and (0 Optionally formulating the particles into a pharmaceutical composition for administration to a .. human or animal for medical use.

[0026] A plurality of transduction particles obtainable by the method.

[0027] A host bacterial cell comprising

28 a) A first DNA; and b) One or more second DNAs;
wherein (i) the DNAs together comprise all genes required to produce a transduction particle comprising a copy of the first DNA packaged by phage structural proteins;
(ii) the first DNA is devoid of at least one functional essential gene (eg, encoding a phage structural protein) required to produce the particle; and wherein the one or more second DNAs comprises said functional essential gene(s);
(iii) the first DNA comprises a phage packaging signal for producing the particle; and (iv) the second DNA is devoid of a nucleotide sequence required for packaging the second DNA
into transduction particles;
wherein the second DNA is required for packaging first DNA to produce particles, wherein the DNAs are operable in the cell for producing transduction particles comprising phage structural proteins that package copies of the first DNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1: The genetic map of P2 genome with non-essential genes boxed;

[0029] Figure 2: Schematic of SaPIbov1;

[0030] Figure 3: (A) Maps of P2 and P4¨ genomic architecture and regulatory network of phage P2 and satellite phage P4; the grey boxed region in P2 was deleted and replaced with a a kanamycin marker sequence, and the box shown in dashed lines shows the region of P4 including the cos site that was included in the CGV; (B) p94 genomic map;

[0031] Figure 4: Schematic view of the SA100 production srain ¨ the defective helper phage will only turn on phage production following induction; since the phage packaging DNA sequence has .. been removed from the helper and placed on the CGVTM, only CGVTM molecules will be packaged into the synthetic phage particles;

[0032] Figure 5: Efficiency of infection ¨ percent infected EMG-2 cells with increasing ratio (MOT) of SA100 particles to EMG-2;

[0033] Figure 6: CGVTM delivery by SA100 ¨ total CFU and CGVTm-containing CFU
after infection of MG1655_pks cells (A) with SA100 containing p114 or p94 or X1-blue_pks cells (B) containing p114;

[0034] Figure 7: Killing of target cells by SA100-delivered CGVTM - killing efficiency of the two target strains by SA100 delivered CGV5TM (2nd, 4th and 6th bars) using wild-type and optimised copy number CGVTM compared to non-infected controls;

[0035] Figure 8: Plotted is the abundance as expressed in colony forming units (CFU) per 5 milliliter faecal sample (n=15). Two groups, CRISPR induced and non-induced are shown. No difference was observed between the groups when the CFU was deteremined on agar plates containing streptomycin;
and

[0036] Figure 9: Plotted is the abundance as expressed in colony forming units (CFU) per 5 milliliter faecal sample (n=15). Two groups, CRISPR induced and non-induced are shown.
The statistical difference is calculated using the Student t-test, showing a statistically significant increase in abundance after 48 hours in the CRISPR non-induced group (p-value=0.011) on agar plates supplemented with spectinomycin and streptomycin. This shows that the CGV
(containing a spectonomycin marker) can be delivered by non-self-replicative particles SA100.

[0037] Figure 10:
1. CRISPR system is inserted into a phage genome containing at least packaging signal and DNA replication module thereby generating a vector, such as a plasmid (we call a CRISPR Guided VectorTM, CGV); the CRISPR system may comprise nucleic acid encoding a Cas (eg, Cas3 or Cas9) and/or one or more crRNAs or gRNAs, optionally also Cascade proteins when the Cas is Cas3;
2. Essential gene(s) are removed or mutated in phage and the function provided by expression in trans from a plasmid (or this may instead be from essential genes integrated in the chromosome of a host bacterial cell (aka the production strain));
3. CGV is replicated and packaged in phage-like transduction particles encoded by functions encoded on CGV, but at least one essential function being expressed in trans from plasmid or chromosome; and 4. Production via propagation cycles on production strain. Propagation on other bacterial strains is advantageously prevented.
DETAILED DESCRIPTION

[0038] The invention relates to tbe production of phage using DNAs (eg, plasmids with helper phage), as well as the phage, helper phage, compositions and methods involving these. The invention finds utility, for example, for containing phage in environments ex vivo and in vivo, reducing the risk of acquisition of antibiotic resistance or other genes by phage, as well as controlling dosing of phage in an environment. The contamination of useful phage populations by helper phage may in examples also be restricted or eliminated, thereby controlling phage propagation and enhancing the proportion of desired phage in phage compositions, such as medicaments, herbicides and other agents where phage may usefully be used. Thus, the invention provides the following embodiments.

[0039] A kit comprising a) A first DNA; and b) One or more second DNAs;
Wherein (i) the DNAs together comprise all phage structural protein genes required to produce a packaged phage particle or a transduction particle comprising a copy of the first DNA;
(ii) the first DNA comprises none or at least one, but not all, of the genes; and wherein the one or more second DNAs comprise the remainder of the genes;
(iii) the first DNA comprises a phage packaging signal for producing the packaged phage particle;
and (iv) the second DNA is devoid of a nucleotide sequence required for packaging the second DNA
into phage particles;
wherein the DNAs are operable when co-existing in a host bacterium for producing packaged phage that comprise the first DNA, wherein the phage require the second DNA for replicaton thereof to produce further phage particles.

[0040] For example the second DNA is devoid of a packaging signal for packaging second DNA.
Additionally or alternatively, the second DNA is devoid of a nucleotide sequence required for replication of helper phage. Optionally, the nucleotide sequence enodes a sigma factor or comprises a sigma factor recognition site, a DNA polymerisation recognition site, or a promoter of a gene required for helper phage DNA replication when the second DNA is comprised by a helper prophage.

[0041] In an example, the second DNA is comprised by an M13 or M13-based helper phage. M13 encodes the following proteins required for phage packaging:-a) pill: host recognition b) pV: coat protein c) pVII, pVIII, pIX: membrane proteins d) pI, pIV, pXI: Channel for translocating the phage to the extracellular space.

[0042] In this example, the second DNA is devoid of one or more of the genes coding for these proteins, eg, is devoid of a gene endoding pill, a gene encoding pV, a gene endoding pVII, a gene endoding pVIII, a gene endoding pIX, a gene endoding pI, a gene endoding pIV
and/or a gene endoding XI.

[0043] In an embodiment, the phage particle of (i) is capable of infecting a target bacterium, the phage comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in the target bacterium, or wherein the NSI comprises a regulatory element that is operable in the target bacterium. In an example, the NSI is capable of recombination with the target cell chromosome or an episome comprised by the target cell to modify the chromosome or episome.
Optionally, this is carried out in a method wherein the chromosome or episome is cut (eg, at a predetermined site using a guided nuclease, such as a Cas, TALEN, zinc finger or meganuclease; or a restriction endonuclease) and simultaneously or sequentially the cell is infected by a phage particle that comprises the first DNA, wherein the DNA is introduced into the cell and the NSI or a sequence thereof is introduced into the chromosome or episome at or adjacent the cut site. In an example the first DNA comprises one or more components of a CRISPR/Cas system operable to perform the cutting (eg, comprising at least a nucleotide sequence encoding a guide RNA or crRNA for targeting the site to be cut) and further comprising the NSI.

[0044] In an embodiment, the presence in the target bacterium of the NSI or its encoded protein or RNA mediates target cell killing, or downregulation of growth or propagation of target cells, or mediates switching off of expression of one or more RNA or proteins encoded by the target cell genome, or downregulation thereof.

[0045] In an embodiment, the presence in the target bacterium of the NSI or its encoded protein or RNA mediates upregulation of growth or propagation of the target cell, or mediates switching on of expression of one or more RNA or proteins encoded by the target cell genome, or upregulation thereof.

[0046] In an embodiment, the NSI encodes a component of a CRISPR/Cas system that is toxic to the target bacterium.

[0047] In an embodiment, the DNA is a first DNA as defined in any preceding paragraph.

[0048] In an embodiment, the first DNA is comprised by a vector (eg, a plasmid or shuttle vector).

[0049] In an embodiment, the second DNA is comprised by a vector (eg, a plasmid or shuttle vector), helper phage (eg, a helper phagemid) or is integrated in the genome of a host bacterial cell.

[0050] An embodiment provides a bacterial cell comprising the first and second DNAs. Optionally, the cell is devoid of a functional CRISPR/Cas system before transfer therein of a first DNA, eg, a first DNA comprising a component of a CRISPR/Cas system that is toxic to the target bacterium. An embodiment provides an antibacterial composition comprising a plurality of cells, wherein each cell is optionally according to this paragraph, for administration to a human or animal subject for medical use.

[0051] A method of producing phage is provided, the method comprising expressing in a host bacterial cell the phage protein genes, wherein packaged phage are produced that comprise the first DNA, wherein the phage require the second DNA for replicaton thereof to produce further phage particles. Optionally, the method comprises isolating the phage particles.

[0052] A composition comprising a population of phage particles obtainable by the method is provided for administration to a human or animal subject for treating an infection of target bacterial cells, wherein the phage are capable of infecting and killing the target cells.

[0053] A method of treating an environment ex vivo, the method comprising exposing the environment to a population of phage particles obtainable by the method is provided, wherein the environment comprises target bacteria and the phage infect and kill the target bacteria. In an example thje subject is further administered an agent simultaneously or sequentially with the phage administration. In an example, the agent is a herbicide, pesticide, insecticide, plant fertilizer or cleaning agent.

[0054] Optionally, the method is for containing the treatment in the environment.

[0055] Optionally, the method is for controlling the dosing of the phage treatment in the environment.

[0056] Optionally, the method is for reducing the risk of acquisition of foreign gene sequence(s) by the phage in the environment.

[0057] A method of treating an infection of target bacteria in a human or animal subject is provided, the method comprising exposing the bacteria to a population of phage particles obtainable by the production method, wherein the phage infect and kill the target bacteria.

[0058] Optionally, the method for treating is for containing the treatment in the subject.

[0059] Optionally, the method for treating is for containing the treatment in the environment in which the subject exists.

[0060] Optionally, the method for treating is for controlling the dosing of the phage treatment in the subject.

[0061] Optionally, the method for treating is for reducing the risk of acquisition of foreign gene sequence(s) by the phage in the subject.

[0062] Optionally, the method for treating is for reducing the risk of acquisition of foreign gene sequence(s) by the phage in the environment in which the subject exists.

[0063] Optionally, target bacteria herein are comprised by a microbiome of the subject, eg, a gut microbiome. Altertnatively, the microbiome is a skin, scalp, hair, eye, ear, oral, throat, lung, blood, rectal, anal, vaginal, scrotal, penile, nasal or tongue microbiome.

[0064] In an example thje subject is further administered a medicament simultaneously or sequentially with the phage administration. In an example, the medicament is an antibiotic, antibody, immune checkpoint inhibitor (eg, an anti-PD-1, anti-PD-Li or anti-CTLA4 antibody), adoptive cell therapy (eg, CAR-T therapy) or a vaccine.

[0065] In an example, the invention employs helper phage for packaging the phage nucleic acid of interest. Thus, the invention provides the following illustrative Aspects:-1. A population of helper phage, wherein the helper phage are capable of packaging first phage nucleic acid to produce first phage particles, wherein the first phage are different from the helper phage and the helper phage are incapable themselves of producing helper phage particles.
2. A composition comprising a population of first phage, wherein the first phage require helper phage according to Aspect 1 for replication of first phage particles; and optionally wherein less than 20, 15, 10, 5, 4, 3,2, 1, 0.5, 0.4, 0.2 or 0.1% of total phage particles comprised by the composition are particles of such helper phage.
In an example the composition comprises helper phage and less than 1% of total phage particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.5% of total phage particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.1% of total phage particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.01% of total phage particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.001% of total phage particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.0001% of total phage particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.00001% of total phage particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.000001% of total phage particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.0000001% of total phage particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.00000001% of total phage particles comprised by the composition are particles of such helper phage.
In an example, the population comprises at least 10,104' 105 or 106 phage particles, as indicated a transduction assay, for example. In an example, the population comprises at least 10 phage particles and eg, no more than 1014 particles. In an example, the population comprises at least 104 phage particles and eg, no more than 1014 particles. In an example, the population comprises at least 105 phage particles and eg, no more than 1014 particles. In an example, the population comprises at least 106 phage particles and eg, no more than 1014 particles. To have a measure of the first phage concentration, for example, one can perform a standard transduction assay when the first phage genome contains an antibiotic marker. Thus, in this case the first phage are capable of infecting target bacteria and in a sample of lml the population comprises at least 103,104' 105 or 106 transducing particles, which can be determined by infecting susceptible bacteria at a multiplicity of infection <0.1 and determining the number of infected cells by plating on a selective agar plate corresponding to the antibiotic marker in vitro at 20 to 37 degrees centigrade, eg, at 20 or 37 degrees centrigrade.
Optionally at least 99.9, 99.8, 99.7, 99.6, 99.5, 99.4, 99.3, 99.2, 99.1, 90, 85, 80, 70, 60, 50 or 40% of total phage particles comprised by the composition are particles of first phage.
In an example, the first phage genome comprises an fl origin of replication.
In an example, the helper phage are E coli phage. In an example, the first phage are E co/i, C dificile, Streptococcus, Klebsiella, Pseudomonas, Acitenobacter, Enterobacteracea, Firmicutes or Bacteroidetes phage. In an example, the helper phage are engineered M13 phage.
In an example, the first phage genome comprises a phagemid, wherein the phagemid comprises a packaging signal for packaging first phage particles in the presence of the helper phage.
The first phage particles may contain a nucleotide sequence of interest (NSI), eg, as defined herein, such as a NSI that encodes a component of a CRISPR/Cas system operable in target bacteria that can be infected by the first phage particles. Once inside the target bacteria, the first phage DNA is incapable of being packaged to form first phage particles in the absence of the helper phage. This usefully contains the activity of the first phage genome and its encoded products (protieins and/or nucleic acid), as well as limits or controls dosing of the NSI and its encoded products in an environment comprising the target bacteria that have been exposed to the first phage. This is useful, for example to control the medical treatment of an environment comprised by a human or animal subject, plant or other environment (eg, soil or a foodstiff or food ingredient).
3. The helper phage or composition of any preceding Aspect, wherein the genome of each first phage is devoid of genes encoding first phage structural proteins.
4. The composition of Aspect 2 or 3, wherein the composition comprises helper phage DNA.
5. The composition of Aspect 4, wherein the DNA comprises helper DNA
fragments.
6. The helper phage or composition of any one preceding Aspect, wherein the helper phage are in the form of prophage.

Thus, the prophage is integrated in the chromosome of a host cell.
Examples of phage structural proteins are phage coat proteins, collar proteins and phage tail fibre proteins.
7. The composition of any one of Aspects 2 or 3, wherein the composition comprises no helper phage DNA comprising a sequence of 20 contiguous nucleotides or more (eg, from 20 to 25, 30, 35, 40, 50 or 100 nucleotides), eg, no helper phage DNA.
This can be determined, for example, using DNA probes (designed on the basis of the known heper phge genome sequence) with PCR, as is conventional. In an example, the composition may comprise residual helper prophage DNA, but essentially otherwise is devoid of helper DNA.
8. The composition of any one of Aspects 2 to 5 and 7, wherein the helper phage are capable of infecting host bacteria and the composition does not comprise host bacteria.
9. The composition of any one of Aspects 2 to 8, wherein the composition is a lysate of host bacterial cells, wherein the lysate comprises helper prophage DNA, eg, such DNA comprises 20 contiguous nucleotides or more (eg, from 20 to 25, 30, 35, 40, 50 or 100 nucleotides) of helper phage DNA.
10. The composition of any one of Aspects 2 to 8, wherein the composition is a lysate of host bacterial cells, wherein the lysate has been processed (eg, filtered) to remove all or some helper phage DNA; or the composition is a lysate of host bacterial cells that is devoid of cellular material.
11. The composition of any one of Aspects 2 to 10, wherein the composition does not comprise helper phage particles.
12. The composition of any one of Aspects 2 to 11, wherein at least 95%
(eg, 100%) of phage particles comprised by the composition are first phage particles.
In another embodiment, the composition comprises second phage particles, wherein the second phage .. are different from the first phage and are not helper phage.
13. The composition of any one of Aspects 2 to 12, wherein the population comprises at least 103, 104, 105 or 106 phage particles, as indicated in a transduction assay.
14. The helper phage or composition of any preceding Aspect, wherein the first phage are capable of replicating in host bacteria in the presence of the helper phage (eg, helper prophage), wherein the first phage comprise antibacterial means for killing target bacteria of a first strain or species, wherein the target bacteria are of a different strain or species and the antibacterial means is not operable to kill the target bacteria.
15. A composition comprising a population of phage, the population comprising (a) A first sub-population of first phage that require a helper phage for packaging the first phage;
(b) A second sub-population of phage comprising the helper phage, wherein the helper phage are as recited in any preceding Aspect.
16. The helper phage or composition of any preceding Aspect, wherein the helper phage are phagemids.
17. A composition comprising (a) A population of helper phage as recited in any preceding Aspect; and (b) A population of nucleic acid vectors comprising vector DNA that comprises a first phage packaging signal;
(c) wherein the helper phage are capable of packaging the vector DNA to produce first phage.
18. The composition of Aspect 17, wherein the vectors are phage.
19. The composition of Aspect 17, wherein the vectors are plasmids or phagemids.
20. The composition of Aspect 19, the vectors are shuttle vectors (eg, pUC
vectors) that can be replicated in first bacteria, wherein the vectors can further be replicated and packaged into first phage in second bacteria (host bacteria) in the presence of the helper phage, wherein the first bacteria are of a strain or species that is different to the strain or species of the host bacteria.
21. The composition of Aspect 20, wherein the first phage are capable of infecting third bacteria .. of a strain or species that is different to the second (and optionally also the first) bacteria.
22. The composition of any one of Aspects 17 to 21, wherein the first phage are capable of replicating in host bacteria in the presence of the helper phage (eg, helper prophage), wherein the first phage comprise antibacterial means for killing target bacteria of a first strain or species, wherein the host bacteria are of a different strain or species and the antibacterial means is not operable to kill the host bacteria.
23. The helper phage or composition of any preceding Aspect, wherein the genome is devoid of a packaging signal (eg, SEQ ID NO: 2 below), wherein the helper phage are incapable of self-replication.
24. The helper phage or composition of Aspect 23, wherein the signal is a pac or cos sequence.

25. The helper phage or composition of any preceding Aspect, wherein the helper phage genome is capable of replication in a host cell.
Thus, the genome is capable of nucleic acid replication but not packaging of helper phage.
26. The helper phage or composition of any one of Aspects 1 to 24, wherein the genome is devoid of a nucleotide sequence required for production of helper phage particles.
27. The helper phage or composition of Aspect 26, wherein the nucleotide sequence enodes a sigma factor (eg, sigma-70) or comprises a sigma factor recognition site, a DNA polymerisation recognition site, or a promoter of a gene required for helper phage DNA
replication.
28. The helper phage or composition of any preceding Aspect, wherein the helper phage are temperate phage.
29. The helper phage or composition of any one of Aspects 1 to 27, wherein the helper phage are lytic phage.
30. The helper phage or composition of any preceding Aspect, wherein the first phage are capable of infecting target bacteria, the first phage comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA (eg, gRNA or crRNA) in target bacteria, or wherein the NSI
comprises a regulatory element that is operable in target bacteria.
31. The helper phage or composition of Aspect 30, wherein the presence in target bacteria of the NSI or its encoded protein or RNA mediates target cell killing, or downregulation of growth or propagation of target cells, or mediates switching off of expression of one or more RNA or proteins encoded by the target cell genomes, or downregulation thereof.
32. The helper phage or composition of Aspect 30, wherein the presence in target bacteria of the NSI or its encoded protein or RNA mediates upregulation of growth or propagation of target cells, or mediates switching on of expression of one or more RNA or proteins encoded by the target cell genomes, or upregulation thereof.
33. An antibacterial composition according to any one of Aspects 2 to 32, wherein the first phage are capable of infecting target bacteria and each first phage comprises engineered antibacterial means for killing target bacteria.
By use of the term "engineered" it will be readily apparent to the skilled addressee that the relevant means has been introduced and is not naturally-occurring in the phage. For example, the means is recombinant, artificial or synthetic.

34. The composition of Aspect 14, 22 or 33, wherein the antibacterial means comprises one or more components of a CRISPR/Cas system.
35. The composition of caim 34, wherein the component(s) comprise (i) a DNA
sequence encoding a guide RNA (eg, a single guide RNA) or comprising a CRISPR array for producing guide RNA, wherein the guide RNA is capable of targeting the genome of target bacteria; (ii) a Cas nuclease-encoding DNA sequence; and/or (iii) a DNA sequence encoding one or more components of Cascade.
In an example, a Cas herein is a Cas9. In an example, a Cas herein is a Cas3.
The Cas may be identical to a Cas encoded by the target bacteria.
36. The composition of any one of Aspects 14, 22 or 33 to 35, wherein the antibacterial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease or meganuclease.
37. The helper phage or composition of any preceding Aspect, wherein the helper phage is for use in medicine practised on a human or animal subject, or the composition is a pharmaceutical composition for use in medicine practised on a human or animal subject.
In an example, the animak is a livestock or companion pet animal (eg, a cow, pig, goat, sheep, horse, dog, cat or rabbit). In an example, the animal is an insect (an insect at any stage of its lifecycle, eg, egg, larva or pupa). In an example, the animal is a protozoan. In an example, the animal is a cephalopod.
38. The composition of any one of Aspects 2 to 36, wherein the composition is a herbicide, pesticide, food or beverage processing agent, food or beverage additive, petrochemical or fuel processing agent, water purifying agent, cosmetic additive, detergent additive or environmental (eg, soil) additive or cleaning agent.
39. The helper phage or composition of any one of Aspects 1 to 37 for use in a contained method of treating a disease or condition of a human or animal subject, wherein the disease or condition is mediated by the target bacteria and the target bacteria are comprised by the subject, the method comprising administering the composition to the subject, whereby the target bacteria are exposed to the antibacterial means and killed and propagation of the first phage is contained.
The inability of the first phage to self-replicate and to require helper phage or second DNA to do this usefully provides containment in the location (eg, gut) of action of the composition and/or in the environment of the subject, eg, when exposed to secretions such as urine and faeces of the subject that otherwise may contain replicated first phage. Inability of the helper phage or second DNA to self-package limits availability of factors required by the first phage to form packaged particles, hence providing containment by limiting first phage propagation. This may be useful, for example, to contain an antibacterial acitivity provided by the first phage, such as a CRISPR/Cas killing principle.
40. A bacterial cell or a plurality of bacterial cells comprising the helper phage or composition of any preceding Aspect, wherein the first phage are capable of replication in the presence of the helper phage in the cell.
The cell may, for example, act as a carrier for the genome of the first phage, wherein the first phage DNA is capable of horizontal transfer from the carrier to the target bacteria once the carrier bacteria have been administered to an environment to be treated, eg, a soil or a human gut or other environment described herein. In an example, the environment is comprised by a human or animal subject and the carrier are commensal or probiotic in the subject. For example the carrier bacteria are Lactobacillus (eg, L reuteri or L lactis), E coli or Streptococcus (eg, S
thermophilus) bacteria. The horizontal transfer can be transfer of a plasmid (such as a conjugative plasmid) to the target bacteria or first phage infection of the target bacteria, wherein the first phage have been prior packaged in the carrier. The use of a carrier is useful too for oral administration or other routes where the carrier can provide protection for the phage, helper or composition from the acid stomach or other harsh environments in the subject. Furthermore, the carrier can be formulated into a beverage, for example, a probiotic drink, eg, an adapted Yakult (trademark), Actimel (trademark), Kevita (trademark), Activia (trademark), Jarrow (trademark) or similar drink for human consumption.
41. The cell(s) of Aspect 40 for administration to a human or animal subject for medical use, comprising killing target bacteria using first phage, wherein the target bacteria mediate as disease or condition in the subject.
In an example, when the subject is a human, the subject is not an embryo.
42. The cell(s) of Aspect 41, wherein the cell(s) comprises helper phage and is symbiotic or probiotic in the subject.

43. A method of killing target bacteria in an environment, optionally wherein the method is not practised on a human or animal body, wherein the method comprises exposing the environment to the cell(s) according to Aspect 42, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65, wherein the environment is or has been exposed to first phage or said vectors to produce first phage in the presence of the helper phage, wherein the first phage are capable of replication in the environment and kill target bacteria.
44. The cell(s) or method of any one of Aspects 40 to 43, wherein the cell is an E coli, Lactobacillus (eg, L lactis or retueri) or Streptococcus (eg, thermophilus) cell.
45. The cell(s) or method of Aspects 40 to 44 wherein the subject is administered or has been administered a cell comprising first phage.
46. The composition of any one of Aspects 2 to 45 in combination with a target bacterial cell wherein the first phage are capable of infecting the target bacterial cell.
47. Use of the helper phage, composition or cell(s) of any one of Aspects 1 to 42 and 44 to 46, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65, in the manufacture of an antibacterial agent that kills target bacteria, for containment of the antibacterial in an environment, eg, containment ex vivo; or containment in a human or animal subject comprising the environment.
48. Use of the helper phage, composition or cell(s) of any one of Aspects 1 to 42 and 44 to 46, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65, in the manufacture of an antibacterial agent that kills the target bacteria, for reducing the risk of acquisition by the first phage of foreign genes.
For example, this is useful for reducing the risk of antibiotic resistance genes by the phage, such as when the phage are in the presence of other phage or plasmids in the environment.
49. Use of the helper phage, composition or cell(s) of any one of Aspects 1 to 42 and 44 to 46, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65, in the manufacture of an antibacterial agent that kills the target bacteria, for reducing the risk of acquisition by the first phage of one or more antibiotic resistance genes.
50. A method of reducing the risk of acquisition by first phage of foreign genes, the method comprising (a) Providing the composition of any one of Aspects 2 to 42 and 44 to 46, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65; and (b) Exposing target bacteria to the composition, wherein the first phage infect the target bacteria;

(c) wherein the helper phage are incapable of self-replication and propagation of first phage is thereby limited, wherein propagation of first phage is prevented or reduced, thereby reducing the risk of acquisition of first phage of foreign genes (eg, antibiotic resistance genes).
51. A method of containing an antibacterial activity in an environment (e.g., ex vivo), the method comprising (a) Providing an antibacterial composition according to any one of Aspects 2 to 42 and 44 to 46, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65; and (b) Exposing target bacteria in the environment to the composition, wherein the bacteria are exposed to the first phage and antibacterial means and are killed;
(c) wherein the helper phage are incapable of self-replication and propagation of first phage is thereby limited, wherein propagation of first phage is prevented or reduced, thereby containing the antibacterial activity.
52. A method of controlling the dosing of first phage in an environment (e.g., ex vivo), the method comprising (a) Providing an antibacterial composition according to any one of Aspects 2 to 42 and 44 to 46, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65; and (b) Exposing target bacteria in the environment to the composition, wherein the bacteria are infected by first phage;
(c) wherein the helper phage are incapable of self-replication and propagation of first phage is thereby limited, wherein propagation of first phage is prevented or reduced, thereby controlling dosing of first phage in the environment.
53. The method of any one of Aspects 43 to 45, 51 and 52, or the use of Aspect 47, wherein the environment is a human or animal microbiome, e.g., a gut microbiome.
54. The method of any one of Aspects 43 to 45, 51 and 52, or the use of Aspect 47, wherein the environment is a microbiome of soil; a plant, part of a part (e.g., a leaf, fruit, vegetable or flower) or plant product (e.g., pulp); water; a waterway; a fluid; a foodstuff or ingredient thereof; a beverage or ingredient thereof; a medical device; a cosmetic; a detergent; blood; a bodily fluid; a medical apparatus; an industrial apparatus; an oil rig; a petrochemical processing, storage or transport apparatus; a vehicle or a container.
55. The method of any one of Aspects 43 to 45, 51 and 52, or the use of Aspect 47, wherein the environment is an ex vivo bodily fluid (e.g., urine, blood, blood product, sweat, tears, sputum or spit), bodily solid (e.g., faeces) or tissue of a human or animal subject that has been administered the composition.
56. The method of any one of Aspects 43 to 45, 51 and 52, or the use of Aspect 47, wherein the environment is an in vivo bodily fluid (e.g., urine, blood, blood product, sweat, tears, sputum or spit), bodily solid (e.g., faeces) or tissue of a human or animal subject that has been administered the composition.
57. A method of producing first phage, wherein the first phage require helper phage to replicate, the method comprising (a) Providing DNA comprising a packaging signal;
(b) Introducing the DNA into a host bacterial cell;
(c) Wherein the host bacterial cell comprises helper phage or wherein helper phage are introduced into the bacterial cell simultaneously or sequentially with step (b);
(d) Wherein the helper phage are according to any preceding Aspect;
(e) Causing or allowing the helper phage to produce phage coat proteins, wherein the packaging signal is recognised in the host cell, whereby first phage are produced using the proteins, the first phage packaging the DNA;
(0 Wherein helper phage particle production in the host cell is inhibited or reduced, thereby limiting the availability of helper phage particles;
(g) Optionally lysing the host cell and obtaining the first phage;
(h) Thereby producing a composition comprising first phage which require the helper phage for replication, wherein further production of first phage particles is prevented or reduced by the limitation of helper phage availability in the composition.
In an embodiment, the DNA is comprised by a phagemid or cloning vector (eg, a shuttle vector, eg, a pUC vector).
There may be a modest amount of helper phage DNA replication to enable first phage protein production efficiently, or should replication of helper phage DNA may be eliminated totally eliminated.
58. The method of Aspect 57, wherein in (c) the helper phage are prophage integrated in the bacterial cell chromosome.
59. The method of Aspect 59, wherein (e) comprises inducing replication of helper phage DNA
and/or expression of the proteins, eg, using UV, mitomycin.

60. The method of any one of Aspects 57 to 59, wherein (g) comprises further separating the first phage from cellular material or helper phage DNA.
61. The method of any one of Aspects 57 to 60, wherein the composition comprises a population of first phage particles, wherein the composition does not comprise helper phage DNA and/or particles.
62. The method of any one of Aspects 57 to 61, wherein the DNA of (a) comprises engineered antibacterial means for killing target bacteria.
63. The method of Aspect 62, wherein the antibacterial means comprises one or more components of a CRISPR/Cas system.
64. The method of Aspect 63, wherein the component(s) comprise (i) a DNA
sequence encoding a guide RNA (eg, a single guide RNA) or comprising a CRISPR array for producing guide RNA, wherein the guide RNA is capable of targeting the genome of target bacteria;
(ii) a Cas (eg, Cas9, Cas3, Cpfl, CasX or CasY) nuclease-encoding DNA sequence; and/or (iii) a DNA
sequence encoding one or more components of Cascade (eg, CasA).
65. The method of any one of Aspects 62 to 64, wherein the antibacterial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease or meganuclease.

66. The helper phage, composition or cell(s) of any one of Aspects 1 to 42 and 44 to 46, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65, for antibacterial treatment of target bacteria in a human or animal subject whereby the antibacterial treatment is contained in the subject.

67. The helper phage, composition or cell(s) of any one of Aspects 1 to 42 and 44 to 46, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65, for antibacterial treatment of target bacteria in a gut of a human or animal subject whereby the antibacterial activity in one or more bodily excretions of the subject is reduced.
This is useful as a safety measure to reduce or eliminate first phage activity outside the subject.

68. The helper phage, composition or cell(s) of Aspect 67, wherein the antibacterial activity in one or more bodily excretions of the subject is eliminated.

69. The helper phage, composition or cell(s) of any one of Aspects 1 to 42 and 44 to 46, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65, for controlling the dosing of antibacterial treatment of target bacteria in a human or animal subject, eg, in the gut of the subject.

Usefully, propagation of the first phage is restricted or eliminated, so dosing in the subject can be controlled, or even pre-determined within a narrow expected range. This is useful, for example, for medicaments comprising the first phage or composition, and may be aid approval of such medicines before FDA and simiar authorities.
Alternatively, the dosing is dosing of an environment, such as soil etc disclosed herein, wherein limitation of the first phage or composition activity is also desirable to limit spread of activities in natural and other terrains.

70. The helper phage, composition or cell(s) of any one of Aspects 1 to 42 and 44 to 46, or a composition obtained or obtainable by the method of any one of Aspects 57 to 65, for fixing the dosing of antibacterial treatment of target bacteria in a human or animal subject, eg, in the gut of the subject.

71. A phage production system, for producing phage (eg, the first phage of any preceding Aspect) comprising a nucleotide sequence of interest (NSI-phage), the system comprising components (i) to (iii):-(a) A first DNA;
(b) A second DNA; and (c) a NSI-phage production factor (NPF) or an expressible nucleotide sequence that encodes a NPF;
Wherein (d) The first DNA encodes a helper phage (eg, said first helper phage recited in any preceding Aspect);
(e) The second DNA comprises the nucleotide sequence of interest (NSI);
(0 When the system is comprised by a bacterial host cell, helper phage proteins are expressed from the first DNA to form phage that package the second DNA in the presence of the NPF, thereby producing NSI-phage; and (g) The system is devoid of a helper phage production factor (HPF) that is required for forming helper phage particles that package the first DNA, or is devoid of an expressible nucleotide sequence that encodes a functional HPF; or the system comprises a nucleotide sequence that comprises or encodes a functional HPF, the system further comprising means for targeted inactivation in the host cell of the HPF sequence to eliminate or minimise production of helper phage comprising the first DNA;
Whereby the system is capable of producing a product comprising a population of NSI-phage, wherein each NSI-phage requires a said helper phage for propagation, optionally wherein the NSI-phage in the product are not mixed with helper phage or less than 20% of total phage comprised by the product are said helper phage.
The invention includes within its concept relatively low level of helper phage particle production if there is a residual capability of helper phage to replicate to produce particles, such as for example in the case that a helper phage packaging signal or other HPF nucleotide sequence in the helper phage genome is mutated (eg, by deletion, substitution or addition of nucleotides therein) to knock down the ability to form phage particles. Preferably, there is no production of helper phage particles, such as by deleting all or part of the sequence from the helper phage genome or inactivating the sequence.

72. A method of producing first phage, wherein the first phage require helper phage to replicate, the method comprising (a) Providing in host cells the system of Aspect 71;
(b) Causing or allowing the helper phage proteins to be produced, whereby the second DNA is packaged to produce first phage; and (c) Optionally lysing the host cells and obtaining a composition comprising first phage.

73. The method of Aspect 72, wherein step (c) comprises separating the first phage from cellular material.

74. The method of Aspect 72 or 73, wherein the composition comprises a population of first phage, wherein less than 20, 10, 5, 4, 3, 2, 1, 0.5 or 0.1% of total phage comprised by the composition are helper phage.

75. The method of any one of Aspects 72 to 74, wherein the second DNA
comprises engineered antibacterial means for killing target bacteria.

76. The method of Aspect 75, wherein the antibacterial means comprises one or more components of a CRISPR/Cas system.

77. The method of Aspect 76 wherein the component(s) comprise (i) a DNA
sequence encoding a guide RNA (eg, a single guide RNA) or comprising a CRISPR array for producing guide RNA, wherein the guide RNA is capable of targeting the genome of target bacteria;
(ii) a Cas nuclease-encoding DNA sequence; and/or (iii) a DNA sequence encoding one or more components of Cascade.

78. The method of any one of Aspects 75 to 77, wherein the antibacterial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease or meganuclease

79. The system of method of any one of Aspects 71 to 78, wherein the first phage are capable of infecting target bacteria, the NSI being capable of expressing a protein or RNA in target bacteria, or wherein the NSI comprises a regulatory element that is operable in target bacteria.

80. The system or method of Aspect 79, wherein the presence in target bacteria of the NSI or its encoded protein or RNA mediates target cell killing, or downregulation of growth or propagation of target cells, or mediates switching off of expression of one or more RNA or proteins encoded by the target cell genomes, or downregulation thereof.

81. The system or method of Aspect 79, wherein the presence in target bacteria of the NSI or its encoded protein or RNA mediates upregulation of growth or propagation of target cells, or mediates switching on of expression of one or more RNA or proteins encoded by the target cell genomes, or upregulation thereof.

82. The system of method of any one of Aspects 71 to 81, wherein each of the NPF and HPF is a packaging signal, eg, SEQ ID NO: 2 or a sequence that is at least 70, 80, 90, 95, 96, 97, 98 or 99%
identical thereto, or is a homologue from a different species.

83. The system of method of Aspect 82, wherein each signal is a pac or cos sequence, or is a homologue.

84. The system of method of any one of Aspects 71 to 81, wherein the HPF is a nucleotide sequence required for replication of helper phage.

85. The system of method of any one of Aspects 71 to 81, wherein the HPF
enodes a sigma factor (eg, sigma-70) or comprises a sigma factor recognition site, a DNA
polymerisation recognition site, or a promoter of a gene required for helper phage DNA replication, a helper phage integrase, a helper phage excissionase or a helper phage origin of replication,

86. A composition comprising a population of first phage obtainable by the method of any one of Aspects 72 to 85, wherein the genome of each first phage is devoid of genes encoding phage proteins.

87. The composition of Aspect 86, wherein the first phage comprise antibacterial means as recited in any one of Aspects 75 to 78.

88. The composition of Aspect 87, comprising DNA identical to the first DNA
or fragments thereof.

89. The composition of Aspect 88, wherein the DNA of the composition is identical to the first DNA and is devoid of a helper phage packaging signal.

90. The composition of any one of Aspects 86 to 89 for antibacterial treatment of target bacteria in a human or animal subject whereby the antibacterial treatment is contained in the subject.

91. The composition of any one of Aspects 86 to 89 for antibacterial treatment of target bacteria in a gut of a human or animal subject whereby the antibacterial activity in one or more bodily excretions of the subject is reduced.

92. The composition of Aspect 91, wherein the antibacterial activity in one or more bodily excretions of the subject is eliminated.

93. The composition of any one of Aspects 86 to 89 for controlling the dosing of antibacterial treatment of target bacteria in a human or animal subject, eg, in the gut of the subject.

94. The composition of any one of Aspects 86 to 89 for fixing the dosing of antibacterial treatment of target bacteria in a human or animal subject, eg, in the gut of the subject.

95. An isolated DNA comprising all structural protein genes of a helper phage genome that are required for producing phage particles, wherein the DNA is devoid of a helper phage production factor (HPF) that is required for producing packaged helper phage, optionally wherein the DNA
comprises one or more promoters for expression of the genes when the DNA is integrated in the genone of a host bacterial cell.

96. The DNA of Aspect 95, wherein the DNA is devoid of any phage packaging signals.

97. The DNA of Aspect 95 or 96, wherein the HPF is a sigma factor-encoding nucleotide sequence or comprises a sigma factor recognition site, a DNA polymerisation recognition site, a promoter of a gene required for helper phage DNA replication, a helper phage integrase-encoding nucleotide sequence, a helper phage excissionase-encoding nucleotide sequence or a helper phage origin of replication.

98. The DNA of any one of Aspects 95 to 97, wherein the DNA comprises a nucleotide sequence encoding a CRISPR/Cas system repressor.

99. The DNA of any one of Aspects 95 to 98, wherein the DNA is integrated in the chromosome of a host bacterial cell, wherein the genes are expressible in the host cell.

100. The DNA of Aspect 99, wherein the cell is devoid of an active CRISPR/Cas system.

101. The DNA of any one of Aspects 95 to 100 in combination with a second DNA, wherein the second DNA comprises the HPF.

102. The DNA of any one of Aspects 95 to 100 in combination with a second DNA, wherein the second DNA comprises a phage packaging signal and optionally the first DNA is devoid of a phage packaging signal.

103. The DNA of Aspect 101 or 102, wherein the second DNA is comprised by a phagemid or a plasmid (eg, a shuttle vector).
[0066] In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a medical container, eg, a syringe, vial, IV bag, inhaler, eye dropper or nebulizer. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a sterile container. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a medically-compatible container. In an example, the kit, DNA(s), first first phage, helper phage or composition is comprised by a fermentation vessel, eg, a metal, glass or plastic vessel.
[0067] In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a medicament, e,g in combination with instructions or a packaging label with directions to administer the medicament by oral, IV, subcutaneous, intranasal, intraocular, vaginal, topical, rectal or inhaled administration to a human or animal subject. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by an oral medicament formulation. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by an intranasal or ocular medicament formulation. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a personal hygiene composition (eg, shampoo, soap or deodorant) or cosmetic formulation. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a detergent formulation. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a cleaning formulation, eg, for cleaning a medical or industrial device or apparatatus. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by foodstuff, foodstuff ingredient or foodstuff processing agent. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by beverage, beverage ingredient or beverage processing agent. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a medical bandage, fabric, plaster or swab. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a herbicide or pesticide. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by an insecticide.
[0068] In an example, the first phage is a is a Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae virus. In an example, the helper phage is a is a Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae virus. In an example, the helper phage is a filamentous M13, a Noviridae, a tailed phage (eg, a Myoviridae, Siphoviridae or Podoviridae), or a non-tailed phage (eg, a Tectiviridae).
[0069] In an example, both the first and helper phage are Corticoviridae. In an example, both the first and helper phage are Cystoviridae. In an example, both the first and helper phage are Inoviridae.
In an example, both the first and helper phage are Leviviridae. In an example, both the first and helper phage are Microviridae. In an example, both the first and helper phage are Podoviridae. In an example, both the first and helper phage are Siphoviridae. In an example, both the first and helper phage are Tectiviridae.

[0070] In an example, the CRISPR/Cas component(s) are component(s) of a Type I
CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type II CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type III CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type IV CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type V CRISPR/Cas system. In an example, the CRISPR/Cas component(s) comprise a Cas9-encoding nucleotide sequence (eg, S pyogenes Cas9, S aureus Cas9 or S thermophilus Cas9). In an example, the CRISPR/Cas component(s) comprise a Cas3-encoding nucleotide sequence (eg, E
coli Cas3, C
difficile Cas3 or Salmonella Cas3). In an example, the CRISPR/Cas component(s) comprise a Cpf-encoding nucleotide sequence. In an example, the CRISPR/Cas component(s) comprise a CasX-encoding nucleotide sequence. In an example, the CRISPR/Cas component(s) comprise a CasY-encoding nucleotide sequence.
[0071] In an example, the first DNA, first phage or vector encode a CRISPR/Cas component or protein of interest from a nucleotide sequence comprising a promoter that is operable in the target bacteria.
[0072] In an example, the host bacteria and/or target bacteria are E coli. In an example, the host bacteria and/or target bacteria are C dificile (eg, the vector is a shuttle vector operable in E coli and the host bacteria are C dificile). In an example, the host bacteria and/or target bacteria are Streptococcus, such as S the rmophilus (eg, the vector is a shuttle vector operable in E coli and the host bacteria are Streptococcus). In an example, the host bacteria and/or target bacteria are Pseudomonas, such as P aeruginosa (eg, the vector is a shuttle vector operable in E coli and the host bacteria are P
aeruginosa). In an example, the host bacteria and/or target bacteria are Klebsiella (eg, the vector is a shuttle vector operable in E coli and the host bacteria are Klebsiella). In an example, the host bacteria and/or target bacteria are Salmonella, eg, S typhimurium (eg, the vector is a shuttle vector operable in E coli and the host bacteria are Salmonella).
[0073] Optionally, host and/or target bacteria is a gram negative bacterium (eg, a spirilla or vibrio).
Optionally, host and/or target bacteria is a gram positive bacterium.
Optionally, host and/or target bacteria is a mycoplasma, chlamydiae, spirochete or mycobacterium. Optionally, host and/or target bacteria is a Streptococcus (eg, pyo genes or thermophilus). Optionally, host and/or target bacteria is a Staphylococcus (eg, aureus, eg, MRSA). Optionally, host and/or target bacteria is an E. coli (eg, 0157: H7) host, eg, wherein the Cas is encoded by the vecor or an endogenous host Cas nuclease activity is de-repressed. Optionally, host and/or target bacteria is a Pseudomonas (eg, aeruginosa).
Optionally, host and/or target bacteria is a Vibro (eg, cholerae (eg, 0139) or vulnificus). Optionally, host and/or target bacteria is a Neisseria (eg, gonnorrhoeae or meningitidis).
Optionally, host and/or target bacteria is a Bordetella (eg, pertussis). Optionally, host and/or target bacteria is a Haemophilus (eg, influenzae). Optionally, host and/or target bacteria is a Shigella (eg, dysenteriae). Optionally, host and/or target bacteria is a Brucella (eg, abortus). Optionally, host and/or target bacteria is a Francisella host. Optionally, host and/or target bacteria is a Xanthomonas host. Optionally, host and/or target bacteria is a Agro bacterium host. Optionally, host and/or target bacteria is a Erwinia host. Optionally, host and/or target bacteria is a Legionella (eg, pneumophila). Optionally, host and/or target bacteria is a Listeria (eg, monocytogenes). Optionally, host and/or target bacteria is a Campylobacter (eg, jejuni). Optionally, host and/or target bacteria is a Yersinia (eg, pestis).
Optionally, host and/or target bacteria is a Borelia (eg, burgdorferi).
Optionally, host and/or target bacteria is a Helicobacter (eg, pylori). Optionally, host and/or target bacteria is a Clostridium (eg, dificile or botulinum). Optionally, host and/or target bacteria is a Erlichia (eg, chaffeensis).
Optionally, host and/or target bacteria is a Salmonella (eg, typhi or enterica, eg, serotype typhimurium, eg, DT 104). Optionally, host and/or target bacteria is a Chlamydia (eg, pneumoniae).
Optionally, host and/or target bacteria is a Parachlamydia host. Optionally, host and/or target bacteria is a Cotynebacterium (eg, amycolatum). Optionally, host and/or target bacteria is a Klebsiella (eg, pneumoniae). Optionally, host and/or target bacteria is an Enterococcus (eg, faecalis or faecim, eg, linezolid-resistant). Optionally, host and/or target bacteria is an Acinetobacter (eg, baumannii, eg, multiple drug resistant).
Further examples of target cells and targeting of antibiotic resistance in such cells using the present invention are as follows:-1. Optionally the target bacteria are Staphylococcus aureus cells, eg, resistant to an antibiotic selected from methicillin, vancomycin, linezolid, daptomycin, quinupristin, dalfopristin and teicoplanin.
2. Optionally the target bacteria are Pseudomonas aeuroginosa cells, eg, resistant to an antibiotic selected from cephalosporins (eg, ceftazidime), carbapenems (eg, imipenem or meropenem), fluoroquinolones, aminoglycosides (eg, gentamicin or tobramycin) and colistin.
3. Optionally the target bacteria are Klebsiella (eg, pneumoniae) cells, eg, resistant to carbapenem.
4. Optionally the target bacteria are Streptoccocus (eg, thermophilus, pneumoniae or pyogenes) cells, eg, resistant to an antibiotic selected from erythromycin, clindamycin, beta-lactam, macrolide, amoxicillin, azithromycin and penicillin.
5. Optionally the target bacteria are Salmonella (eg, serotype Typhi) cells, eg, resistant to an antibiotic selected from ceftriaxone, azithromycin and ciprofloxacin.
6. Optionally the target bacteria are Shigella cells, eg, resistant to an antibiotic selected from ciprofloxacin and azithromycin.

7. Optionally the target bacteria are mycobacterium tuberculosis cells, eg, resistant to an antibiotic selected from Resistance to isoniazid (INH), rifampicin (RMP), fluoroquinolone, amikacin, kanamycin and capreomycin and azithromycin.
8. Optionally the target bacteria are Enterococcus cells, eg, resistant to vancomycin.
9. Optionally the target bacteria are Enterobacteriaceae cells, eg, resistant to an antibiotic selected from a cephalosporin and carbapenem.
10. Optionally the target bacteria are E. coli cells, eg, resistant to an antibiotic selected from trimethoprim, itrofurantoin, cefalexin and amoxicillin.
11. Optionally the target bacteria are Clostridium (eg, dificile) cells, eg, resistant to an antibiotic selected from fluoroquinolone antibiotic and carbapenem.
12. Optionally the target bacteria are Neisseria gonnorrhoea cells, eg, resistant to an antibiotic selected from cefixime (eg, an oral cephalosporin), ceftriaxone (an injectable cephalosporin), azithromycin and tetracycline.
13. Optionally the target bacteria are Acinetoebacter baumannii cells, eg, resistant to an antibiotic selected from beta-lactam, meropenem and a carbapenem.
14. Optionally the target bacteria are Campylobacter cells, eg, resistant to an antibiotic selected from ciprofloxacin and azithromycin.
15. Optionally, the target cell(s) produce Beta (13)-lactamase.
16. Optionally, the target cell(s) are bacterial cells that are resistant to an antibiotic recited in any one of examples 1 to 14.
Mobile Genetic Elements, Genomic Islands, Pathogenicity Islands etc.
[0072] Genetic variation of bacteria and archaea can be achieved through mutations, rearrangements and horizontal gene transfers and recombinations. Increasing genome sequence data have demonstrated that, besides the core genes encoding house-keeping functions such as essential metabolic activities, information processing, and bacterial structural and regulatory components, a vast number of accessory genes encoding antimicrobial resistance, toxins, and enzymes that contribute to adaptation and survival under certain environmental conditions are acquired by horizontal gene transfer of mobile genetic elements (MGEs). Mobile genetic elements are a heterogeneous group of molecules that include plasmids, bacteriophages, genomic islands, chromosomal cassettes, pathogenicity islands, and integrative and conjugative elements. Genomic islands are relatively large segments of DNA ranging from 10 to 200 kb often integrated into tRNA
gene clusters flanked by 16-20 bp direct repeats. They are recognized as discrete DNA segments acquired by horizontal gene transfer since they can differ from the rest of the chromosome in terms of GC content (%G+C) and codon usage.

[0073] Pathogenicity islands (PTIs) are a subset of horizontally transferred genetic elements known as genomic islands. There exists a particular family of highly mobile PTIs in Staphylococcus aureus that are induced to excise and replicate by certain resident prophages.
These PTIs are packaged into small headed phage-like particles and are transferred at frequencies commensurate with the plaque-forming titer of the phage. This process is referred to as the SaPI
excision replication-packaging (ERP) cycle, and the high-frequency SaPI transfer is referred to as SaPI-specific transfer (SPST) to distinguish it from classical generalized transduction (CGT). The SaPIs have a highly conserved genetic organization that parallels that of bacteriophages and clearly distinguishes them from all other horizontally acquired genomic islands. The SaPThencoded and SaPIbov2-encoded integrases are used for both excision and integration of the corresponding elements, and it is assumed that the same is true for the other SaPIs. Phage 80a can induce several different SaPIs, including SaPI1, SaPI2, and SaPIbovl, whereas 911 can induce SaPIbovl but neither of the other two SaPIs.
[0074] Reference is made to "Staphylococcal pathogenicity island DNA packaging system involving cos-site packaging and phage-encoded HNH endonucleases", Quiles-Puchalt et al, PNAS April 22, 2014. 111(16) 6016-6021. Staphylococcal pathogenicity islands (SaPIs) are highly mobile and carry and disseminate superantigen and other virulence genes. It was reported that SaPIs hijack the packaging machinery of the phages they victimise, using two unrelated and complementary mechanisms. Phage packaging starts with the recognition in the phage DNA of a specific sequence, termed "pac" or "cos" depending on the phage type. The SaPI strategies involve carriage of the helper phage pac- or cos-like sequences in the SaPI genome, which ensures SaPI
packaging in full-sized phage particles, depending on the helper phage machinery. These strategies interfere with phage reproduction, which ultimately is a critical advantage for the bacterial population by reducing the number of phage particles.
[0075] Staphylococcal pathogenicity islands (SaPIs) are the prototypical members of a widespread family of chromosomally located mobile genetic elements that contribute substantially to intra- and interspecies gene transfer, host adaptation, and virulence. The key feature of their mobility is the induction of SaPI excision and replication by certain helper phages and their efficient encapsidation into phage-like infectious particles. Most SaPIs use the headful packaging mechanism and encode small terminase subunit (TerS) homologs that recognize the SaPI-specific pac site and determine SaPI
packaging specificity. Several of the known SaPIs do not encode a recognizable TerS homolog but are nevertheless packaged efficiently by helper phages and transferred at high frequencies. Quiles-Puchalt et al report that one of the non¨terS-coding SaPIs, SaPIbov5, and found that it uses two different, undescribed packaging strategies. SaPIbov5 is packaged in full-sized phage-like particles either by typical pac-type helper phages, or by cos-type phages¨i.e., it has both pac and cossites and uses the two different phage-coded TerSs. This is an example of SaPI packaging by a cos phage, and in this, it resembles the P4 plasmid of Escherichia coll. Cos-site packaging in Staphylococcus aureus is additionally unique in that it requires the HNH nuclease, carried only by cos phages, in addition to the large terminase subunit, for cos-site cleavage and melting.
[0076] Characterization of several of the phage-inducible SaPIs and their helper phages has established that the pac (or headful) mechanism is used for encapsidation. In keeping with this concept, some SaPIs encode a homolog of TerS, which complexes with the phage-coded large terminase subunit TerL to enable packaging of the SaPI DNA in infectious particles composed of phage proteins. These also contain a morphogenesis (cpm) module that causes the formation of small capsids commensurate with the small SaPI genomes. Among the SaPI sequences first characterized, there were several that did not include either a TerS homolog or a cpm homolog, and the same is true of several subsequently identified SaPIs from bovine sources and for many phage-inducible chromosomal islands from other species. It was assumed, for these several islands, either that they were defective derivatives of elements that originally possessed these genes, or that terS
and cpm genes were present but not recognized by homology.
[0077] Quiles-Puchalt et al observed that an important feature of 4SLT/SaPIbov5 packaging is the requirement for an HNH nuclease, which is encoded next to the OSLT terminase module. Proteins carrying HNH domains are widespread in nature, being present in organisms of all kingdoms. The HNH motif is a degenerate small nucleic acid-binding and cleavage module of about 30-40 aa residues and is bound by a single divalent metal ion. The HNH motif has been found in a variety of enzymes playing important roles in many different cellular processes, including bacterial killing;
DNA repair, replication, and recombination; and processes related to RNA. HNH
endonucleases are present in a number of cos-site bacteriophages of Gram-positive and -negative bacteria, always adjacent to the genes encoding the terminases and other morphogenetic proteins. Quiles-Puchalt et al have demonstrated that the HNH nucleases encoded by 4)12 and the closely related OSLT have nonspecific nuclease activity and are required for the packaging of these phages and of SaPIbov5.
Quiles-Puchalt et al have shown that HNH and TerL are jointly required for cos-site cleavage.
Quiles-Puchalt et al have also observed that only cos phages of Gram-negative as well as of Gram-positive bacteria encode HNH nucleases, consistent with a special requirement for cos-site cleavage as opposed to pac-site cleavage, which generates flush-ended products. The demonstration that HNH
nuclease activity is required for some but not other cos phages suggests that there is a difference between the TerL proteins of the two types of phages¨one able to cut both strands and the other needing a second protein to enable the generation of a double-stranded cut.
[0078] The invention, also involves, in certain configurations the use of mobile genetic elements (MGEs). Thus, there are provided the following Clauses. Any of the other configurations, Aspects, Examples or description of the invention above or elsewhere herein are combinable mutatis mutandis with any of these Clauses:-1. A composition for use in antibacterial treatment of bacteria, the composition comprising an engineered mobile genetic element (MGE) that is capable of being mobilised in a first bacterial host cell of a first species or strain, the cell comprising a first phage genome, wherein in the cell the MGE
is mobilised using proteins encoded by the phage and replication of first is inhibited, wherein the MGE encodes an antibacterial agent or encodes a component of such an agent.
In the alternative, instead of a bacteria, the host cell is a archaeal cell and instead of a phage there is a virus that is capable of infecting the archaeal cell.
In an example, the MGE is capable of integration into the genome of the host cell comprising the genome of a first phage, for example integration in the chromosome of the host cell and/or an episome thereof.
Optionally, the MGE inhibits first phage replication.
In an example, first phage replication is totally inhibited. In an example, it is reduced by at least 50, 60, 70, 80 or 90% compared to replication in the absence of the MGE in host cells. This can be assessed by a standard in vitro plaque assay to determine the relative amount of first phage plaque formation.
Optionally, in the presence of the agent, (i)host cells are killed by the antibacterial agent;
(ii) growth or proliferation of host cells is reduced; and/or (iii)host cells are sensitised to an antibiotic, whereby the antibiotic is toxic to the cells.
2. The composition of Clause 1, wherein the agent is toxic to cells of the same species or strain as the host cell.
3. The composition of Clause 1 or 2, wherein the agent is toxic to cells of a species or strain that is different from the strain or species of the host cell.
4. The composition of Clause 1, wherein the agent is toxic to cells of the same species as the host cell, and wherein the host cell has been engineered so that the agent is not toxic to the host cell.
5. The composition of Clause 4, wherein the agent is a guided nuclease system (optionally a CRISPR/Cas system) and cells of the same species as the host cell comprise a target sequence that is cut by the nuclease, wherein the target sequence has been removed or altered in the host cell whereby the nuclease is not capable of cutting the target sequence.

Viruses undergo lysogenic and lytic cycles in a host cell. If the lysogenic cycle is adopted, the phage chromosome can be integrated into the bacterial chromosome, or it can establish itself as a stable plasmid in the host, where it can remain dormant for long periods of time. If the lysogen is induced, the phage genome is excised from the bacterial chromosome and initiates the lytic cycle, which culminates in lysis of the cell and the release of phage particles. The lytic cycle leads to the production of new phage particles which are released by lysis of the host.
6. The composition of any preceding Clause, wherein the first phage is a temperate phage.
7. The composition of any preceding Clause, wherein the first cell comprises the first phage as a prophage.
8. The composition of any one of Clauses 1 to 5, wherein the first phage is a lytic phage.
9. The composition of any preceding Clause, wherein in the presence of a first phage the mobilisation of the MGE causes host cell lysis.
10. The composition of any preceding Clause, wherein the MGE is capable of being packaged in transduction particles that comprise some, but not all, structural proteins of the first phage.
"Transduction particles" may be phage or smaller than phage and are particles that are capable of transducing nucleic acid encoding the antibiotic or component thereof or other DNA into target bacterial cells.
In some examples, instead of a nucleic acid encoding an antibiotic or component thereof, there is used a nucleic acid sequence of interest (eg, any NSI disclosed herein) that encodes a protein or RNA of interest for expression in the cell into which transduction from a transduction particle can take place.
Examples of structural proteins are phage proteins selected from one, more or all of the major head and tail proteins, the portal protein, tail fibre proteins, and minor tail proteins.
The MGE comprises a packaging signal sequence operable with proteins encoded by the first phage to package the MGE (or at least nucleic acid thereof encoding the agent or one or more components thereof) into transduction particles that are capable of infecting host cells of the same species or strain as the first host cell.
11. The composition of any preceding Clause, wherein mobilisation of the MGE comprises packaging of copies of the MGE or nucleic acid encoding the agent or component into transduction particles that are capable of transferring the copies into target bacterial cells for antibacterial treatment of the target cells.

12. The composition of Clause 10 or 11, wherein the transduction particles are particles of second phage that are capable of infecting cells of said first species or strain.
13. The composition of any one of Clauses 10 to 12, wherein the transduction particles are non-self replicative particles.
A "non-self replicative transduction particle" refers to a particle, (eg, a phage or phage-like particle;
or a particle produced from a genomic island (eg, a SaPI) or a modified version thereof) capable of delivering a nucleic acid molecule encoding an antibacterial agent or component (or any protein or RNA) into a bacterial cell, but does not package its own replicated genome into the transduction particle. In an alternative herein, instead of a phage, there is used or packaged a virus that infects an animal, human, plant or yeast cell. For example, an adenovirus when the cell is a human cell.
14. The composition of any preceding Clause, wherein the MGE is devoid of genes encoding phage structural proteins.
Optionally, the MGE is devoid of one or more phage genes rinA, terS and terL.
In an example, in a host cell a protein complex comprising the small terminase (encoded by terS) and large terminase (encoded by terL) proteins is able to recognise and cleave a double-stranded DNA
molecule of the MGE at or near the pac site (cos site or other packaging signal sequence comprised by the MGE), and this allows the MGE or plasmid DNA molecule to be packaged into a phage capsid.
When first phage as prophage in the host cell is induced, the lytic cycle of the phage produces the phage's structural proteins and the phage's large terminase protein. The MGE
or plasmid is replicated, and the small terminase protein encoded by the MGE or plasmid is expressed.
The replicated MGE or plasmid DNA containing the terS (and the nucleotide sequence encoding the antibacterial agent or component) are packaged into phage capsids, resulting in non-self replicative transduction particles carrying only MGE or plasmid DNA.
15. The composition of any one of Clauses 1 to 13, wherein the MGE
comprises phage structural genes and a packaging signal sequence and the first phage is devoid of a packaging signal sequence.
16. The composition of any preceding Clause, wherein the MGE is a modified version of a MGE
that is naturally found in bacterial cells of the first species or strain.
17. The composition of any preceding Clause, wherein the MGE comprises a modified genomic island.
Optionally, the genomic island is an island that is naturally found in bacterial cells of the first species or strain. In an example, the genomic island is selected from the group consisting of a SaPI, a SaPI1, a SaPI2, a SaPIbov 1 and a SaPibov2 genomic island.

18. The composition of any preceding Clause, wherein the MGE comprises a modified pathogenicity island.
Optionally, the pathogenicity island is an island that is naturally found in bacterial cells of the first species or strain, eg, a Staphylococcus SaPI or a Vibro PLE or a P. aeruginosa pathogenicity island (eg, a PAPI or a PAGI, eg, PAPI-1, PAGI-5, PAGI-6, PAGI-7, PAGI-8, PAGI-9, PAGI-10, or PAGI-19. The composition of Clause 18, wherein the pathogenicity island is a SaPI (S aureus pathogenicity island).
20. The composition of Clause 19, wherein the first phage is 011, 80a, 012 or 0SLT.
Staphylococcus phage 80a appears to mobilise all known SaPIs. Thus, in an example, the MGE
comprises a modified SaPI and the first phage is a 80a.
21. The composition of Clause 18, wherein the pathogenicity island is a V.
cholerae PLE (phage-inducible chromosomal island-like element) and optionally the first phage is ICP1.
22. The composition of Clause 18, wherein the pathogenicity island is a E
coli PLE.
23. The composition of any one of Clauses 1 to 16, wherein the MGE
comprises P4 DNA, eg, a P4 packaging signal sequence.
24. The composition of Clause 23, wherein the first phage are P2 phage or a modified P2 phage that is self-replicative defective; optionally present as a prophage.
25. The composition of any preceding Clause, wherein the MGE comprises a pacA gene of the Enterobacteriaceae bacteriophage Pl.
26. The composition of any preceding Clause, wherein the MGE comprises a packaging initiation site sequence, optionally a packaging initiation site sequence of Pl.
27. The composition of any preceding Clause, wherein the MGE comprises a nucleotide sequence that is beneficial to cells of the first species or strain, optionally encoding a protein that is beneficial to cells of the first species or strain.
This is useful where, not only does the presence of the MGE reduce first phage replication in the host cell, but also the MGE is taken up and may provide a survival, growth or other benefit to the host cell, promoting uptake and/or retention of MGEs by host cells. In an example, expression of the antibacterial agent in the host cell is under the control of an inducible promoter or weak promoter to allow for a period where uptake of MGEs into host cells may be favoured owing to the presence of the nucleotide sequence that is beneficial to cells of the first species or strain.
28. The composition of any preceding Clause, wherein the MGE is devoid of rinA.
29. The composition of any preceding Clause, wherein the MGE is is devoid of terL.

30. The composition of any preceding Clause, wherein the MGE comprises a terS or a homologue thereof, and optionally is devoid of any other terminase gene.
The terS homologues are sequences which, like terS, recognise the SaPI-specific pac site (or other packaging sequence) comprised by the MGE or plasmid and determine packaging specificity for packaging the MGE.
Examples of terminase genes are pacA, pacB, terA, terB and terL.
31. The composition of any preceding Clause, wherein the first phage is a pac-type phage (eg, 011 or 80a) operable with a pac comprised by the MGE.
32. The composition of any one of Clauses 1 to 30, wherein the first phage is a cos-type phage (eg, 012 or 0SLT) operable with a cos comprised by the MGE.
Optionally, the phage is P2. Optionally, the first phage is a T7 or T7-like phage that recognises direct repeat sequences comprised by the MGE for packaging.
33. The composition of any preceding Clause, wherein the plasmid or MGE
comprises a pac and/or cos sequence or a homologue thereof.
34. The composition of any preceding Clause, wherein the plasmid or MGE
comprises a terS or a homologue thereof and optionally devoid of terL.
The terS homologues are sequences which, like terS, recognise the SaPI-specific pac site (or other packaging sequence) comprised by the MGE or plasmid and determine packaging specificity for packaging the MGE.
In an example, the terS comprises the sequence of SEQ ID NO: 1.
35. The composition of Clause 34, wherein the terS is a S aureus bacteriophage 980a terS or a bacteriophage 911 terS.
36. The composition of any preceding Clause, wherein the MGE is a modified SaPIbov 1 or SaPIbov5 and is devoid of a terS.
37. The composition of any preceding Clause, wherein the first phage is devoid of a functional packaging signal sequence and the MGE comprises a packaging signal sequence operable with proteins encoded by the first phage for producing transduction particles that package copies of the MGE or copies of a nucleic acid encoding the agent or component.
38. The composition of any preceding Clause, wherein the MGE or plasmid comprises a Ppi or homologue, which is capable of complexing with first phage TerS, thereby blocking function of the TerS.

39. The composition of any preceding Clause, wherein the MGE comprises a morphogenesis (cpm) module.
40. The composition of any preceding Clause, wherein the MGE comprises cpmA
and/or cpmB.
Optionally the cpmA and B are from any SaPI disclosed herein. In an example any SaPI is a SaPI
disclosed in Table 2 and optionally the host cell or target cell is any corresponding Staphylococcus disclosed in the table.
41. The composition of any preceding Clause, wherein the MGE or first phage comprises one, more or all genes cpl, cp2, and cp3.
In an example, the MGE comprises a modified SaPI and comprises one, more or all genes cpl, cp2, and cp3.
42. The composition of any preceding Clause, wherein the MGE or first phage encodes a HNH
nuclease.
43. The composition of any preceding Clause, wherein the MGE or first phage comprises an integrase gene that encodes an integrase for excising the MGE and integrating the MGE into a bacterial cell genome.
44. The composition of any preceding Clause, wherein the MGE is devoid of a functional integrase gene, and the first phage or host cell genome (eg, bacterial chromosome or a bacterial episome) comprises a functional integrase gene.
45. The composition of any preceding Clause, wherein the transcription of MGE nucleic acid is under the control of a constitutive promoter, for transcription of copies of the agent or component in a host cell.
Optionally, Constitutive transcription and production of the agent in target cells may be used where the target cells should be killed, eg, in medical settings.
Optionally, the transcription of MGE nucleic acid is under the control of an inducible promoter, for transcription of copies of the agent or component in a host cell. This may be useful, for example, to control switching on of the antibacterial activity against target bacterial cells, such as in an environment (eg, soil or water) or in an industrial culture or fermentation container containing the target cells. For example, the target cells may be useful in an industrial process (eg, for fermentation, eg, in the brewing or dairy industry) and the induction enables the process to be controlled (eg, stopped or reduced) by using the antibacterial agent against the target bacteria.

46. The composition of Clause 45, wherein the promoter is foreign to the host cell.
47. The composition of Clause 45 or 46, wherein the promoter comprises a nucleotide sequence that is at least 80% identical to an endogenous promoter sequence of the host cell.
48. The composition of any preceding Clause comprising a nucleic acid that is separate from the MGE, wherein the nucleic acid comprises all genes necessary for producing first phage particles.
49. The composition of any one of Clauses 1 to 47 comprising a nucleic acid that is separate from the MGE, wherein the nucleic acid comprises less than, all genes necessary for producing first phage particles, but comprises genes encoding structural proteins for production of transduction particles that package MGE nucleic acid encoding the antibacterial agent or one or more components thereof.
When the agent comprises a plurality of components, eg, wherein the agent is a CRISPR/Cas system, or is a CRISPR array encoding crRNA or a nucleic acid encoding a guide RNA
(eg, single guide RNA) operable with a Cas in host cells, wherein the crRNA or gRNA guides the Cas to a target sequence in the host cell to modify the target (eg, cut it or repress transcription from it).
50. The composition of Clause 48 or 49, wherein the genes are comprised by the host cell chromosome and/or one or more host cell episome(s).
51. The composition of Clause 50, wherein the genes are comprised by a chromosomally-integrated prophage of the first phage.
52. The composition of any preceding Clause, wherein the agent is a guided nuclease system or a component thereof, wherein the agent is capable of recognising and cutting host cell DNA (eg, chromosomal DNA).
In examples, such cutting causes one or more of the following:-(i)The host cell is killed by the antibacterial agent;
(ii) growth or proliferation of the host cell is reduced; and/or (iii)The host cell is sensitised to an antibiotic, whereby the antibiotic is toxic to the cell.
53. The composition of Clause 52, wherein the guided nuclease system is selected from a CRISPR/Cas system, TALEN system, meganuclease system or zinc finger system.
54. The composition of Clause 52, wherein the system is a CRISPR/Cas system and each MGE
encodes a (a) CRISPR array encoding crRNA or (b) a nucleic acid encoding a guide RNA (gRNA, eg, single guide RNA), wherein the crRNA or gRNA is operable with a Cas in target bacterial cells, wherein the crRNA or gRNA guides the Cas to a target nucleic acid sequence in the host cell to modify the target sequence (eg, cut it or repress transcription from it).

Optionally, the Cas is a Cas encoded by a functional endogenous nucleic acid of a host cell. For example, the target is comprised by a DNA or RNA of the host cell.
55. The composition of Clause 52, wherein the system is a CRISPR/Cas system and each MGE
encodes a Cas (eg, a Cas nuclease) that is operable in a target bacterial cells to modify a target nucleic acid sequence comprised by the target cell.
56. The composition of Clause 53, 54 or 55, wherein the Cas is a Cas3, Cas9, Cas13, CasX, CasY
or Cpfl.
57. The composition of any one of Clauses 52 to 56, wherein the system is a CRISPR/Cas system and each MGE encodes one or more Cascade Cas (eg, Cas, A, B, C, D and E).
58. The composition of any one of Clauses 52 to 57, wherein each MGE
further encodes a Cas3 that is operable in a target bacterial cell with the Cascade Cas.
59. The composition of any preceding Clause, wherein the first species or strain is a gram positive species or strain.
60. The composition of any one of Clauses 1 to 58, wherein the first species or strain is a gram negative species or strain.
61. The composition of any preceding Clause, wherein the first species or strain is selected from Table 1.
In an example, the first species of strain is a Staphylococcus (eg, S aureus) species or strain and optionally the MGE is a modified SaPI; and optionally the first phage is a 980a or 911. In an example, the first species of strain is a Vibrio (eg, V cholerae) species or strain and optionally the MGE is Vibrio (eg, V cholerae) PLE.
62. The composition of any preceding Clause, wherein the first species or strain is selected from Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas and Enterobacter.
These are species that P2 phage can infect. Thus, in an embodiment, the MGE
comprises one or more P4 sequences (eg, a P4 packaging sequence) and the first phage is P2. Thus, the MGE is packaged by P2 structural proteins and the resultant transduction particles can infect a broad spectrum of species, ie, two or more of Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas and Enterobacter.
63. A nucleic acid vector comprising a MGE integrated therein, wherein the MGE is according to any preceding Clause and the vector is capable of transferring the MGE or a copy thereof into a host bacterial cell.

Suitable vectors are plasmids (eg, conjugative plasmids) or viruses (eg, phage or packaged phagemids).
64. The vector of Clause 63, wherein the vector is a shuttle vector.
A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types.
65. The vector of Clause 63, wherein the vector is a plasmid, wherein the plasmid is capable of being transformed into a host bacterial cell comprising a first phage.
66. A non-self replicative transduction particle comprising said MGE or vector of any preceding .. Clause.
By "non-replicative" it is meant that the MGE is not capable by itself of self-replicating. For example, the MGE is devoid of one or more nucleotide sequences encoding a protein (eg, a structural protein) that is necessary to produce a transduction particle comprising a copy of the MGE.
67. A composition comprising a plurality of transduction particles, wherein each particle comprises a MGE or vector according to any one of Clauses 1 to 65, wherein the transduction particles are capable of transferring the MGEs, or nucleic acid encoding the agent or component, or copies thereof into target bacterial cells, wherein a. target cells are killed by the antibacterial agent;
b. growth or proliferation of target cells is reduced; or c. target cells are sensitised to an antibiotic, whereby the antibiotic is toxic to the cells.
In an example, the reduction in growth or proliferation of host cells is at least 50, 60, 70, 80, 90 or 95%. The antibiotic can be any antibiotic disclosed herein.
68. The composition of Clause 67, wherein the agent is a guided nuclease system or a component thereof, wherein the agent is capable of recognising and cutting host cell DNA
(eg, chromosomal DNA) whereby a. target cells are killed by the antibacterial agent;
b. growth or proliferation of target cells is reduced; or c. target cells are sensitised to an antibiotic, whereby the antibiotic is toxic to the cells.
69. A composition comprising a plurality of non-self replicative transduction particles, wherein each particle comprises a MGE or plasmid according to any one of Clauses 1 to 65, wherein the transduction particles are capable of transferring the MGEs, or nucleic acid encoding the agent or component, or copies thereof into target bacterial cells, wherein the agent is a CRISPR/Cas system and the component comprises a nucleic acid encoding a crRNA or a guide RNA
that is operable with a Cas in a target bacterial cell to guide the Cas to a target nucleic acid sequence of the cell to modify the sequence, whereby a. target cells are killed by the antibacterial agent;
b. growth or proliferation of target cells is reduced; or c. target cells are sensitised to an antibiotic, whereby the antibiotic is toxic to the cells.
In an example, the reduction in growth or proliferation of host cells is at least 50, 60, 70, 80, 90 or 95%. The antibiotic can be any antibiotic disclosed herein.
70. A kit comprising the composition of Clause 69 and said antibiotic.
71. The composition of Clause 69, wherein the composition comprises said antibiotic.
72. The composition of any one of Clauses 67 to 69, wherein less than 10%
of transduction particles comprise by the composition are first phage particles.
73. The composition of any one of Clauses 67 to 69, wherein no first phage particles are present in the composition.
74. The MGE, vector, particle, composition or kit of any preceding Clause for medical use in a human or animal patient.
75. The MGE, vector, particle, composition or kit of any preceding Clause for treating or preventing an infection by target bacterial cells in a human or animal patient, wherein the antibacterial agent is toxic to the target cells.
76. The MGE, vector, particle, composition or kit of any preceding Clause for treating or preventing an infection by target bacterial cells in a human or animal patient, wherein in the presence of the antibacterial agent a. target cells are killed by the antibacterial agent;
b. growth or proliferation of target cells is reduced; and/or c. target cells are sensitised to an antibiotic, whereby the antibiotic is toxic to the cells.
77. A method of producing a plurality of transduction particles, the method comprising combining the composition of any one of Clauses 1 to 62, 67 to 69 and 71 to 76 with host bacterial cells of said first species, wherein the cells comprise the first phage, allowing a plurality of said MGEs to be introduced into host cells and culturing the host cells under conditions in which first phage-encoded proteins are expressed and MGE copies are packaged by first phage proteins to produce a plurality of transduction particles, and optionally separating the transduction particles from cells and obtaining a plurality of transduction particles separated from cells.

78. The method of Clause 77, comprising separating the transduction particles from any first phage, optionally by filtering or centrifugation, thereby obtaining a plurality of transduction particles in the absence of first phage.
79. The method of Clause 77 or 78, wherein the particles encode a guided nuclease system (optionally a CRISPR/Cas system) or component thereof for cutting a target nucleic acid sequence comprised by target bacterial cells.
80. The method of Clause 79, wherein the sequence is comprised by an antibiotic resistance gene and the method comprises combining the plurality of particles with said antibiotic in a kit or a mixture.
81. The method of any one of Clauses 77 to 80, wherein said conditions comprise induction of a lytic cycle of the first phage.
82. A bacterial host cell comprising a first phage and a MGE, vector or particle as recited in any one of Clauses 1 to 66, wherein the agent is toxic to cells of the same species as the host cell, and wherein the host cell has been engineered so that the agent is not toxic to the host cell.
83. A bacterial host cell comprising a first phage, wherein the cell is comprised by a kit, the kit further comprising a composition as recited in any one of Clauses 1 to 62, 67 to 69 and 71 to 76, wherein the agent is toxic to cells of the same species as the host cell, and wherein the host cell has been engineered so that the agent is not toxic to the host cell.
84. The cell of Clause 83, wherein the agent is a guided nuclease system (optionally a CRISPR/Cas system) and cells of the same species as the host cell comprise a target sequence that is cut by the nuclease, wherein the target sequence has been removed or altered in the host cell whereby the nuclease is not capable of cutting the target sequence.
85. A bacterial host cell comprising a first phage and a MGE, vector or particle as recited in any one of Clauses 1 to 66, wherein the agent is not toxic to the host cell, but the agent is toxic to second cells of a species or strain that is different from the species or strain of the host cell, wherein the MGE
is mobilizable in transduction particles producible by the host cell that are capable of transferring the MGE or a copy thereof into a said second cell, whereby the second cell is exposed to the antibacterial agent.
86. A bacterial host cell comprising a first phage, wherein the cell is comprised by a kit, the kit further comprising a composition as recited in any one of Clauses 1 to 62, 67 to 69 and 71 to 76, wherein the agent is not toxic to the host cell, but the agent is toxic to second cells of a species or strain that is different from the species or strain of the host cell, wherein the MGE is mobilizable in transduction particles producible by the host cell that are capable of transferring the MGE or a copy thereof into a said second cell, whereby the second cell is exposed to the antibacterial agent.
87. The cell of Clause 86, wherein the first phage is a prophage.

88. A bacterial host cell comprising a MGE, vector or particle as recited in any one of Clauses 1 to 66 and nucleic acid under the control of one or more inducible promoters, wherein the nucleic acid encodes all structural proteins necessary to produce a transduction particle that packages a copy of the MGE or plasmid, wherein the agent is not toxic to the host cell, but the agent is toxic to second cells of a species or strain that is different from the species or strain of the host cell, wherein the MGE is mobilizable in transduction particles producible by the host cell that are capable of transferring the MGE or a copy thereof into a said second cell, whereby the second cell is exposed to the antibacterial agent.
89. The cell of Clause 88, wherein the structural proteins are structural proteins of a lytic phage.
90. The cell of Clause 88 or 89, wherein the nucleic acid comprises terS
and/or terL.
91. The cell of any one of Clauses 88 to 90, wherein the host and second cells are of the same species and the host cell has been engineered so that the antibiotic is not toxic to the host cell.
92. The cell of any one of Clauses 88 to 91, wherein the nucleic acid is comprised by a plasmid.
93. The cell of any one of Clauses 88 to 92, wherein the agent is a guided nuclease system (optionally a CRISPR/Cas system) and the second cells comprise a target sequence that is cut by the nuclease, wherein the target sequence is absent in the genome of the host cell whereby the nuclease is not capable of cutting the host cell genome.
94. The composition, vector, particle, kit or method of any preceding Clause, wherein the cell, host cell or target cell is selected from a Staphylococcal, Vibrio, Pseudomonas, Clostridium, E coli, Helicobacter, Klebsiella and Salmonella cell.
95. A plasmid comprising a. A nucleotide sequence encoding an antibacterial agent or component thereof for expression in target bacterial cells;
b. A constitutive promoter for controlling the expression of the agent or component;
c. An optional terS nucleotide sequence;
d. An origin of replication (on); and e. A phage packaging sequence (optionally pac, cos or a homologue thereof);
and f. the plasmid being devoid of g. All nucleotide sequences encoding phage structural proteins necessary for the production of a transduction particle (optionally a phage), or the plasmid being devoid of at least one of such sequences; and h. Optionally terL.
96. The plasmid of Clause 95, wherein the antibacterial agent is a CRISPR/Cas system and the plasmid encodes a crRNa or guide RNA (eg, single gRNA) that is operable with a Cas in the target cells to guide the Cas to a target nucleotide sequence to modify (eg, cut) the sequence, whereby a. target cells are killed by the antibacterial agent;
b. growth or proliferation of target cells is reduced; or c. target cells are sensitised to an antibiotic, whereby the antibiotic is toxic to the cells.
97. The plasmid of Clause 95 or 96, wherein the antibacterial agent is a CRISPR/Cas system and the plasmid encodes a Cas that is operable with a crRNa or guide RNA (eg, single gRNA) in the target cells to guide the Cas to a target nucleotide sequence to modify (eg, cut) the sequence, whereby a. target cells are killed by the antibacterial agent;
b. growth or proliferation of target cells is reduced; or c. target cells are sensitised to an antibiotic, whereby the antibiotic is toxic to the cells.
98. The plasmid of Clause 97, wherein the plasmid further encodes said crRNA or gRNA.
99. A host cell comprising the plasmid of any one of Clauses 95 to 98, wherein the host cell does not comprise the target nucleotide sequence.
100. The host cell of Clause 99, wherein the cell is capable of replicating the plasmid and packaging the replicated plasmid in transduction particles that are capable of infecting target bacterial cells.
101. The host cell of Clause 99 or 100, wherein the host cell comprises, integrated in the cell chromosome and/or one or more episomes of the cell, a. A terL;
b. An optional terS; and c. Expressible nucleotide sequences encoding all structural proteins necessary for the production of transduction particles that package copies of the plasmid;
d. wherein the chromosome and episomes of the cell (other than said plasmid) are devoid of a phage packaging sequence, wherein the phage packaging sequence comprised by the plasmid is operable together with the product of said terS and terL in the production of packaged plasmid.
102. The cell of Clause 101, wherein the terL, optional terS and nucleotide sequences encoding the structural proteins are comprised by a phage (optionally a prophage) genome in the host cell.
103. A bacterial host cell comprising the genome of a helper phage that is incapable of self-replication, optionally wherein the genome is present as a prophage, and a plasmid according to any one of Clauses 95 to 98, wherein the helper phage is operable to package copies of the plasmid in transduction particles, wherein the particles are capable of infecting bacterial target cells to which the antibacterial agent is toxic.

104. The cell of Clause 103, wherein the host cell is a cell of first species or strain and the target cells are of the same species or strain, and optionally wherein the hosts cell is an engineered cell that to which the antibacterial agent is not toxic.

105. The cell of Clause 103, wherein the host cell is a cell of first species or strain and the target cells are of a different species or strain, wherein the antibacterial agent is not toxic to the host cell.

106. A method of making a plurality of transduction particles, the method comprising culturing a plurality of host cells according to any one of Clauses 103 to 105, optionally inducing a lytic cycle of the helper phage, and incubating the cells under conditions wherein transducing particles comprising packaged copies of the plasmid are created, and optionally separating the particles from the cells to obtain a plurality of transduction particles.

107. A plurality of transduction particles obtainable by the method of Clause 106 for use in medicine, eg, for treating or preventing an infection of a human or animal subject by target bacterial cells, wherein transducing particles are administered to the subject for infecting target cells and killing the cells using the antibacterial agent.

108. A method of making a plurality of transduction particles, the method comprising i.Producing host cells whose genomes comprise nucleic acid encoding structural proteins necessary to produce transduction particles that can package first DNA, wherein the genomes are devoid of a phage packaging signal, wherein the expression of the proteins is under the control of inducible promoter(s);
ii.Producing first DNA encoding an antibacterial agent or a component thereof (eg, as defined in any preceding Clause), wherein the DNA comprises a phage packaging signal;
iii.Introducing the DNA into the host cells;
iv.Inducing production of the structural proteins in host cells, whereby transduction particles are produced that package the DNA;
v.Optionally isolating a plurality of the transduction particles; and vi.Optionally formulating the particles into a pharmaceutical composition for administration to a human or animal for medical use.

109. The method of Clause 108, wherein the DNA comprises a MGE as defined in any preceding Clause.

110. The method of Clause 108 or 109, wherein the structural proteins are P2 phage proteins and optionally the packaging signal is a P4 phage packaging signal.

111. The method of Clause 108 or 109, wherein the DNA comprises a modified SaPI or a genomic island DNA.

112. The method of any one of Clauses 108 to 111, wherein the cells in step (iv) comprise a gene encoding a helper phage activator, optionally wherein the activator is a P4 phage delta or ogr protein when the structural proteins are P2 proteins; or the activator is a SaPI rinA, ptiA, ptiB or ptiM when the MGE comprises a modified SaPI; and optionally the expression of the activator(s) is controlled by an inducible promoter, eg, a T7 promoter.

113. The method of any one of Clauses 108 to 112, wherein the packaging signal is P4 phage Sid and/or psu; or the signal is SaPI cpmA and/or cpmB.
This is useful for packaging DNAs into smaller capsids.

114. The method of any one of Clauses 108 to 113, wherein the cell genomes comprise prophages, wherein each prophage comprises said nucleic acid encoding structural proteins.

115. The method of Clause 114, wherein the prophages are P2 prophages devoid of cos and optionally one, more or all genes selected from int, cox orf78, B, oif80, oif81, oif82, orf83, A, oif91, tin, old, orf30 andfun(Z); and optionally the packaging signal of (ii) is a cos or P4 packaging signal.

116. The method of Clause 114 or 115, wherein the prophages are P2 prophages devoid of cos and comprising genes from Q to S, V to G and Fr to ogr.

117. The method of Clause 114, wherein the prophages are phill prophages devoid of a packaging signal and comprising gene 29 (terS) to gene 53 (lysin); and optionally the packaging signal of (ii) is a phill packaging signal.

118. A plurality of transduction particles obtainable by the method of any one of Clauses 108 to 117.

119. The particles of Clause 118 for administration to a human or animal for medical use.
Further Concepts of the invention are as follows:-[0079] The present invention is optionally 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, aeorspace 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.
[0080] The present invention is optionally for use in an industry or the environment is an industrial environment, wherein the industry is an industry of a field selected from the group consisting of the medical and healthcare; pharmaceutical; human food; animal food; plant fertilizers; beverage; dairy;
meat processing; agriculture; livestock farming; poultry farming; fish and shellfish farming;
veterinary; oil; gas; petrochemical; water treatment; sewage treatment;
packaging; electronics and computer; personal healthcare and toiletries; cosmetics; dental; non-medical dental; ophthalmic; non-medical ophthalmic; mineral mining and processing; metals mining and processing; quarrying;

aviation; automotive; rail; shipping; space; environmental; soil treatment;
pulp and paper; clothing manufacture; dyes; printing; adhesives; air treatment; solvents; biodefence;
vitamin supplements; cold storage; fibre retting and production; biotechnology; chemical; industrial cleaning products; domestic cleaning products; soaps and detergents; consumer products; forestry; fishing;
leisure; recycling;
plastics; hide, leather and suede; waste management; funeral and undertaking;
fuel; building; energy;
steel; and tobacco industry fields.
[0081] In an example, the ifirst DNA, first phage or vector comprises a CRISPR
array that targets target bacteria, wherein the array comprises one, or two or more spacers (eg, 2, 3, 4, 5, 6, 7, 8, 9 ,10, 20, 30, 40, 50 or more spacers) for targeting the genome of target bacteria.
[0082] In an example, the target bacteria are comprised by an environment as follows. In an example, the environment is a microbiome of a human, eg, the oral cavity microbiome or gut microbiome or the bloodstream. In an example, the environment is not an environment in or on a human. In an example, the environment is not an environment in or on a non-human animal. In an embodiment, the environment is an air environment. In an embodiment, the environment is an agricultural environment. In an embodiment, the environment is an oil or petroleum recovery environment, eg, an oil or petroleum field or well. In an example, the environment is an environment in or on a foodstuff or beverage for human or non-human animal consumption.
[0083] In an example, the environment is a a human or animal microbiome (eg, gut, vaginal, scalp, armpit, skin or oral cavity microbiome). In an example, the target bacteria are comprised by a human or animal microbiome (eg, gut, vaginal, scalp, armpit, skin or oral cavity microbiome).
[0084] In an example, the DNAs, phage or composition of the invention are administered intranasally, topically or orally to a human or non-human animal, or is for such administration. The skilled person aiming to treat a microbiome of the human or animal will be able to determine the best route of administration, depending upon the microbiome of interest. For example, when the microbiome is a gut microbiome, administration can be intranasally or orally.
When the microbiome is a scalp or armpit microbiome, administration can be topically. When the microbiome is in the mouth or throat, the administration can be orally.
[0085] In any use or method herein, in an embodiment the first phage are contacted with the target bacteria at a multiplicity of infection (MOI) of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600 or 700. For example, the MOI is from 20 to 200, from 20 to 100, fro 50 to 200, from 50 to 100, from 75 to 150, 100 or about 100, or 200 or about 200. In an example, this may be determined by obtaining a sample of the microbiome containing the target bacteria (eg, a sample of a waterway or gut microbiome of a subject) and determining the number of CFU/ml or mg in the sample and using this to titrate the phage dose at the desired MOT to be exposed to the microbiome or administered to the environment or subject to be treated.
[0086] In an example, the environment is harboured by a beverage or water (eg, a waterway or drinking water for human consumption) or soil. The water is optionally in a heating, cooling or industrial system, or in a drinking water storage container.
[0087] In an example, the host and/or target bacteraia are Finnicutes selected from Anaerotruncus, Acetanaerobacterium, Acetitomaculum, Acetivibrio, Anaerococcus, Anaerofilum, Anaerosinus, Anaerostipes, Anaerovorax, Butyrivibrio, Clostridium, Capracoccus, Dehalobacter, Dialister, Dorea, Enterococcus, Ethanoligenens, Faecalibacterium, Fusobacterium, Gracilibacter, Guggenheimella, Hespellia, Lachnobacterium, Lachnospira, Lactobacillus, Leuconostoc, Megamonas, Mitsuokella, Oribacterium, Oxobacter, Papillibacter, Proprionispira,Pseudobutyrivibrio, Pseudoramibacter, Roseburia, Ruminococcus, Sarcina, Seinonella, Shuttleworthia, Sporobacter, Sporobacterium, Streptococcus, Subdoligranulum, Syntrophococcus, Thermobacillus, Turibacter and Weisella.
[0088] In an example, the kit, DNA(s), first phage, helper phage, composition, use or method is for reducing pathogenic infections or for re-balancing gut or oral microbiota eg, for treating or preventing obesity or disease in a human or animal. For example, the first phage, helper phage, composition, use or method is for knocking-down Clostridium dificile or E coli bacteria in a gut microbiota of a human or animal.
[0089] In an example, the packaging signal, NPF and/or HPF consists or comprises SEQ ID NO: 2 or a structural or functional homologue thereof.
[0090] In an example, the packaging signal, NPF and/or HPF consists or comprises SEQ ID NO: 2 or a nucleotide sequence that is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.
[0091] In an example, the disease or condition is a cancer, inflammatory or autoimmune disease or condition, eg, obesity, diabetes IBD, a GI tract condition or an oral cavity condition.
[0092] Optionally, the environment is comprised by, or the target bacteria are comprised by, a gut microbiota, skin microbiota, oral cavity microbiota, throat microbiota, hair microbiota, armpit microbiota, vaginal microbiota, rectal microbiota, anal microbiota, ocular microbiota, nasal microbiota, tongue microbiota, lung microbiota, liver microbiota, kidney microbiota, genital microbiota, penile microbiota, scrotal microbiota, mammary gland microbiota, ear microbiota, urethra microbiota, labial microbiota, organ microbiota or dental microbiota.
Optionally, the environment is comprised by, or the target bacteria are comprised by, a plant (eg, a tobacco, crop plant, fruit plant, vegetable plant or tobacco, eg on the surface of a plant or contained in a plant) or by an environment (eg, soil or water or a waterway or acqueous liquid).

[0093] Optionally, the disease or condition of a human or animal subject is selected from (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, eg, heart attack, stroke or atrial fibrillation;
(0 A liver disease or condition;
(g) A kidney disease or condition, eg, chronic kidney disease (CKD);
(h) A pancreas disease or condition;
(i) A lung disease or condition, eg, cystic fibrosis or COPD;
(i) A gastrointestinal disease or condition;
(k) A throat or oral cavity disease or condition;
(1) An ocular disease or condition;
(m) A genital disease or condition, eg, a vaginal, labial, penile or scrotal disease or condition;
(n) A sexually-transmissible disease or condition, eg, gonorrhea, HIV
infection, syphilis or Chlamydia infection;
(o) An ear disease or condition;
(13) A skin disease or condition;
(q) A heart disease or condition;
(r) A nasal disease or condition (s) A haematological disease or condition, eg, anaemia, eg, anaemia of chronic disease or cancer;
(t) A viral infection;
(u) A pathogenic bacterial infection;
(v) A cancer;
(w) An autoimmune disease or condition, eg, SLE;
(x) An inflammatory disease or condition, eg, 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;
(cc) Miscarriage;
(ft) A blood clotting condition;

(gg) Bronchitis;
(hh) Dry or wet AMD;
(ii) Neovascularisation (eg, of a tumour or in the eye);
Common cold;
(kk) Epilepsy;
(11) Fibrosis, eg, liver or lung fibrosis;
(mm) A fungal disease or condition, eg, thrush;
(nn) A metabolic disease or condition, eg, obesity, anorexia, diabetes, Type I or Type II diabetes.
(oo) Ulcer(s), eg, gastric ulceration or skin ulceration;
(pp) Dry skin;
(qq) Sjogren's syndrome;
(a) 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, eg, infertility or low fertility;
(vv) Jaundice;
(ww) Skin rash;
(xx) Kawasaki Disease;
(yy) Lyme Disease;
(zz) An allergy, eg, 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;
A bone disorder;
(kkk) Sepsis or septic shock;
(111) Sinusitus;
(mmm) Stress (eg, occupational stress);
(nnn) Thalassaemia, anaemia, von Willebrand Disease, or haemophilia;

(000) Shingles or cold sore;
(ppp) Menstruation;
(qqq) Low sperm count.
[0094] NEURODEGENERATIVE OR CNS DISEASES OR CONDITIONS FOR
TREATMENT OR PREVENTION BY THE INVENTION
[0095] In an example, the neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer disease , geriopsychosis, Down syndrome, Parkinson's disease, Creutzfeldt-j akob 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.
[0096] 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 (eg, 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 (eg, 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 W02015136541 and/or the method can be used with any method disclosed in W02015136541 (the disclosure of this document is incorporated by reference herein in its entirety, eg, for providing disclosure of such methods, diseases, conditions and potential therapeutic agents that can be administered to the subject for effecting treatement and/or prevention of CNS and neurodegenerative diseases and conditions, eg, agents such as immune checkpoint inhibitors, eg, anti-PD-1, anti-PD-L1, anti-TIM3 or other antibodies disclosed therein).
[0097] CANCERS FOR TREATMENT OR PREVENTION BY THE METHOD
[0098] 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.
[0099] Haematologic 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, myeiodysplastic syndrome, hairy cell leukaemia and myelodysplasia.
[00100] 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, medu!loblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).
[00101] AUTOIMMUNE DISEASES FOR TREATMENT OR PREVENTION BY THE
METHOD
1. Acute Disseminated Encephalomyelitis (ADEM) 2. Acute necrotizing hemorrhagic leukoencephalitis 3. Addison's disease 4. Agammaglobulinemia 5. Alopecia areata 6. Amyloidosis 7. Ankylosing spondylitis 8. Anti-GBM/Anti-TBM nephritis 9. Antiphospholipid syndrome (APS) 10. Autoimmune angioedema 11. Autoimmune aplastic anemia 12. Autoimmune dysautonomia 13. Autoimmune hepatitis 14. Autoimmune hyperlipidemia 15. Autoimmune immunodeficiency 16. Autoimmune inner ear disease (AIED) 17. Autoimmune myocarditis 18. Autoimmune oophoritis 19. Autoimmune pancreatitis 20. Autoimmune retinopathy 21. Autoimmune thrombocytopenic putpura (ATP) 22. Autoimmune thyroid disease 23. Autoimmune urticaria 24. Axonal & neuronal neuropathies 25. Balo disease 26. Behcet's disease 27. Bullous pemphigoid 28. Cardiomyopathy 29. Castleman disease 30. Celiac disease 31. Chagas disease 32. Chronic fatigue syndrome 33. Chronic inflammatory demvelinating polyneuropathv (CIDP) 34. Chronic recurrent multifocal ostomvelitis (CRMO) 35. Churg-Strauss syndrome 36. Cicatricial pemphigoid/benign mucosal pemphigoid 37. Crohn's disease 38. Cogans syndrome 39. Cold agglutinin disease 40. Congenital heart block 41. Coxsackie mvocarditis 42. CREST disease 43. Essential mixed cryoglobulincm i a 44. Demyelinating neuropathics 45. Dermatitis herpetiformis 46. Dermatomyositis 47. Devic's disease (neuromyelitis optica) 48. Discoid lupus 49. Dressler's syndrome 50. Endometriosis 51. Eosinophilic esophagitis 52. Eosinophilic fasciitis 53. Erythema nodosum 54. Experimental allergic encephalomyelitis 55. Evans syndrome 56. Fibromyalgia 57. Fibrosing alveolitis 58. Giant cell arteritis (temporal artcritis) 59. Giant cell myocarditis 60. Glomerulonephritis 61. Goodpasture's syndrome 62. Granulomatosis with Polyangiitis (GPM (formerly called Wegener's Granulomatosis) 63. Graves' disease 64. Guillain-Barre syndrome 65. Hashimoto's encephalitis 66. Hashimoto's thyroiditis 67. Hemolytic anemia 68. Hcnoch-Schonlein putpura 69. Herpes gestationis 70. Hvpogammaglobulinemia 71. Idiopathic thrombocvtopenic purpura (ITP) 72. IgA nephropathy 73. IgG4-related sclerosing disease 74. Immunoregulatoty lipoproteins 75. Inclusion body myositis 76. Interstitial cystitis 77. Juvenile arthritis 78. Juvenile diabetes (Type 1 diabetes) 79. Juvenile myositis 80. Kawasaki syndrome 81. Lambert-Eaton syndrome 82. Leukocvtoclastic vasculitis 83. Lichen planus 84. Lichen sclerosus 85. Ligneous conjunctivitis 86. Linear IgA disease (LAD) 87. Lupus (SLE) 88. Lyme disease, chronic 89. Meniere's disease 90. Microscopic polyangiitis 91. Mixed connective tissue disease (MCTD) 92. Mooren's ulcer 93. Mucha-Habermann disease 94. Multiple sclerosis 95. Myasthenia gravis 96. Myositis 97. Narcolepsy 98. Neuromvelitis optica (Devic's) 99. Neutropenia 100. Ocular cicatricial pemphigoid 101. Optic neuritis 102. Palindromic rheumatism 103. PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus) 104. Paraneoplastic cerebellar degeneration 105. Paroxysmal nocturnal hemoglobinuria (PNH) 106. Parry Romberg syndrome 107. Parsonnage-Turner syndrome 108. Pars planitis (peripheral uveitis) 109. Pemphigus 110. Peripheral neuropathy 111. Perivenous encephalomyelitis 112. Pernicious anemia 113. POEMS syndrome 114. Polyarteritis nodosa 115. Type I. H. & III autoimmune polvglandular syndromes 116. Polvinyalgia rheumatica 117. Polymyositis 118. Postmvocardial infarction syndrome 119. Postpericardiotomv syndrome

120. Progesterone dermatitis

121. Primary biliary cirrhosis

122. Primary sclerosing cholangitis

123. Psoriasis

124. Psoriatic arthritis

125. Idiopathic pulmonary fibrosis

126. Pyoderma gangrenosum

127. Pure red cell aplasia

128. Raynauds phenomenon

129. Reactive Arthritis

130. Reflex sympathetic dystrophy

131. Reiter's syndrome

132. Relapsing polvchondritis

133. Restless legs syndrome

134. Retroperitoneal fibrosis

135. Rheumatic fever

136. Rheumatoid arthritis

137. Sarcoidosis

138. Schmidt syndrome

139. Scleritis

140. Scleroderma

141. Sjogren's syndrome

142. Sperm & testicular autoimmunity

143. Stiff person syndrome

144. Subacute bacterial endocarditis (SBE)

145. Susac's syndrome

146. Sympathetic ophthalmia

147. Takayasu's arteritis

148. Temporal arteritis/Giant cell arteritis

149. Thrombocytopenic purpura (TTP)

150. Tolosa-Hunt syndrome

151. Transverse myelitis

152. Type 1 diabetes

153. Ulcerative colitis

154. Undifferentiated connective tissue disease (UCTD)

155. Uveitis

156. Vasculitis

157. Vesiculobullous dermatosis

158. Vitiligo

159. Wegener's granulomatosis (now termed Granulomatosis with Polyangiitis (GPA).
[00102] INFLAMMATORY DISEASES FOR TREATMENT OR PREVENTION BY THE
METHOD
1. Alzheimer 2. ankylosing spondylitis 3. arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis) 4. asthma 5. atherosclerosis 6. Crohn's disease 7. colitis 8. dermatitis 9. diverticulitis 10. fibromyalgia 11. hepatitis 12. irritable bowel syndrome (IBS) 13. systemic lupus erythematous (SLE) 14. nephritis 15. Parkinson's disease 16. ulcerative colitis.

[00103] PARAGRAPHS:
By way of example, the invention provides the following Paragraphs (which may optionally be combined with any of the disclosure above):-1. An antibacterial composition comprising a population of first phage, wherein the first phage require helper phage for replication of first phage particles, wherein the helper phage are capable of packaging first phage nucleic acid to produce first phage particles, wherein the first phage are different from the helper phage and the helper phage are incapable themselves of producing helper phage particles, wherein the first phage are capable of infecting target bacteria and each first phage comprises antibacterial means for killing bacteria.
2. The composition of Paragraph 1, wherein at least 95% of phage particles comprised by the composition are first phage particles.
Optionally, the composition comprises helper phage. Optionally, the composition comprises no more than 1 helper phage particle per 1 x 106 or more of phage particles.
Optionally, the composition comprises no more than 1 helper phage particle per 1 x 10' or more of phage particles. Optionally, the composition comprises no more than 1 helper phage particle per 1 x 109 or more of phage particles. Optionally, the composition comprises no more than 1 helper phage particle per 1 x 1010 or more of phage particles.
3. The composition of Paragraph 1 or 2, wherein each first phage comprises one or more components of a CRISPR/Cas system, wherein the component(s) comprise a DNA
sequence encoding a guide RNA (optionally a single guide RNA) or comprising a CRISPR array for producing guide RNA, wherein the guide RNA is capable of targeting the genome of target bacteria.
4. The composition of any preceding Paragraph, wherein each first phage genome is devoid of genes encoding phage proteins.
5. A composition according to any preceding Paragraph for use in antibacterial treatment of bacteria, the composition comprising an engineered mobile genetic element (MGE) that is capable of being mobilised in a first bacterial host cell of a first species or strain, the cell comprising a helper phage genome, wherein in the cell the MGE is mobilised using proteins encoded by the helper phage and replication of helper phage is inhibited, wherein the MGE encodes an antibacterial agent or encodes a component of such an agent, wherein the MGE comprises a modified genomic island, modified pathogenicity island, SaPI (S aureus Pathogenicity Island), V
cholerae PLE (Phage-Like Inducible Chromosomal Island-Like Element) or E coli PLE.
6. A kit comprising (a) A first DNA; and (b) One or more second DNAs;
Wherein (i) the DNAs together comprise all phage structural protein genes required to produce a packaged phage particle comprising a copy of the first DNA;
(ii) the first DNA comprises none or at least one, but not all, of the genes; and wherein the one or more second DNAs comprise the remainder of the genes;
(iii) the first DNA comprises a phage packaging signal for producing the packaged phage particle;
and (iv) the second DNA is devoid of a nucleotide sequence required for packaging the second DNA
into phage particles;
wherein the DNAs are operable when co-existing in a host bacterium for producing packaged phage (first phage) that comprise the first DNA, wherein the first phage require the second DNA for replication thereof to produce further first phage particles.
7. The kit of Paragraph 6, wherein the phage particle of (i) is capable of infecting a target bacterium, the phage comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in the target bacterium, wherein the presence in the target bacterium of the NSI-encoded protein or RNA mediates target cell killing, or downregulation of target cell growth or propagation.
8. The kit of Paragraph 6, wherein the phage particle of (i) is capable of infecting a target bacterium, the phage comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in the target bacterium, or wherein the NSI comprises a regulatory element that is operable in the target bacterium.
9. The kit of Paragraph 8, wherein the presence in the target bacterium of the NSI or its encoded protein or RNA mediates target cell killing, or downregulation of target cell growth or propagation, or mediates switching off of expression of one or more RNA or proteins encoded by the target cell genome, or downregulation thereof.

10. The kit of Paragraph 8, wherein the presence in the target bacterium of the NSI or its encoded protein or RNA mediates upregulation of growth or propagation of the target cell, or mediates switching on of expression of one or more RNA or proteins encoded by the target cell genome, or upregulation thereofy.
11. The kit of any one of Paragraphs 7, 8 or 9, wherein the NSI encodes a component of a CRISPR/Cas system that is toxic to the target bacterium.
12. The kit of any one of Paragraphs 7, 8 or 9, wherein the NSI comprises engineered antibacterial means for killing target bacteria.
13. The kit of any one of Paragraphs 6 to 12, wherein the packaged phage particle genome is devoid of genes encoding phage proteins.
14. The kit of any one of Paragraphs 6 to 13, wherein the first DNA
comprises none of the structural protein genes.
15. The kit of any one of Paragraphs 6 to 14, wherein the second DNA is devoid of a phage packaging signal.
16. The kit of any one of Paragraphs 6 to 15, wherein each signal is a pac or cos sequence, or is a homologue thereof.
17. The kit of any one of Paragraphs 6 to 16, wherein each signal comprises SEQ ID NO: 2 or a sequence that is at least 70, 80, 90, 95, 96, 97, 98 or 99% identical thereto, or is a homologue from a different species.
18. An isolated DNA, wherein the DNA is a first DNA as defined in any one of Paragraphs 6 to 17.
19. The kit or DNA of any one of Paragraphs 6 to 18, wherein the first DNA
is comprised by a vector (optionally, a plasmid, phagemid or shuttle vector).
20. The kit or DNA of any one of Paragraphs 6 to 19, wherein the second DNA
is comprised by a vector (optionally a plasmid, phagemid or shuttle vector), helper phage (optionally a helper phagemid) or is integrated in the genome of a host bacterial cell.
21. A host bacterial cell comprising the first and second DNAs as defined in any one of Paragraphs 6 to 17, 19 and 20; or comprising the DNA of Paragraph 18, 19 or 20.
22. A method of producing a phage composition, the method comprising expressing in a cell of Paragraph 21 the phage proteins, wherein packaged first phage particles are produced that comprise the first DNA, wherein the first phage require the second DNA for replication thereof to produce further first phage; and optionally separating an amount of first phage from cellular material wherein an amount of purified phage is obtained.
In an example, the purified phage are mixed with a pharmaceutically-acceptable excipient, carrier or diluent (eg, an aqueous liquid or water) to produce a pharmaceutical composition.
In an example any composition or kit of the invention is in combination with a label or instructions for use to treat and/or prevent a disease or condition in a human; optionally wherein the label or instructions comprise a marketing authorisation number (eg, an FDA or EMA
authorisation number);
optionally wherein the kit comprises an injection pen or IV container that comprises the first DNA or first phage.
23. The method of Paragraph 22, comprising isolating the first phage particles.
24. A composition (eg, antibacterial composition, eg, for medcial use) comprising a population of first phage particles obtainable by the method of Paragraph 22 or 23.
In an example, the first phage particles are obtained by the method.
In an example, any composition of the invention comprises at least 1 x 103 first phage per ml or mg, such as when the composition is comprised by a fluid (eg, a liquid) or solid.
In an example, any composition of the invention comprises at least 1 x 104 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 105 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 106 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 107 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 10' first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 109 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 1010 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 1011 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 1012 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 1013 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 1014 first phage per ml or mg.
In an example, any composition of the invention comprises up to 1 x 1014 first phage per ml or mg, such as when the composition is comprised by a fluid (eg, a liquid) or solid.
In an example, any composition of the invention comprises up to 1 x 1013 first phage per ml or mg. In an example, any composition of the invention comprises up to 1 x 1012 first phage per ml or mg. In an example, any composition of the invention comprises up to 1 x 1011 first phage per ml or mg. In an example, any composition of the invention comprises up to 1 x 1010 first phage per ml or mg. In an example, any composition of the invention comprises up to 1 x 109 first phage per ml or mg.
In an example, any composition of the invention comprises at least 1 x 103 to 1 x 1010, 1 x 1011, 1 x 1012, 1 x 10" or 1 x 1014 first phage per ml or mg, such as when the composition is comprised by a fluid (eg, a liquid) or solid. In an example, any composition of the invention comprises at least 1 x 104 to 1 x 1010, 1 x 1011, 1 x 1012, 1 x 10" or 1 x 1014 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 105 to 1 x 1010, 1 x 1011, 1 x 1012, 1 x 10" or 1 x 1014 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 106 to 1 x 1010, 1 x 1011, 1 x 1012, 1 x 10" or 1 x 1014 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 107 to 1 x 1010, 1 x 1011, 1 x 1012, 1 x 10" or 1 x 1014 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 108 to 1 x 1010, 1 x 1011, 1 x 1012, 1 x 10" or 1 x 1014 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 109 to 1 x 1010, 1 x 1011, 1 x 1012, 1 x 10" or 1 x 1014 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 1010 to 1 x le, 1 x 1011, 1 x 1012, 1 x 10" or 1 x 1014 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 1011 to 1 x le, 1 x 1011, 1 x 1012, 1 x 1013 or 1 x 1014 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 1012 to 1 x le, 1 x 1011, 1 x 1012, 1 x 10" or 1 x 1014 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 1013 to 1 x le, 1 x 1011, 1 x 1012, 1 x 1013 or 1 x 1014 first phage per ml or mg. In an example, any composition of the invention comprises at least 1 x 1014 to 1 x le, 1 x 1011, 1 x 1012, 1 x 10" or 1 x 1014 first phage per ml or mg.

In an example, the composition comprises one or more doses of the first phage for administration to a subject for medical use, eg, to treat or prevent a disease or condition in the subject. In an example, the composition comprises a single dose. In an example, the composition comprises (or comprises at least) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 doses. In an example, each dose is (or is at least) a 0.5, 1, 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 75, 100, 125, 200 or 250mg or ml dose comprising said phage (ie, the dose is said amount and comprises phage and an excipient, diluent or carrier for example).
In an example, the composition comprises one or more doses of the first phage for administration to a subject for non-medical use, eg, for agricultural use. In an example, the composition comprises a single dose. In an example, the composition comprises (or comprises at least) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 doses. In an example, each dose is (or is at least) a 0.5, 1, 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 75, 100, 125, 200, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 10000, 50000, 100000 mg or ml dose comprising said phage (ie, the dose is said amount and comprises phage and an excipient, diluent or carrier for example). The dose may be dissolved or diluted in a solvent (eg, an aqueous solvent or water) before use for contacting with target bacteria. In an example 1 imperial gallon comprises one dose of the first phage, eg, for agricultural use, such as crop spraying, or for animal or livestock use, such as use as a beverage.
25. The method of Paragraph 22 or 23 or the composition of Paragraph 24, wherein the second DNA is comprised by helper phage DNA and less than 5% of total phage particles comprised by the composition are helper phage particles.
26.
The method or composition of any one of Paragraphs 22 to 24, wherein the second DNA is comprised by helper phage DNA and the composition comprises no more than 1 helper phage particle per 1 x 106 or more of phage particles.
This has been demonstrated in Example 3 and shown to be efficacious for target bacteria killing in Example 4. In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 107 or more of phage particles. In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 10' or more of phage particles. In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 109 or more of phage particles. In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 1010 or more of phage particles.

In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 106 of phage particles. In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 107 of phage particles. In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 108 of phage particles. In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 109 of phage particles.
In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 106 to 1 x 109 of phage particles. In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 106 to 1 x 108 of phage particles. In an embodiment, the composition comprises no .. more than 1 helper phage particle per 1 x 106 to 1 x 107 of phage particles. In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 107 to 1 x 109 of phage particles.
In an embodiment, the composition comprises no more than 1 helper phage particle per 1 x 107 to 1 x 108 of phage particles.
In an example, the proportion of helper phage is determined as plaque forming units (PFU), eg, PFU/ml of the phage composition comprising said number of phage particles. In example, the proportion of non-helper phage (ie, first phage) is determined as number of transduced units of target bacteria (TFU), eg, TFU/ml of the phage composition. PFU is determined on lawns of a susceptible indicator bacterium while TFU is determined in a transduction assay in which a culture of susceptible .. indicator bacteria is infected with the phage composition ensuring surplus of indicator cells (i.e. at a low multiplicity of infection (MOI < 0.01) and number of transduced cells are determined by plating on selective plates.
Thus, the composition may comprise one or more helper phage particles. The level of helper particles is, however, extremely low. This is beneficial as the composition is relatively pure (and useful, for example, therefore as a medicament). It is also useful as the chances of the first phage being replicated is extremely low, providing the advantages of dosing control of phage, containment of phage (eg, in a human or animal body or an environment), and the lack of phage replication reduces the chances of acquiring undesirable genes (eg, antibiotic resistance genes) by the phage.
27. The composition of any one of Paragraphs 1 to 5, the method of Paragraph 22, 23, 25 or 26 or the composition of Paragraph 24, 25 or 26, wherein each first phage particle comprises a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in a target bacterium, wherein the presence in the target bacterium of the NSI-encoded protein or RNA
mediates target cell killing, or downregulation of target cell growth or propagation.
28. The method or composition of Paragraph 27, wherein the NSI encodes a component of a CRISPR/Cas system that is toxic to target cells.
29. The method or composition of Paragraph 28, wherein the component comprises (i) a DNA
sequence encoding a guide RNA (eg, a single guide RNA) or comprising a CRISPR
array for producing guide RNA, wherein the guide RNA is capable of targeting the genome of target bacteria;
(ii) a Cas nuclease-encoding DNA sequence; and/or (iii) a DNA sequence encoding one or more components of Cascade.
30. The method or composition of Paragraph 29, wherein the NSI encodes a Cas nuclease and a guide RNA of the system (or wherein the NSI encodes a Cas nuclease and a CRISPR array for producing guide RNA), wherein the guide RNA is capable of targeting the genome of target bacteria, wherein the guide RNA is capable of guiding the Cas in target cells to mediate target cell killing, or downregulation of target cell growth or propagation.
Optionally, the NSI encodes a Cas9, and a tracrRNA and a CRISPR array for producing guide RNA.
31. The method or composition of any one of Paragraphs 1 to 5 and 22 to 30, wherein the first phage particles comprise no phage structural protein genes.
32. The kit, DNA, method or composition of any preceding Paragraph, wherein the first DNA or first phage DNA is comprised by a high copy number plasmid.
Optionally, the first DNA or first phage DNA is comprised by a medium copy number plasmid.
The meaning of low, medium and high copy number on and plasmids is known to the skilled addressee and these are terms of art. As is known by the skilled person, copy number denotes the average number of plasmid copies per cell. For example, a low copy number plasmid is a plasmid that exists in from 1 to 10 copies per bacterial cell in which the plasmid is harboured; a medium copy number plasmid exists in from 11 to 50 (eg, 11 to 40 or 20 to 30 or 40) copies per cell; and a high copy number is >50 (eg, up to 100, 200, 250, 300, 400, 500, 600 or 700) copies per cell. In an example, the plasmid or vector comprising first DNA is a medium copy number plasmid or vector. In an example, the plasmid or vector comprising first DNA is a high copy number plasmid or vector. An example of common on and plasmids is shown in Table 7.
33. The method or composition of Paragraph 27 or any one of Paragraphs 28 to 32 when dependent from Paragraph 27, wherein the NSI comprises engineered antibacterial means for killing target bacteria.
34. The method of composition of Paragraph 33, wherein the antibacterial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease or meganuclease 35. The composition of any one of Paragraphs 1 to 5 and 24 to 34 for administration to a human or animal subject for medical use.
36. The composition of any one of Paragraphs 1 to 5 and 24 to 34 for administration to a human or animal subject for treating an infection of target bacterial cells, wherein the first phage are capable of infecting and killing the target cells, optionally wherein the infection is a gut microbiome infection.
Optionally, the target cells are E coli cells the first DNA is comprised by a high copy number plasmid.
In an example, the gut microbiome is an upper GI tract microbiome. In an example the target cells are comprised by the upper GI tract of the subject. In an example, the first phage are delivered to the upper GI tract of the subject.
In an example, the gut microbiome is a stomach or small intestine microbiome.
In an example the target cells are comprised by the stomach or small intestine of the subject.
In an example, the first phage are delivered to the stomach or small intestine of the subject.
37. The composition of Paragraph 33 or 34 for use in a contained method of treating a disease or condition of a human or animal subject, wherein the disease or condition is mediated by target bacteria and the target bacteria are comprised by the subject (optionally comprised by a gut microbiome), the method comprising administering the composition to the subject, whereby the target bacteria are exposed to the antibacterial means and killed and propagation of the first phage is contained.

38. A method of treating an environment ex vivo, the method comprising exposing the environment to a composition comprising a population of first phage particles, wherein the composition is obtainable by the method of any one of Paragraphs 22, 23 and 25 to 34, or the composition is according to any one of Paragraphs 1 to 5 and 24 to 34, wherein the environment comprises target bacteria and the first phage infect and kill the target bacteria.
39. The method of Paragraph 38, wherein the method is for containing the treatment in the environment.
40. The composition of any one of Paragraphs 1 to 5 and 24 to 34 for controlling the dosing of the first phage treatment in the subject; or the method of Paragraph 38 or 39 for controlling the dosing of the first phage treatment in the environment.
41. The composition of any one of Paragraphs 1 to 5 and 24 to 34 for reducing the risk of acquisition of foreign gene sequence(s) by the first phage in the subject; or the method of Paragraph 38 or 39 for reducing the risk of acquisition of foreign gene sequence(s) by the first phage in the environment.
CONCEPTS:
[00104] The invention provides the following Concepts, as exemplified by Example 6.
1. A host bacterial cell comprising c) A first DNA; and d) One or more second DNAs;
wherein (v) the DNAs together comprise all genes required to produce a transduction particle comprising a copy of the first DNA packaged by phage structural proteins;
(vi) the first DNA is devoid of at least one functional essential gene (eg, encoding a phage structural protein) required to produce the particle; and wherein the one or more second DNAs comprises said functional essential gene(s);
(vii) the first DNA comprises a phage packaging signal for producing the particle; and (viii) the second DNA is devoid of a nucleotide sequence required for packaging the second DNA
into transduction particles;

wherein the second DNA is required for packaging first DNA to produce particles, wherein the DNAs are operable in the cell for producing transduction particles comprising phage structural proteins that package copies of the first DNA.
2. The cell of Concept 1, wherein the host bacterial cell comprises a) said first DNA; and b) said one or more second DNAs;
wherein (i) the DNAs together comprise encode all phage structural proteins required to produce a packaged transduction particle comprising a copy of the first DNA;
(ii) the first DNA encodes none or at least one, but not all, of the structural proteins; and wherein the one or more second DNAs encode the remainder of the structural proteins;
(iii) the first DNA comprises a phage packaging signal for producing the particle; and (iv) the second DNA is devoid of a nucleotide sequence required for packaging the second DNA
into transduction particles;
wherein the second DNA is required for packaging first DNA to produce particles, wherein the DNAs are operable in the cell for producing transduction particles comprising phage structural proteins that package copies of the first DNA.
3. The cell of Concept 1, wherein (i) the first DNA is comprised by an episome (eg, a plasmid) that is devoid of said essential or structural protein gene(s) and/or the second DNA is comprised by an episome (eg, a plasmid) or a chromosome of the cell.
4. The cell of Concept 1, 2 or 3, wherein all of said essential genes or phage structural protein genes are comprised by the second DNA and the first DNA is devoid of said genes.
5. The cell of any preceding Concept, wherein the first DNA encodes a guided nuclease or a component of a CRISPR/Cas system (optionally, a crRNA or a guide RNA).
6. The cell of any preceding Concept, wherein the first DNA comprises a phage origin of replication and/or the first DNA comprises phage replication genes and/or phage lysis genes; and/or (ii) the first DNA comprises a phage origin of replication but no bacterial origin of replication.

7. The cell of any preceding Concept, wherein each transduction particle is a non-self replicative transduction particle.
8. The cell of any preceding Concept, wherein each particle comprises a phage tail fibre.
9. The cell of any preceding Concept, wherein the essential genes or structural protein genes;
and packaging signal are genes and a packaging signal of a tailed phage, eg, a P2, T4, T7, Phi92, lambda, K1-5 or 933w phage.
10. The cell of any preceding Concept, wherein the first DNA is comprised by a phage genome, wherein the phage genome is integrated in a plasmid; optionally wherein each particle is capable of infecting a target bacterium, the first DNA comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in the target bacterium, wherein the NSI replaces the essential gene(s) or structural protein gene(s) of the phage.
11. The cell of any preceding Concept, wherein each particle is capable of infecting a target bacterium, the first DNA comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in the target bacterium, wherein the presence in the target bacterium of the NSI-encoded protein or RNA mediates target cell killing, or downregulation of target cell growth or propagation; or (ii) wherein each particle is capable of infecting a target bacterium, the first DNA
comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in the target bacterium, or wherein the NSI comprises a regulatory element that is operable in the target bacterium.
12. The cell of Concept 11, wherein the presence in the target bacterium of the NSI or its encoded protein or RNA mediates target cell killing, or downregulation of target cell growth or propagation, or mediates switching off of expression of one or more RNA or proteins encoded by the target cell genome, or downregulation thereof.
13. The kit of Concept 11, wherein the presence in the target bacterium of the NSI or its encoded protein or RNA mediates upregulation of growth or propagation of the target cell, or mediates switching on of expression of one or more RNA or proteins encoded by the target cell genome, or upregulation thereof.

14. The cell of any preceding Concept, wherein the packaging signal is a pac or cos sequence, or is a homologue thereof; or is a direct terminal repeat (DTR).
15. The cell of any preceding Concept, wherein the packaging signal comprises SEQ ID NO: 2 or a sequence that is at least 70, 80, 90, 95, 96, 97, 98 or 99% identical thereto, or is a homologue from a different phage.
16. An isolated DNA, comprising a first DNA as defined in any preceding Concept; or comprising a second DNA as defined in any preceding Concept.
17. The first DNA or the second DNA of any one of Concepts 1 to 15, wherein the DNA is comprised by a plasmid.
18. The second DNA of Concept 16 or 17, wherein the DNA is comprised by a cell, wherein the cell does not comprise the first DNA.
19. A kit comprising a cell as recited in Concept 18, wherein the kit comprises a vector (optionally a plasmid) comprising the first DNA, wherein the vector is not comprised by the cell.
20. The kit of Concept 18 or 19, wherein the cell is a bacterial or archaeal cell.
21. A method of producing a transduction particle composition, the method comprising expressing in a cell of any one of Concepts 1 to 15 phage structural proteins and replicating the first DNA, wherein transduction particles are produced that comprise packaged first DNA; and optionally separating an amount of transduction particles from cellular material wherein an amount of purified transduction particles is obtained.
22. The method of Concept 21, comprising isolating transduction particles.
23. A composition comprising a population of transduction particles obtainable by the method of Concept 21 or 22.
24. The method of Concept 21 or 22 or the composition of Concept 23, wherein the second DNA
is comprised by plasmid DNA and less than 5% of total DNA comprised by the composition is DNA
of said plasmid.

25. The method or composition of any one of Concepts 21 to 24, wherein each transduction particle comprises a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in a target bacterium, wherein the presence in the target bacterium of the NSI-encoded protein or RNA mediates target cell killing, or downregulation of target cell growth or propagation.
26. The method or composition of Concept 25, wherein the NSI encodes a guided nuclease or a component of a CRISPR/Cas system that is toxic to target cells.
27. The method or composition of Concept 26, wherein the component comprises (i) a DNA
sequence encoding a guide RNA (eg, a single guide RNA) or comprising a CRISPR
array for producing guide RNA, wherein the guide RNA is capable of targeting the genome of target bacteria;
(ii) a Cas nuclease-encoding DNA sequence; and/or (iii) a DNA sequence encoding one or more components of Cascade.
28. The method or composition of Concept 26, wherein the NSI encodes a Cas nuclease and a guide RNA of the system (or wherein the NSI encodes a Cas nuclease and a CRISPR array for producing guide RNA), wherein the guide RNA is capable of targeting the genome of target bacteria, wherein the guide RNA is capable of guiding the Cas in target cells to mediate target cell killing, or downregulation of target cell growth or propagation.
29. The method or composition of Concept 25, wherein the NSI comprises engineered antibacterial means for killing target bacteria.
30. The method or composition of Concept 29, wherein the antibacterial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease or meganuclease 31. The cell of any one of Concepts 1 to 15, the DNA of any one of Concepts 16 to 18, or the composition of any one of Concepts 23 to 30 for administration to a human or animal subject for medical use.
32. The cell of any one of Concepts 1 to 15, the DNA of any one of Concepts 16 to 18, or the composition of any one of Concepts 23 to 30 for administration to a human or animal subject for treating an infection of target bacterial cells, wherein the particles are capable of infecting and killing the target cells, optionally wherein the infection is a gut, blood, lung or uterine tract microbiome infection.
33. The composition of Concept 31 or 32 for use in a contained method of treating a disease or condition of a human or animal subject, wherein the disease or condition is mediated by target bacteria and the target bacteria are comprised by the subject (optionally comprised by a gut, blood, lung or uterine tract microbiome), the method comprising administering the composition to the subject, whereby the target bacteria are exposed to antibacterial means encoded by the first DNA and killed, and propagation of the transduction particles is contained.
34. A method of treating an environment ex vivo, the method comprising exposing the environment to a composition comprising a population of particles, wherein the particles are capable of transducing first DNA into the target cells comprised by the environment, the first DNA encoding antibacterial means that is toxic to taraget cells whereby target cells are killed, wherein the composition is obtainable by the method of any one of Concepts 21, 22 and 24 to 30, or the composition is according to any one of Concepts 23 to 30.
35. The method of Concept 34, wherein the method is for containing the treatment in the environment.
36. The composition of any one of Concepts 23 to 30 for controlling in a human or animal subject the dosing of transduction particle treatment of a target bacterial cell infection in the subject, wherein the particles are capable of transducing first DNA into the target cells, the first DNA encoding antibacterial means that is toxic to taraget cells whereby target cells are killed; or the method of Concept 34 or 35 for controlling the dosing of the particle treatment in the environment.
37. The composition of any one of Concepts 23 to 30 for reducing in a human or animal subject the risk of acquisition of foreign gene sequence(s) by the particles in the subject; or the method of Concept 34 or 35 for reducing the risk of acquisition of foreign gene sequence(s) by the particles in the environment.
[00105] Optionally, each particle is capable of infecting a target bacterium, the first DNA comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in the target bacterium. For example, the NSI encodes an antimibacterial agent. For example, the NSI encodes a guided nuclease (eg, a Cas, TALEN, zinc finger nuclease or meganuclease). For example, the NSI

encodes a component of a CRISPR/Cas system. For example, the nuclease or system is operable in the target cell to cut a target nucleic acid (DNA or RNA) sequence comprised by the target cell (eg, comprised by a chromosome or episome thereof).
[00106] In an embodiment, an essential gene may be omitted from the first DNA, whereby the first .. DNA is devoid of the essential gene. In another embodiment, the first DNA
may comprise a mutant of the essential gene which does not provide the essential gene function (ie, this is not a functional essential gene). In another embodiment, first DNA may comprise a non-expressible form of the essential gene, eg, wherein a regulatory element of the gene has been deleted or mutated so that the gene does not function, eg, does not express its encoded protein.
.. [00107] Optionally, the essential genes are not phage terminase genes.
Optionally, the first DNA is not devoid of all phage terminase genes. Optionally, the first DNA is not devoid of phage structural protein genes. Optionally, the essential genes are not phage structural protein genes. Optionally, the essential genes are not phage terminase genes and not phage structural protein genes. Optionally, the first DNA does not comprise an origin of replication (on) operable in a bacterial host cell for .. replication of the first DNA (and optionally the first DNA further comprises a pahge orgin of replication).
[00108] Optionally, each particle comprises a tail fibre (eg, a tail fibre comprising one or more tail fibre domains of a wild-type phage).
[00109] Optionally, each particle comprises phage capsid proteins, a packaging signal (comprised by the first DNA) and optionally phage replication gene(s), wherein all of these components are proteins, packaging signal and gene(s) of the same phage (eg, a wild-type phage).
[00110] Optionally, each particle comprises phage capsid proteins, a packaging signal (comprised by the first DNA) and optionally phage replication gene(s), wherein the packaging signal and gene(s) are components of the same phage (eg, a wild-type phage) and the capsid proteins are proteins of a different phage (eg, a wild-type phage).
[00111] Optionally, the first DNA is devoid of all phage structural protein genes and the second DNA
comprises all phage structural protein genes required for packaging first DNA
to produce particles, and for example the packaging signal is a packaging signal of a phage selected from a Caudovirales, Myovirideae, Podovirideae or Siphovirideae phage. Optionally, the first DNA is devoid of all phage structural protein genes and the second DNA comprises all phage structural protein genes required for packaging first DNA to produce particles, and for example the packaging signal is a packaging signal of a phage selected from a P2, Phi92, T7, lambda, 933w, K1-5 and T4 phage.
[00112] Optionally, the first DNA is devoid of all phage structural protein genes and the second DNA
comprises all phage structural protein genes required for packaging first DNA
to produce particles, and for example the phage structural protein genes of the second DNA ae genes of a phage selected from a Caudovirales, Myovirideae, Podovirideae or Siphovirideae phage. Optionally, the first DNA is devoid of all phage structural protein genes and the second DNA comprises all phage structural protein genes required for packaging first DNA to produce particles, and for example the phage structural protein genes of the second DNA ae genes of a phage selected from a P2, Phi92, T7, lambda, 933w, K1-5 and T4 phage.
[00113] The essential or structural protein gene(s) and packaging signal may be structural protein genes and a packaging signal of a tailed phage. Alternatively, the essential or structural protein gene(s) and packaging signal may be essential or structural protein gene(s) and a packaging signal of a temperate phage. Alternatively, the essential or structural protein gene(s) and packaging signal may be essential or structural protein gene(s) and a packaging signal of a lytic phage.
[00114] Optionally, the essential or structural protein gene(s) and packaging signal may be essential or structural protein gene(s) and packaging signal of a P2, T4, T7, Phi92, lambda, K1-5 or 933w phage.
Optionally, the essential or structural protein genes and packaging signal may be essential or structural protein genes and a packaging signal of a tailed phage. Optionally, the essential or structural protein genes and packaging signal may be essential or structural protein genes and a packaging signal of a Caudovirales phage.
Optionally, the essential or structural protein genes and packaging signal may be essential or structural protein genes and a packaging signal of a Myovirideae, Podovirideae or Siphovirideae phage. Optionally, the essential or structural protein genes and packaging signal may be essential or structural protein genes and a packaging signal of a P2 phage. Optionally, the essential or structural protein genes and packaging signal may be essential or structural protein genes and a packaging signal of a T7 phage. Optionally, the essential or structural protein genes and packaging signal may be essential or structural protein genes and a packaging signal of a Phi92 phage. Optionally, the essential or structural protein genes and packaging signal may be essential or structural protein genes and a packaging signal of a lambda phage. Optionally, the essential or structural protein genes and packaging signal may be essential or structural protein genes and a packaging signal of a 933w phage.
Optionally, the essential or structural protein genes and packaging signal may be essential or structural protein .. genes and a packaging signal of a K1-5 phage. Optionally, the essential or structural protein genes and packaging signal may be essential or structural protein genes and a packaging signal of a T4 phage.
[00115] Optionally, any phage herein may be a tailed phage. Optionally, any phage herein may be a Caudovirales phage. Optionally, any phage herein may be a Myovirideae, Podovirideae or Siphovirideae phage.
Optionally, any phage herein may be a P2, T4, T7, Phi92, lambda, K1-5 or 933w phage. Optionally, any phage herein may be a P2 phage. Optionally, any phage herein may be a T7 phage.
Optionally, any phage herein may be a Phi92 phage. Optionally, any phage herein may be a lambda phage.
Optionally, any phage herein may be a 933w phage. Optionally, any phage herein may be a K1-5 phage. Optionally, any phage herein may be a T4 phage.
[00116] An essential gene as per the invention may be any nucleic acid sequence (not necessarily encoding a protein) that is required to produce the particle or a phage. In an example, an essential gene encodes a protein.
In an example, the essential gene(s) are not a packaging signal or phage origin of replication.
[00117] For example, each essential gene is selected from = a phage gene encoding a phage structural protein;

= a phage gene encoding a gene expression activator (eg, a RNA polymerase, such as a RNA
polymerase of coliphage T4, T3 or K1-5); gene Q (eg, gene Q of coliphage lambda); gene Rin (eg, Rin of staphylococcal phage NM1), gene ogr (eg, ogr of coliphage P2) or gene delta (eg, delta of coli satellite phage P4));
= a phage RNA metabolism gene (ie, encoding a protein that is comprised by RNA metabolism system of a phage);
= a phage DNA metabolism gene ie, encoding a protein that is comprised by DNA metabolism system of a phage);
= a phage DNA packaging gene (ie, encoding a protein that is comprised by DNA packaging .. system of a phage);
= a phage gene encoding a protein necessary for bacterial cell lysis.
[00118]For example, each essential gene is selected from the following P2 genes (function is given in brackets):
= Gene B (DNA replication);
= Gene Q (portal protein);
= Genes P and M (terminase);
= Genes 0, L and N (capsid);
= Genes X, R, S, v, W, J, I, H, G, Fl, FIT, E, E', T, U, D (tail and tail fiber);
= Genes X, Y, lysA, lysB, lysC (lysis cassette); and = Ogr (activator of late gene expression).
[00119]For example, each essential gene is selected from the following T7 genes (function is given in brackets):
= Gene 1 (RNA polymerase);
= Genes 1.3, 2.5, 3, 3.5, 4a, 5, 6 (DNA and RNA metabolism);
= Genes 6.7, 7.3, 8, 9, 10A, 10b, 14, 15, 16 (capsid and internal core);
= Genes 11, 12, 17 (tail);
= Genes 17.5, 18.5, 18.6, 18.6 (lysis); and = Genes 18 and 19 (terminase).
[00120] The phage is a virus that is capable of infecting a target bacterial cell or the transduction particle is capable of transducing a target bacterial cell, ie, is capable of introducing first DNA or a portion thereof into the target cell.

[00121] In an embodiment, the kit comprises a cell (eg, a bacterial cell) comprising the first and second DNAs. For example, the cell is a bacterial cell, the first DNA is comprised by an episome (eg, plasmid) that is devoid of said essential or structural protein gene(s) and the second DNA is comprised by a chromosome of the cell; and optionally all essential or phage structural protein genes are comprised by the second DNA and the first DNA and episome is devoid of said genes.
Optionally, the first DNA (or portion thereof) encodes a guided nuclease or a component of a CRISPR/Cas system (eg, a Cas, Cascade protein, crRNA, guide RNA or tracrRNA).
Optionally, the first DNA (or portion thereof) encodes a crRNA or a guide RNA.
[00122] Optionally, the first DNA comprises a phage origin of replication.
[00123] Optionally, the transduction particle is a non-self replicative transduction particle.
[00124] Optionally, the first DNA comprises phage replication genes and/or phage lysis genes.
[00125] Optionally, the phage or particle comprises a tail fibre, eg, a tail fibre (or domain thereof) of a wild-type phage that comprises the structural proteins.
[00126] In an alternative to a bacterial cell, the cell is an archaeal cell and the phage is a virus that is capable of infecting archaea or the transduction particle is capable of transducing archaea.
[00127] Optionally, a packaging signal herein is a pac or cos sequence, or is a homologue thereof; or a direct terminal repeat (DTR).
[00128] 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 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. US Patent Application number 15/985,658, PCT/EP2018/082053, all publications and patent applications and all US equivalent patent applications and patents 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.
[00105] 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.
[00106] The term "or combinations thereof" or similar 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, CABABB, 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.
[00107] Any part of this disclosure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.
[00108] 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
Example 1: Efficient phage CRISPR delivery vehicle production [00109] Background We designed a strategy for efficient production of phage particles comprising components of a CRISPR/Cas system for killing target E coli Nissle strain bacteria. So our phage composition will consist of a lysate primarily containing CRISPR/Cas system components packaged in phage particles which will be devoid of phage protein-encoding sequences and which will have no or a very low proportion of helper phage. Also the strategy will work alternatively in less well 79amster7979ius79 phage/bacterial strain combinations. Worked exemplification is provided in Examples 3 and 4.
[00110] Outline of strategy for CRISPR/Cas component packaging in hitherto unknown phages (a) Identify a high or medium copy number cloning/lshuttle vector (capable of cloning and propagation in a first E coli strain (cloning production strain) and then transfer to a second bacterial host strain of interest (target host strain)), the vector containing an E coli on for replication in the E coli cloning production strain;
(b) Isolate temperate phage against the target host (second) bacterium;
(c) To produce the cloning production strain, identify or engineer a phage production strain of the host target bacteria (or other bacteria) that has an inactive CRISPR/Cas system (eg, a repressed Cas3 or other nuclease) and/or lacks the target protospacer found in the second strain, and which can be infected and lysogenized with the temperate phage; or repress or inactivate the system in the production strain;
(d) In that production strain make a lysogen using the temperate phage (helper phage) .. and test that it can be induced;
(e) Identify the packaging sequence (pac or cos) using PhageTerm (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557969/) on whole genome sequenced phage;
Delete the pac/cos packaging signal sequence in the helper phage in the production strain bacteria;
(g) Incorporate the packaging signal in the shuttle vector along with a CRISPR-array or single gRNA-encoding sequence (and optionally other components of the CRISPR/Cas system, such as a Cas9-encoding nucleotide sequence and optionally tracrRNA-encoding sequence, or Cas3 and/or Cascade-encoding sequence);
(h) Transform the vector into production host strain;
(i) Induce (eg, UV or mitomycin C induce) and harvest phage comprising the CRISPR/Cas component(s). Alternatively, use a system with inducible RecA in trans to simulate SOS
(needs to be activated RecA).
[00111] Example of the above specifically for E coli Nissle using phage P2:
[00112] Nissle is useful due to its GRAS (Generally Regarded as Safe) status and P2 has a relatively broad host range (most E coli, Shigella, Klebsiella, Salmonella in 79amster79 to DNA delivery into e.g. Pseudomonas; Kahn et al 1991, "Bacteriophage P2 and P4", Methods in Enzymology, vol 204, pp264-280).

[00113]We will use pUC19 or other high or medium copy number cloning vector.
Temperate phage P2 can lysogenize Nissle. Most E coli K strains have an inactive CRISPR/Cas system and can be infected by P2 and thus all regular cloning hosts can be used (here exemplified by E coli TOP10).
[00114]P2 is introduced into TOP10 to produce a lysogen. P2 cannot be induced with mitomycin C or UV but we will use the epsilon anti-repressor from the parasite phage P4 that derepresses P2 and makes it go into lytic phase. We will express this gene from an inducible promoter in the production host strain.
[00115] The 325 bp packaging signal sequence of SEQ ID NO: 2 will be used.
[00116] The packaging sequence will be deleted in the P2 prophage of the lysogenic production TOP10 strain.
[00117] A pUC19 shuttle vector encoding a guide RNA that targets the genome of the target Nissle strain (or alternatively comprising a CRISPR array for producing such a guide RNA) will be constructed and the packaging signal SEQ ID NO: 2 will be added. If the target Nissle harbours its own endogenous CRISPR/Cas system, we will use an activation strategy to activate the endogenous Cas3 by including Cas activating genes in the vector. If not, we will include an exogenous Cas3-encoding nucleotide sequence (and optionally one or more nucleotide sequences encoding one or more required Cascade components) in the vector for expression in the target Nissle. We will transform the vector into the TOP10 production strain, induce the P4 anti-repressor and harvest phage comprising the CRISPR/Cas component(s).
[00118] Since the induced (helper) phage DNA does not contain a packaging signal we will be able to isolate particles with only the vector DNA packaged. Thus, we will obtain a composition comprising such phage which can be used to infect target Nissle E coli bacteria and introduce the CRISPR/Cas component(s) therein for killing the target bacteria.
Example 2: MGEs, Genomics Islands etc [00119] Overview of possible different MGE packaging strategies follow.
[00120] Applicable to different types of phages:
= Identify packaging signal and structural genes in the helper phage = Delete packaging signal in helper phage and place on plasmid comprising MGE
= Place both helper and plasmid in production strain = Induce structural gene transcription of helper to get production of helper-phage-packaged MGEs [00121]For using parasitic mobile elements (P4 phage or SaPI etc) activation of helper phage structural genes is done by induction of a helper phage activator obtained from the parasitic element Delta in P4 or one, more or al of ptiA/B/M in SaPI.
[00122]If one wants smaller size particles one can choose to package in a parasite-size capsid (typically 10-20 kb) by including in the MGE or vector P4 Sid and psu or cpmA/B from a SaPI.
[00123] One can use defective helper phages where at least the packaging signal has been removed and structural genes are either on a plasmid or integrated as a cryptic prophage in the production host.
If for some reason one cannot use this approach and need to use functional helper phages, one will include in the MGE or vector the genes on the parasite that hijack the phage packaging machinery to preferentially package parasite DNA (in our case CGVTM) over phage DNA.
[00124]List of the minimal genes one could include on a plasmid vector from P4.
[00125]P4 sequence: see https://www.ncbi.nlm.nih.gov/nuccore/x51522 [00126]Cos packaging site: SEQ ID NO: 3 [00127] The homologous sequence between P2 and P4; this may be used as an alternative packaging signal in the MGE or vector: SQ ID NO: 4 [00128]For small capsid size (packages 11.4kb instead of 33.5 kb) Sid and/or Psu can be included in the MGE or vector:-Sid: SEQ ID NO: 5 Psu: SEQ ID NO: 6 [00129] To activate helper phage P2, Delta from P4 can be included in a host cell genome (provided separately in a host cell, not on the MGE or vector to be packaged) Delta: SEQ ID NO: 7 [00130] Minimum genes to include in the host chromosome/episome from P2.
P2 sequence (acc.number: NC_001895) [00131]Figure 1 shows the genetic map of P2 genome with non-essential genes boxed ¨ one, more or all of these can be excluded (but Cos is always deleted). Cos is deleted and preferably the whole region from int through Cos. This region may, for example, be swapped with a resistance marker while the orf30 and fun(Z) genes are left intact or may be deleted.
[00132] Thus in an embodiment of any aspect herein of the invention, int through to Cos (ie, including int and Cos) are omitted from the helper phage DNA or second DNA or a homologous region is omitted when different phage are used as the basis for the helper phage DNA or second DNA.
The sequences of various stretches of the P2 genome are as follows:-[00133]"Q" through "S": SEQ ID NO: 8 [00134]"V" through "G": SEQ ID NO: 9 [00135] "Fl" through "ogr": SEQ ID NO: 10 [00136]Minimal genes to include from a SaPI on a vector or MGE.
Several different SaPI systems exist. Figure 2 shows one of the well characterized SaPIs (SaPIbov1), which exploits phages phill or phi80a1pha as helper phage. SaPIbov 1 sequence (acc.number:
AF217235.1) Packaging signal [00137]If one uses a defective helper phage with deleted packaging signal one can use that signal from the helper phage and include it in the DNA to be packaged. An example from S. aureus phill (acc. Number: AF424781) is SEQ ID NO: 11.
[00138]For small capsid size (packages 15.8 kb instead of 43.6 kb), one can include cpmA and/or cpmB in the MGE or vector (SEQ ID Nos: 12 and 13).
[00139] To activate helper phage phill one can include one, more or all of ptiA, B and M (provided separately in the production host cell and not on the MGE or vector to be packaged) (SEQ ID Nos:
14-16).
[00140]Minimum genes to include in the host chromosome/episome from phill.
Phill sequence (acc.number: AF424781) [00141]gene #29 (terS) through gene #53 (lysin): SEQ ID NO: 17 [00142]A list of phage that work with SaPIs [00143] Different SaPIs are linked to different helper phages (see Table 2 below) [00144] One can mutate the helper phage to only contain structural genes to direct the phage to package in smaller capsids. If only looking at the genes responsible for small capsid packaging (cpmA
and cpmB) these are highly conserved among staphylococci indicating that they will function to redirect packaging in a variety of phages broader than the list below (Table 2).
Example 3: Design, construction and in vitro efficacy of a synthetic transduction particle delivery platform Executive summary [00145] SA100 is a synthetic DNA delivery vehicle comprising non-self-replicative transduction particles comprising phage coat proteins packaging DNA plasmids that comprise nucleic acid sequences endoding CRISPR/Cas components, wherein the particles are capable of infecting target E
coli host cells to introduce the DNAs therein. Introduced DNAs are then able to be used in the host cells to produce the components. We call such DNAs CRISPR-Guided Vectors (CGVsTm). As shown in this example, we made such particles using a defective helper phage.
Upon production of the synthetic particles we obtain a pure synthetic lysate devoid of any phage nucleic acid encoding phage proteins. We show that this lysate is capable of very efficient delivery of a CGVTM to E. coli in vitro. The particles are non-self-replicative and require the helper phage for replication; thus usefully the lysate is pure and removes the possibility of replication of the particles containing the CGVTM.
This is useful to contain the action of the antibacterial, to allow for more controlled (eg, predetermined) dosing for administration to a human or animal subject or an environement for example, and the elimination of replication in the subject or environment reduces the chances of phage acquiring undesirable genes or traits from surrounding phage or bacteria (eg, undesirable antibiotic resistance genes) ¨ and even then the limitation of particle replication of the invention affords a way of reducing or eliminating spread of such genes or traits. This may be of interest to authorities such as the US FDA or USDA who consider such aspects when approving medicines or antibacterials for use in the environment.
Study objectives [00146] Objective 1: Design and engineer the fully synthetic phage-based CGV
delivery platform SA100.

[00147] Objective 2: Use SA100 to demonstrate CGV delivery to E.coli.
Materials and methods Bacterial strains and growth conditions [00148] We made a bacterial production strain for producing the synthetic SAE100 transduction partiles. E coli strain C-2323 harboring phage P2 was used as starting material for engineering our SA100/CGV production strain (gift from Gail E. Christie at the Virginia Commonwealth University).
Other strains used for testing SA100 and infectivity were EMG-2, C-la and C-1792, (purchased from the Coli Genetic Stock Center http://cgsc2.bio1ogy.ya1e.edu/).
[00149] All bacterial cultures were grown in LB medium supplemented with 2mM
MgCl2. When appropriate, cultures were supplemented with 50 g/mL kanamycin or 50 g/mL
spectinomycin for selection of defective prophage and CGV, respectively. In SA100 infection studies, the medium was supplemented with 2.5 mM CaCl2 to aid adsorption of the SA100 particles to bacterial cells. For induction of the CRISPR/Cas system, cells were exposed to 0.5mM IPTG and 2mM
theophylline.
Construction of defective helper prophage [00150] The pTK-red recombineering plasmid (p'72) was transformed in strain C-2323.
A KamR kanamycin marker gene was amplified using oligos with 50 bp homology to the regions of the P2 prophage immediately up- and downstream of the approximately 10 kbp region of P2 that we wanted to delete (see Figure 3). Following successful replacement of the desired region with the KanR marker gene, the thermosensitive pTK-red recombineering plasmid was cured.
Engineering CGV (p94) for packaging in the SA100 particles [00151] CGV p77 was constructed using plasmid p53 as a template. Plasmid p53 is a plasmid encoding components of a CRISPR/Cas system, the plasmid comprising nucleic acid sequences endoding a tracrRNA and the Cas9 protein from Streptococcus pyogenes (SpCas).
Nucleic acid sequence for produing crRNAs was obtained, expression driven by a plac promoter. The two fragments were assembled using Gibson assembly (NEB E55 10S) and transformed into E. coli competent cells. Assembled p77 plasmid was verified by full sequencing.
[00152] p77 harboring the inducible CRISPR/Cas system was used as the backbone for insertion of a fragment comprising an arabinose inducible promoter transcribing the region sid through cos from the satellite phage P4 (Figure 3).
[00153] Both fragments were PCR amplified and cloned by restriction enzyme cloning. The resulting SA100 packable CGV (p94) was sequence verified.

Production of SA100 packaged CGVs [00154] CGV p94 was transformed into the previously constructed strain harboring the defective prophage thereby generating production strain #189.
[00155] The production strain was grown exponentially for approximately 10 generations in the presence of kanamycin and spectinomycin, ensuring balanced growth of the population. At optical density (0D600) of 1 the culture was induced with 1% arabinose and incubation was continued until optical density measurements were low and stable. After spinning away cell debris the lysate was filtered (0.45 tim) and stored at +4C until further use.
Transduction protocol to determine SA100 titers.
[00156] Determination of titers was done by mixing the SA100 lysate with a suitable bacterial strain ensuring at least 100-fold surplus of bacterial cells in presence of 2.5 mM
CaCl2. Following 30 min incubation, the bacteria were diluted and plated on LB containing spectinomycin for enumeration of bacteria that had received the p94 CGV by transduction. To verify purity of the SA100 lysate, undiluted lysate was spotted on a lawn of bacteria (C-1a) known to support P2 proliferation using standard soft-agar overlay. Absence of plaque formation was used to verify purity.
DNA delivery assay [00157] A transduction protocol was used in which the SA100 lysate was diluted before infecting the bacterial culture to obtain multiplicities of infection (MOT) ranging from 0.01 to 100 effectively spanning a 4 log-ratio of SA100 to bacterial cells.
[00158] Following 30 min infection in the presence of 2.5 mM CaCl2 the bacteria were enumerated on LB plates with and without spectinomycin selection for delivery of the p94 CGV. The obtained numbers were used to calculate the percentage of the population infected at a given MOT.
Results Design of the SA100 vehicle [00159] The overall design of the SA100 E. coli production strain harboring CGV and defective prophage is outlined schematically in Figure 4. We used the native E. coli phage P2 integrated as a prophage on the chromosome of the production strain as our starting material.
In the prophage, we deleted genes that are essential for phage to initiate and carry out its own proliferation thus generating a defective helper phage.

[00160] We then placed expression of the structural genes needed to produce SA100 transduction particles under the control of an inducible promoter allowing us to turn on production of particles.

Finally, we removed the packaging sequence normally used by the phage to package its DNA into the phage particles from the phage DNA and placed it on our CGV (see cos sites shown in Figure 3).
Upon induction of the phage structural genes we got packaging of CGV DNA into the particles that were subsequently released by lysis of the production cells.
Engineering defective helper phage

[00161] E. coli strain C-2323 harboring phage P2 on the chromosome was used as starting material for engineering the production strain with the defective helper phage. A region on the P2 phage comprising integrase, promoters for initiation of phage proliferation, origin of replication, DNA
replication genes and the cos site (DNA sequence 86amster8686i when packaging DNA into phage particles) (see boxed P2 DNA shown in Figure 3), was replaced by a kanamycin marker using recombineering. The site-specific integration of the marker and deletion of approximately 10 kbp of phage DNA was verified by sequencing.
Engineering CGV to enable SA100 packaging

[00162] We used a single vector (p77) with the CRIPSR/Cas system expressed from inducible promoters for engineering our SA100 packable CGV.

[00163] To enable activation of the defective P2 helper phage and CGV
packaging into synthetic transduction particles, we cloned the genetic region comprising sid-delta-psu-cos (cos packaging site) from the satellite phage P4 into the p77 plasmid. The cloned P4 region (SEQ ID
NO: 18), which is known to be able to activate the P2 phage was already cloned in another vector p93 where it was expressed from an arabinose inducible promoter. The resulting CGV (p94), containing all elements necessary for P2 activation and SA100 packaging, was verified by complete sequencing. P4 genomic architecture and the region cloned in p94 is shown in the lower box in Figure 3A and the p94 genomic map is shown in Figure 3B.
Production of SA100 packaged CGVs

[00164] The p94 CGV was electroporated into the strain harboring the defective prophage resulting in production strain #189.

[00165] SA100 packaged CGVs were produced as described above. Titers of the SA100 packaged CGV was approximately 1010 TFU/mL determined by transduction of SA100 packaged p94 into 3 strains of E. coli. As expected, no contamination of the native P2 phage could be observed (Table 3 below). Accordingly, we can assume a native phage contamination of less than 1 in 1e9 per synthetic particle.

CGV delivery to E. coli using SA100

[00166] To determine the number of phages required for 100% infection of an E.
coli population, we performed an infection experiment varying the ratio of SA100 particles to bacterial cells, the so-called multiplicity-of-infection (MOT) from 0.01 to 100.

[00167]We observed that a MOT of 1 meaning one SA100 particle per E. coli cell, was sufficient for 100% infection of the bacterial population (Figure 4).
Discussion and conclusions

[00168] SA100, a fully synthetic phage-based CGV delivery vehicle was designed and constructed.
Upon expression in a production strain also harboring a compatible CGV, a high titer lysate of SA100 packaged CGVs devoid of native phage contamination was produced. Finally, the obtained SA100 packaged CGVs were efficiently delivered into E. coli.
Example 4: CGV in vitro killing of E. coli following delivery by the SA100 synthetic transduction particle delivery platform Executive summary

[00169]We used the SA100 synthetic DNA delivery vehicle to deliver 87amster8787 CRISPR guided vectors (CGVTM) to two E. coli target strains. Upon induction of the system we could demonstrate up to 4 logs killing of the target population.
Introduction

[00170]We have previously engineered the phage-based synthetic DNA delivery vehicle SA100 (Example 3) for delivery of CGVs to E. coli target cells. Here, we 87amster8787 a CGV for delivery by the SA100 vehicle and subsequently tested the efficacy in killing E. coli target cells.
Study objectives

[00171] Objective 1: Optimize CGV for delivery by SA100.

[00172] Objective 2: kill E.coli by SA100 delivered CGVs Materials and methods Bacterial strains and culture conditions

[00173] All bacterial cultures were grown in LB medium supplemented with 2mM
MgCl2. When appropriate, cultures were supplemented with 50 g/mL kanamycin or 50 g/mL
spectinomycin for .. selection of defective prophage and CGV, respectively. In SA100 infection studies, the medium was supplemented with 2.5 mM CaCl2 to aid adsorption of the SA100 particles to bacterial cells. For induction of the CRISPR/Cas system, cells were exposed to 0.5mM IPTG and 2mM
theophylline.
Target strains MG1655_pks and XL1-blue_pks were constructed by recombineering of a 20-nucleotide sequence complementary to the guide RNA spacer sequence in our CGVs into the lacZ
gene of MG1655 and XL1-blue thereby generating target strains MG1655_pks and XL1-blue_pks, respectively.
Production of SA100 packaged CGVs

[00174] CGVs (p94 and p114) were separately transformed into a respective strain harboring the defective prophage thereby generating production strain #189 and #226, respectively.

[00175] The production strains were grown exponentially for approximately 10 generations in the presence of kanamycin and spectinomycin ensuring balanced growth of the population. At optical density (0D600) of 1 cultures were concentrated x10 and induced with 1%
arabinose and incubation was continued until optical density measurements were low and stable. After spinning away cell debris the lysate was filtered (0.45 tim), further concentrated approximately x4 using Amicon Ultra-15 centrifugal filters and stored at +4C until further use.
CGV delivery and killing

[00176] Exponentially growing cultures of the E. coli target strains were infected with SA100 packaged CGVs p94 or p114 at a MOI of 20 in the presence of 2.5mM CaCl2.

[00177] Following 30 min incubation at 37 degreees centigrade, the bacterial cultures were serially diluted and plated on LB plates containing IPTG and theophylline (for inducing the expression of CRISPR/Cas components) to investigate CGV mediated killing or on LB+spectinomycin to investigate CGV delivery to the target populations.

Results Optimising CGV DNA by increasing copy number

[00178] CGV p94 was previously shown to be efficiently delivered to E. coli (Example 3). This CGV
contains the CloDF13 origin of replication, in which a single SNP will increase copy number from 7 to 80 according to Stuitje et al. 1981, "Identification of mutations affecting replication control of plasmid Clo DF13", Nature, vol 290, pp264-267. By incorporating the SNP into an oligo used for PCR amplification of the whole CGV, we created p114 containing the mutated CloDF13 on (SEQ ID
NO: 23). Sequence verification was carried out.
CGV delivery by SA100

[00179] We previously showed efficient CGV delivery using p94 packaged in SA100 (Example 3).
Here, we compared delivery of CGVs p94 and p114 to MG1655_pks (Fig 6A and additionally p114 delivery to XL1-blue_pks (Fig 6B. Using a multiplicity of infection (MOI) of 20 we demonstrate 100% delivery to the target populations.
Killing efficacy of SA100 delivered CGV

[00180] Using the same experimental setup as above (target strains, SA100 packaged CGVs and MOI
of 20) we investigated the ability of the delivered CGVs to kill the target cell upon induction of the expression of the CRISPR/Cas system.

[00181] The p94 CGV targeting the pks DNA sequence in MG1655_pks was able to reduce the target strain approximately 2 logs while p114 targeting the same sequence reduced the target population approximately 4 logs (Fig 7). The same magnitude of killing was observed when p114 was targeting pks in the XL1-blue_pks target population (Fig 7).
Discussion and conclusions Following SA100 delivery of CGVs to target cells we were able to increase killing of the target population from approximately 2 logs using p94 to 4 logs using p114 probably due to a high copy number CGV.

Example 5: In vivo antibacterial delivery to gut microbiome using non-self-replicative particles & target cell killing in microbiome setting Executive summary

[00182]E. coli AMG1655-pks was used to colonize the murine gut. We show statistically significant delivery of CGVTM pSNP114 into MG1655-PKS by SA100 delivery vehicle (synthetic non-self-replicative phage particles, see Examples 3 and 4). We observered a reduction in survival upon activation of the CGVTM targeting E. coli MG1655-pks.
Introduction

[00183] We have previously shown that E. coli ATCC43888 colonizes the a murine gut of NMRI
mice model (data not shown) and that activating of a CRISPR array in E. coli ATCC43888 in the mouse gut reduces survival, where guide RNA produced using the array target the E coli genome for Cas cutting (data not shown). In this Example we set out to assess the survival of a CRISPR/Cas-based antibacterial (a CGVTM) delivered by a Synthetic NanoboioticTM, namely SA100, into MG1655-pks.
Study objective:

[00184] Establish survival reduction of E. coli MG1655-pks in the murine gut microbiome by delivery of CGVTM pSNP114 with Synthetic Nanoboiotic TM SA100.
Materials and methods

[00185]E coli was E coli MG1655 with pks fragment inserted, with a strep resistance marker, phylo group A.

[00186] Cells were grown overnight in LB media supplemented with 50 g/mL
spectinomycin. Next day the OD was measured and the cultures were diluted down to 108 CFU/mL.
Vehicle, induced and uninduced groups are shown in Table 4.
= 15 female NMRI mice, 26-30 gram (Taconic) = Inducer solution containing theophylline and L-arabinose Laboratory animal facilities and housing of mice

[00187] The temperature was 21 C +/- 2 C and could be regulated by heating and cooling, and light/dark period was in 12-hours intervals of 6 a.m.- 6 p.m./6 p.m ¨ 6 a.m.
Mice had free access to food and to domestic quality drinking water until 3 days prior to the study.
Mice were housed 6-7 mice /cage in the standard facility with bedding from Aspen Wood from Tapvei and paper strands from Sizzle-nest as nesting material until 1 day prior to the study and there after moved into the GMO
facility 1 mouse per cage. The From day 0 the wooden bedding was changed to white paper bedding to facilitate collection of faeces from the cages Preparation of streptomycin drinking water

[00188] 6 liters of 5 g/L of streptomycin was prepared by dissolving 6.94 g streptomycin sulphate / L
of sterile water. Mice were given streptomycin water starting day -3 and throughout the study.
Inoculation of mice

[00189] Inocula was formulated prior to inoculation as ready to use suspensions. Mice were inoculated with 0.25 ml by oral gavage at 8 am day 0. A titre of approximately 10" TFU/ml was used.
Treatment of mice

[00190] Mice were dosed with inducers and 10" TFU/mL SA100 by oral gavage at 7 am and 1 pm day 1 and again at 7 am day 2. Inducers were mixed immediately prior to each treatment time point prior to the treatment mice were gavaged with antacid.
Collection of faecal samples from cages

[00191] Immediately prior to inoculation 0.5 ml faecal samples were collected from cages into three 15 ml Nunc tubes. Day 1 and 2 after inoculation, 0.5 ml faecal samples were collected prior to treatment at 7 am and mice were moved to a new cage. Day 2 after inoculation faecal samples were also collected 4 hours after treatment CFU determination [0019210.5 ml faecal matter was dissolved in 5 ml sterile saline by vortexing multiple times during 1-4 hours. Colony counts were determined by 10 fold serially dilution of the faecal samples in sterile 0.9% NaCl and 20 111_, spots were applied to agar plates. The lower detection limit of faecal samples was 50 CFU/ml. All agar plates were incubated 18-22 hrs at 35 C in ambient air.
[00193] Delivery of the CGV was enumerated by plating on LB + streptomycin +
spectinomycin plates plates. Abudance of the target strain was enumerated by plating on LB
with streptomycin only.

Clinical monitoring of mice [00194] The body weight of the mice were monitored throughout the study and the mice were scored 0 ¨ 6 based on their behavior and clinical signs (Table 5). Mice were euthanized if reaching score 3 or 20% weight loss.
Results [00195]Microbiome analysis of mice prior to inoculation No bacteria was detected in 2 out of 3 of the pooled samples collected prior to inoculation. One of the pooled samples had 6.54 10g10 CFU/ml of small white colonies. MALDI analysis indicated Paenibacillus odorifer with a score value of 1.71 and as second most likely Staphylococcus xylosus with a score value of 1.43 both with a low rank quality. Also in the 2 other pooled samples 3-4 10g10 CFU/ml of small white colonies appeared after 48 hours of incubation.
[00196] At 24 h after inoculation the CFU levels in the faecal samples ranged from 6.1 ¨ 8.6 10g10 CFU/sample as determined on the LB+ strep agar plates. Similar levels were observed day 2 after inoculation. No significant differences were observed with in each of the 2 inoculation groups.
[00197] On the LB+ strep + spec agar plates, no colonies were observed day 1 in in any of the groups.
Day 2 CFU counts ranging from 1 to 5 log10 CFU/sample was observed in the inducer treated groups but not in the vehicle treated groups.
Layout of animal study [00198] Twenty four mice were used in each murine intervention study.
Clinical mice score [00199]Mice did not show any clinical signs of infection or discomfort during the entire study period.
Induction killing of MG1655-pks [00200]Faecal matter extracts were plated out on on plates containing streptomycin to determine the CFU of the E. coli MG1655-PKS strain (Fig 8). Streptomycin CFU counts show no difference between the various treatment groups.
[00201]When plating the faecal samples on spectomycin and streptomycin to determine delivery of CGV pSNP114 (because this CGV carries a spectomycin resistance marker) we observe a significant increase in CFU, and thus CGV delivery, after 48 hours (Fig 9). When the CRISPR system was induced we observed a significant decrease in the mean CFU counts.

Discussion and conclusions [00202]We successfully demonstrated guided nuclease antibacterial (CGVTM) delivery using Synthetic NanobioticTM platform, SA100, into E. coli MG1655-pks in the murine gut model. Thus, delivery of an antibacterial using non-self-replicative particles to a microbiome in vivo was .. established. A statistically significant reduction in CFU counts was observed using the activated CRISPR system on the CGVTM.
Example 6: Production of Transduction Particles By Essential Functions in Trans [00203]Native phage are engineered to convert them into DNA carrier vehicles (transduction particles) for our CRISPR-Guided VectorsTM (CGVs). The particles can transduce target bacterial to introduce the vectors, from which CRISPR/Cas systems or one or more components thereof are expressed.We make use of lytic propagation in which essential helper functions are provided only by the host cell (SA800 & SA900).
SA800 & SA900 [00204] Production of SA800 & SA900 particles is based on lytic propagation on a production host bacterial strain. We engineer native (wild-type) phage by inactivating essential phage functions (e.g.
by deleting structural genes) and adding nucleotide sequences encoding the CRISPR-Cas systems.
[00205]By providing the essential genes on a separate helper plasmid, we can make the CGV-phage hybrid propagate on the production strain. However, CGV propagation is not possible on any other bacterial strains due to the lack of the helper plasmid.
[00206]We refer to Figure 10. To construct a non-self replicative particle comprising a CGV
comprising nucleotide sequences encoding components of a CRISPR-Cas system, we engineer a native phage to contain a phage packaging signal and genes needed for phage DNA replication. The CGV may contain multiple phage genes as long as one or more essential genes are deleted or mutated to make the CGV-phage hybrid non-self replicative on strains not expressing the essential function(s).
In the production strain, this function(s) is provided in trans expressed from a plasmid or the bacterial cell chromosome.
[00207] In one example, the production cell will contain a plasmid with a gene encoding a structural .. phage protein, that is expressed during CGV propagation and packaging. A
vector is constructed comprising a native phage genome in which this gene is mutated and into which are inserted nucleotide sequences encoding components of a CRISPR-Cas system, forming a CGV-phage hybrid.
Thus, the CGV-phage hybrid is non-functional to package the CGV DNA into transduction particles in the absence of the helper function provided in trans.

[00208] In a minimal version, all essential functions (ie, essential for packaging) are carried by the production host cell (eg, on a chromosome or one or more plasmids) except for the phage packaging signal and a phage DNA replication machinery (including phage origin of replication) which are instead carried on the CGV. Importantly, the DNA replication machinery may not need originate from the same phage as the essential genes on the helper plasmid or chromosome. For example, the DNA
replication machinery from phage lambda may be included on a CGV (and used to replicate this) carrying a phage P2 packaging signal and packaged into P2 phage capsids (eg, wherein the capsid proteins are encoded by a helper plasmid or chromosome of the production strain cell).
[00209] The CGV is capable of replicating as a lytic phage (i.e. propagation by infection-lysis-reinfection cycles) on a suitable production host strain carrying the helper function(s). However, in non-production cells such as clinical target bacteria, the phage cannot propagate by the lytic cycle but merely functions as a DNA delivery vehicle, thereby delivering sequences encoding one or more components encoding a CRISPR/Cas component(s) (or alternatively another antibacterial agent or other protein or RNA of interest).

Table 1: Example Bacteria t..) Optionally, the host cells are selected from this Table and/or the target cells are selected from this Table (eg, wherein the host and target cells are of a o t..) o different species; or of the same species but are a different strain or the host cells are engineered but the target cells are wild-type or vice versa).
For example .6.
.6.
the host cells are E coli cells and the target cells are C dificile, E coli, Akkermansia, Enterobacteriacea, Ruminococcus, Faecalibacterium, Firmicutes, n.) oe Bacteroidetes, Salmonella, Klebsiella, Pseudomonas, Acintenobacter or Streptococcus cells.
Abiotrophia Acidocella Actinomyces Alkalilimnicola Aquaspirillum Abiotrophia defectiva Acidocella aminolytica Actinomyces bovis Alkalilimnicola ehrlichii Aquaspirillum polymorphum Acidocella facilis Actinomyces denticolens Aquaspirillum Acaricomes Alkaliphilus Actinomyces europaeus putridiconchylium P
Acaricomes phytoseiuli Acidomonas Alkaliphilus oremlandii Actinomyces georgiae Aquaspirillum serpens 2 Acidomonas methanolica Alkapus ransvaaenss o Actinomyces gerencseriae ;3'lihil t l i vi ?.
Acetitomaculum .
Actinomyces Aquimarina N) Acetitomaculum ruminis Acidothermus Allochromatium , hordeovulneris Aquimarina late rcula Acidothermus cellulolyticus Allochromatium vinosum r., N) Acetivibrio Actinomyces howellii Arcanobacterium li i h Actinomyces yovagnas Acetivibrio cellulolyticus Acidovorax Ac Alloiococcus Arcanobacterium lii i Actinomyces srae Acetivibrio ethanolgignens Acidovorax anthurii Act Alloiococcus otitis haemolyticum Acetivibrio multivorans Acidovorax caeni Actinomyces johnsonii Arcanobacterium pyo genes Actinomyces meyeri Allokutzneria Acidovorax cattleyae Iv n Acetoanaerobium Actinomyces naeslundii Allokutzneria albata 1-3 Acidovorax citrulli Archangium t=1 1-d Acetoanaerobium noterae Actinomyces neuii t..) Acidovorax defluvii Archangium gephyra o t..) o Acidovorax delafieldii Actinomyces odontolyticus 'a o .6.
t..) t..) un Acidovorax facilis Actinomyces oris o t..) Acetobacter Altererythrobacter Arcobacter o Acidovorax konjaci Actinomyces radingae t..) o Acetobacter aceti Altereiythrobacter Arcobacter butzleri Acidovorax temperans Actinomyces slackii .6.
Acetobacter cerevisiae ishigakiensis Arcobacter ciyaerophilus .6.
t...) Acidovorax valerianellae Actinomyces turicensis oe Acetobacter cibinongensis Arcobacter halophilus Actinomyces viscosus Altermonas Acetobacter estunensis Acinetobacter Arcobacter nitrofigilis Altermonas haloplanktis Acetobacter fabarum Acinetobacter baumannii Actinoplanes Arcobacter ski rrowii Altermonas macleodii Acetobacter ghanensis Acinetobacter baylyi Actinoplanes auranticolor Arhodomonas Acetobacter indonesiensis Acinetobacter bouvetii Actinoplanes brasiliensis Alysiella Arhodomonas aquaeolei P
Acetobacter lovaniensis Acinetobacter calcoaceticus Actinoplanes consettensis Alysiella crassa Acetobacter malo rum Acinetobacter gerneri Actinoplanes deccanensis ;3' o Alysiella filifonnis Arsenophonus o, ..
Acetobacter nitrogenifigens Acinetobacter haemolyticus Actinoplanes derwentensis Arsenophonus nasoniae 2' , Acetobacter oeni Acinetobacter johnsonii Actinoplanes digitatis Aminobacter , ""
Acetobacter orientalis Acinetobacter junii Actinoplanes durhamensis Aminobacter aganoensis Acetobacter orleanensis Acinetobacter lwoffi Actinoplanes ferrugineus Aminobacter aminovorans Acetobacter pasteurianus Acinetobacter parvus Actinoplanes globisporus Arthrobacter Aminobacter niigataensis Acetobacter pornorurn Acinetobacter radioresistens Actinoplanes humidus Arthrobacter agilis Acetobacter senegalensis Acinetobacter schindleri Actinoplanes italicus Aminobacterium Arthrobacter albus Iv Acetobacter xylinus Acinetobacter soli Actinoplanes liguriensis Aminobacterium mobile Arthrobacter aurescens n ,-i m Acinetobacter tandoii Actinoplanes lobatus Arthrobacter Iv t..) o Acetobacterium Aminomonas r..) Acinetobacter tjernbergiae Actinoplanes missouriensis chlorophenolicus o 'a Acetobacterium bakii Aminomonas paucivorans o .6.
t..) t..) un Acetobacterium carbinolicum Acinetobacter towneri Actinoplanes palleronii Arthrobacter citreus o t..) Ammoniphilus Acetobacterium dehalogenans Acinetobacter ursingii Actinoplanes philippinensis Arthrobacter ciystallopoietes 2 =
Ammoniphilus oxalaticus Acetobacterium fimetarium Acinetobacter venetianus Actinoplanes rectilineatus Arthrobacter cumminsii c,.) .6.
.6.
hilus oxalivorans r..) Acetobacterium malicum Actinoplanes regularis Ammonip Arthrobacter globiformis oe oraosp Acetobacterium paludosum Acrocarp Actinoplanes Arthrobacter Amphibacillus Acrocarpospora corrugata Acetobacterium tundrae teichomyceticus histidinolovorans Amphibacillus xylanus Acetobacterium wieringae Acrocarpospora Actinoplanes utahensis Arthrobacter ilicis l haa Acetobacterium woodii macrocep Arthrobacter luteus Amphritea Acrocarpospora Actinopolyspora Arthrobacter methylotrophus Amphritea balenae P
Acetofilamentum pleiomorpha Actinopolyspora halophila Arthrobacter mysorens Amphritea japonica Acetofilamentum rigidum Actinopolyspora o Arthrobacter nicotianae 03-' --.1 Actibacter .
mortivallis r., Amycolatopsis Arthrobacter nicotinovorans r.,0 Acetohalobium Actibacter sediminis , Amycolatopsis alba Arthrobacter oxydans Acetohalobium arabaticum Actinosynnema r.,7 r., Amycolatopsis albidoflavus Arthrobacter pascens Actinoalloteichus Actinosynnema mirum Acetomicrobium Actinoalloteichus Amycolatopsis azurea Arthrobacter Acetomicrobium faecale cyanogriseus Actinotalea Amycolatopsis coloradensis phenanthrenivorans Amycolatopsis lurida Arthrobacter Acetomicrobium flavidum Actinoalloteichus Actinotalea fennentans Amycolatopsis mediterranei polychromo genes hymeniacidonis Iv n Acetonema Aerococcus Amycolatopsis rifamycinica Atrhrobacter protophormiae Actinoalloteichus spitiensis t=1 Iv Acetonema longum Aerococcus sanguinicola Amycolatopsis rubida Arthrobacter w o t..) o Aerococcus urinae psychrolactophilus 'a o .6.
r..) t..) un Aerococcus urinaeequi Amycolatopsis sulphurea Arthrobacter ramosus o t..) Acetothermus Actinobaccillus o Aerococcus urinaehominis Amycolatopsis tolypomycina Arthrobacter sulfonivorans t..) o Acetothermus paucivorans Actinobacillus capsulatus Aerococcus viridans Arthrobacter sulfureus c,.) .6.
.6.
Actinobacillus delphinicola Anabaena t..) Arthrobacter uratoxydans oe Acholeplasma Actinobacillus hominis Aeromicrobium Anabaena cylindrica Arthrobacter ureafaciens Acholeplasma axanthum Actinobacillus indolicus Aeromicrobium mythreum Anabaena flos-aquae Arthrobacter viscosus Acholeplasma brassicae Actinobacillus lignieresii Anabaena variabilis Arthrobacter woluwensis Acholeplasma cavigenitalium Aeromonas Actinobacillus minor Acholeplasma equifetale Aeromonas Anaeroarcus Actinobacillus muris Asaia Acholeplasma granularum allosaccharophila Anaeroarcus burkinensis P
Actinobacillus Asaia bogorensis Acholeplasma hippikon Aeromonas bestiarum pleuropneumoniae ;3' o Anaerobaculum .?.
oe Acholeplasma laidlawii Aeromonas caviae Asanoa Actinobacillus porcinus r., Anaerobaculum mobile ,30 , Acholeplasma modicum Aeromonas encheleia Asanoa ferruginea Actinobacillus rossii N) Acholeplasma morum Aeromonas r., Actinobacillus scotiae Anaerobiospirillum Asticcacaulis Acholeplasma multilocale enteropelo genes Actinobacillus seminis Anaerobiospirillum Asticcacaulis biprosthecium Acholeplasma oculi Aeromonas eucrenophila Actinobacillus succinogenes succiniciproducens Asticcacaulis excentricus Acholeplasma palmae Aeromonas ichthiosmia Actinobaccillus suis Anaerobiospirillum thomasii Acholeplasma parvum Aeromonas jandaei Actinobacillus ureae Atopobacter 1-d Acholeplasma pleciae Aeromonas media n Anaerococcus Atopobacter phocae 1-3 Acholeplasma vituli Actinobaculum Aeromonas popoffii t=1 Anaerococcus hydrogenalis Iv t..) o Actinobaculum massiliense Aeromonas sobria t..) Anaerococcus lactolyticus o 'a Actinobaculum schaalii Aeromonas veronii o .6.
t..) t..) un Actinobaculum suis Anaerococcus prevotii o t..) Achromobacter Agrobacterium Atopobium o Actinomyces urinale Anaerococcus tetradius t..) o Achromobacter denitrificans Agrobacterium Atopobium fossor Anaerococcus vaginalis .6.
.6.
Achromobacter insolitus Actinocatenispora gelatinovorum Atopobium minutum r..) oe Achromobacter piechaudii Actinocatenispora rupis Anaerofustis Atopobium parvulum Achromobacter ruhlandii Actinocatenispora Agrococcus Anaerofustis stercorihominis Atopobium rimae Agrococcus citreus Achromobacter spanius thailandica Atopobium vaginae Agrococcus jenensis Anaeromusa Actinocatenispora sera Acidaminobacter Anaeromusa acidaminophila Aureobacterium romonas Acidaminobacter Actinocorallia Ag Aureobacterium barkeri P
Agromonas oligotrophica Anaeromyxobacter hydrogenoformans Actinocorallia aurantiaca ;'3 o Anaeromyxobacter Aurobacterium ..
o Actinocorallia aurea r., Acidaminococcus Agromyces ens r.
dehalogenans Aurobacterium liquefaci3O
, Actinocorallia cavernae Agromyces fucosus Acidaminococcus fermentans Actinocorallia glomerata r.,^' Acidaminococcus intestini Agromyces hippuratus Anaerorhabdus Avibacterium Actinocorallia herbida Agromyces luteolus Anaerorhabdus furcosa Avibacterium avium Actinocorallia libanotica Acidicaldus Agromyces mediolanus Avibacterium gallinarum Actinocorallia longicatena Anaerosinus Acidicaldus organivorans Agromyces ramosus Avibacterium paragallinarum Anaerosinus glycerini Agromyces rhizospherae Avibacterium volantium Actinomadura 1-d Acidimicrobium n ,-i Actinomadura alba Anaerovirgula m Acidimicrobium ferrooxidans Akkermansia Azoarcus 1-d t..) Actinomadura atramentaria o i Anaerovrgula multivorans t..) Akkermansia muciniphila Azoarcus indigens o Actinomadura 'a o .6.
t..) t..) un ban gladeshensis Azoarcus tolulyticus 0 Acidiphilium Albidiferax Ancalomicrobium t..) o Actinomadura catellatispora Azoarcus toluvorans t..) o Acidiphilium acidophilum Actinomadura chibensis Albidiferax ferrireducens Ancalomicrobium adetum .6.
.6.
Acidiphilium angustum Actinomadura chokoriensis Azohydromonas t..) oe Albidovulum Ancylobacter Acidiphilium ciy Azohydromonas australica ptum Actinomadura citrea Albidovulum inexpectatum Ancylobacter aquaticus Azohydromonas lata Acidiphilium multivorum Actinomadura coerulea Acidiphilium organovorum Actinomadura echinospora Alcaligenes Aneurinibacillus Azomonas Acidiphilium rubrum Actinomadura fibrosa Alcaligenes denitrificans Aneurinibacillus Azomonas agilis Actinomadura formosensis Alcaligenes faecalis aneurinilyticus Acidisoma Azomonas insignis P

Actinomadura hibisca i i Aneurinibacillus mgulanus Acidisoma sibiricum Azomonas macrocyto genes 1-, Actinomadura ktjaniata Alcanivorax ;3' o Aneurinibacillus .?.
o Acidisoma tundrae r., Actinomadura latina Alcanivorax borkumensis the rmoaerophilus Azorhizobium ,30 , Actinomadura livida Alcanivorax jadensis o Acidisphaera Azorhizobium caulinodans r, N) Actinomadura Angiococcus Acidisphaera rubrifaciens Algicola luteofluorescens Angiococcus discifonnis Azorhizophilus Algicola bacteriolytica Acidithiobacillus Actinomadura macra Azorhizophilus paspali Angulomicrobium Acidithiobacillus albertensis Actinomadura madurae Alicyclobacillus Angulomicrobium tetraedrale Azospirillum Acidithiobacillus caldus Actinomadura oligospora Iv Alicyclobacillus n Azospirillum brasdense Acidithiobacillus ferrooxidans Actinomadura pelletieri disulfidooxidans Anoxybacillus t=1 Azospirillum halopraeferens Iv Acidithiobacillus thiooxidans Actinomadura rubrobrunea t..) Alicyclobacillus Anoxybacillus pushchinoensis o t..) Azospirillum irakense o Actinomadura rugatobispora 'a o .6.
t..) t..) un Actinomadura umbrina sendaiensis o t..) Acidobacterium Aquabacterium Azotobacter o Actinomadura Alicyclobacillus vulcanalis t..) o Acidobacterium capsulatum Aquabacterium commune Azotobacter beijerinckii verrucosospora .6.
.6.
Alishewanella Aquabacterium parvum Azotobacter chroococcum t...) Actinomadura vinacea oe Alishewanella fetalis Azotobacter nigricans Actinomadura viridilutea Azotobacter salinestris Actinomadura viridis Alkalibacillus Azotobacter vinelandii Actinomadura yumaensis Alkalibacillus haloalkaliphilus P

, 1¨ Bacillus Bacteroides Bibersteinia Borrelia Brevinema o 2 1-, r., [see below] Bacteroides caccae Bibersteinia trehalosi Borrelia afzelii Brevinema andersonii 2 '7 Bacteroides coagulans Borrelia americana 0"
N) Bifidobacterium Brevundimonas "
Bacteroides eggerthii Borrelia burgdorferi Bifidobacterium ado lescentis Brevundimonas alba Bacteroides fragilis Borrelia carolinensis Bacteriovorax Bifidobacterium angulatum Brevundimonas aurantiaca Bacteroides galacturonicus Borrelia coriaceae Bacteriovorax stolpii Bifidobacterium animalis Brevundimonas diminuta Bacteroides helco genes Borrelia garinii Bifidobacterium asteroides Brevundimonas intermedia Bacteroides ovatus Borrelia japonica Iv Bifidobacterium bifidum Brevundimonas subvibrioides n Bacteroides pectinophilus t=1 Bifidobacterium boum Brevundimonas vancanneytii Iv Bacteroides pyo genes t..) o Bifidobacterium breve t..) o Bacteroides salyersiae 'a o .6.
t..) t..) un Bacteroides stercoris Bifidobacterium catenulatum Brevundimonas variabilis 0 t..) Bosea o Bacteroides suis Bifidobacterium choerinum Brevundimonas vesicularis t..) o Bosea minatitlanensis Bacteroides tectus Bifidobacterium coiyneforme c...) .6.
.6.
Bosea thiooxidans Brochothrix t..) Bacteroides thetaiotaomicron Bifidobacterium cuniculi oe Bacteroides uniformis Bifidobacterium dentium Brochothrix campestris Brachybacterium Brochothrix thermosphacta Bacteroides ureolyticus Bifidobacterium gallicum Brachybacterium Bacteroides vulgatus Bifidobacterium gallinarum alimentarium BruceIla Bifidobacterium indicum Balnearium Brachybacterium faecium BruceIla canis Bifidobacterium longum P
Balnearium lithotrophicum Bifi Brachybacterium BruceIla neotomaedobacterium 2 paraconglomeratum 1-, magnumBifidobacterium o .? Bryobacter .
w Balneatrix Brachybacterium rhamnosum r., meiycicum atusre obacter aggg , Balneatrix alpica Brachybacterium Biy , Bifidobacterium minimum 0"
, tyrofermentans r.,"
Balneola Bifidobacterium Burkholderia pseudocatenulatum Burkholderia ambifaria Balneola vulgaris Brachyspira Bifidobacterium Brachyspira alvinipulli Burkholderia andropogonis Barnesiella pseudolongum Burkholderia anthina Brachyspira hyodysenteriae Barnesiella viscericola Bifidobacterium pullorum Brachyspira innocens Burkholderia caledonica Iv n Bifidobacterium ruminantium Burkholderia caiyophylli 1-3 Brachyspira murdochii t=1 Bartonella 1-d Bifidobacterium saeculare Brachyspira pilosicoli Burkholderia cenocepacia t..) o Bartonella alsatica t..) o Bifidobacterium subtile Burkholderia cepacia 'a o .6.
t..) t..) un Bartonella bacilliformis Bifidobacterium Burkholderia cocovenenans o t..) o Bartonella clarridgeiae the rmophilum Burkholderia dolosa t..) o Bartonella doshiae Burkholderia fungorum c,.) .6.
Bradyrhizobium Bilophila .6.
t..) Bartonella elizabethae Burkholderia glathei oe Bilophila wadsworthia Bradyrhizobium canariense Bartonella grahamii Burkholderia glumae Bradyrhizobium elkanii Bartonella henselae Burkholderia graminis Biostraticola Bradyrhizobium japonicum Bartonella rochalimae Burkholderia kururiensis Biostraticola tofi Bradyrhizobium liaoningense Bartonella vinsonii Burkholderia multivorans Bizionia Brenneria Burkholderia phenazinium P
Bavariicoccus Burkholderia plantarii 2 Bizionia argentinensis Brenneria alni Bavariicoccus seileri 1-, Burkholderia pyrrocinia ;3' o Brenneria nigrifluens .2 r., Blastobacter Burkholderia silvatlantica 2' Brenneria quercina Bdellovibrio , Blastobacter capsulatus Burkholderia stabilis Brenneria quercina Bdellovibrio bacteriovorus r.3^' Blastobacter denitrificans Burkholderia thailandensis Brenneria salicis Bdellovibrio exovorus Burkholderia tropica Blastococcus Brevibacillus Burkholderia unamae Beggiatoa Blastococcus aggregatus Brevibacillus agri Burkholderia vietnamiensis Beggiatoa alba Blastococcus saxobsidens Brevibacillus borstelensis Iv n Beijerinckia Brevibacillus brevis Buttiauxella Blastochloris t=1 Buttiauxella agrestis Iv Betjerinckia derxii Brevibacillus centrosporus t..) Blastochloris viridis o t..) b ll iauxea rennerae o Betjerinckia fluminensis Brevibacillus choshinensis Butt 'a o .6.
t..) t..) un Betjerinckia indica Brevibacillus invocatus Buttiauxella ferragutiae o t..) Blastomonas o Betjerinckia mobilis Brevibacillus late rosporus Buttiauxella gaviniae t..) o Blastomonas natatoria Brevibacillus parabrevis Buttiauxella izardii c,.) .6.
Belliella .6.
t..) Brevibacillus reuszeri Buttiauxella noackiae oe Belliella baltica Blastopirellula Buttiauxella warmboldiae Blastopirellula marina Brevibacterium Bellilinea Brevibacterium abidum Butyrivibrio Blautia Bellilinea caldifistulae Brevibacterium album Butyrivibrio fibrisolvens Blautia coccoides Brevibacterium aurantiacum Butyrivibrio hungatei Belnapia Blautia hansenii P
Brevibacterium celere Butyrivibrio proteoclasticus Belnapia moabensis Blautia producta Brevibacterium epidermidis 1-, ;33' o Blautia wexlerae .2 .6.
Brevibacterium Bergeriella , Bergeriella denitrificans Bogoriella frigoritolerans N) Brevibacterium halotolerans r., Bogoriella caseilytica Beutenbergia Brevibacterium iodinum Beutenbergia cavernae Bordetella Brevibacterium linens Bordetella avium Brevibacterium lyticum Bordetella bronchiseptica Brevibacterium mcbrellneri Iv Bordetella hinzii Brevibacterium otitidis n ,-i m Bordetella holmesii Brevibacterium oxydans Iv t..) o Bordetella parapertussis t..) o 'a o .6.
t..) t..) un Bordetella pertussis Brevibacterium paucivorans o tµ.) o Bordetella petrii Brevibacterium stationis tµ.) o Bordetella trematum c,.) .6.
.6.
n.) oe Bacillus B. acidiceler B. aminovorans B. glucanolyticus B. taeanensis B. lautus B. acidicola B. amylolyticus B. gordonae B. tequilensis B. lehensis B. acidiproducens B. andreesenii B. gottheilii B. the rmantarcticus B. lentimorbus P
B. acidocaldarius B. aneurindyncus B. graminis B. the rmoaerophilus B. lentus , ...]
o h li B l th B l l h B
i th B. acidoterrestris B. anracs B. amapaus B.
ermoamyovorans B. ceniformis ..
un r., B. aeolius B. aquimaris B. haloalkaliphilus B. the rmocatenulatus B. ligniniphilus , , , B. aerius B. arenosi B. halochares B. the rmocloacae B. litoralis ' r., r., B. aerophilus B. arseniciselenatis B. halodenitrificans B. the rmocopriae B. locisalis B. agaradhaerens B. arsenicus B. halodurans B. thermodenitrificans B. luciferensis B. agri B. aurantiacus B. halophilus B. the rmoglucosidasius B. luteolus B. aidingensis B. arvi B. halosaccharovorans B. the rmolactis B. luteus B. akibai B. cnyabhattai B.
hemicellulosilyticus B. the rmoleovorans B. macauensis Iv n ,-i B. alcalophilus B. asahii B. hemicentroti B. the rmophilus B. macerans t=1 Iv B. algicola B. atrophaeus B. herbersteinensis B. the rmoruber B. macquariensis tµ.) o tµ.) o B. alginolyticus B. axarquiensis B. horikoshii B. the rmosphaericus B. macyae 'a o .6.
tµ.) tµ.) un B. alkalidiazotrophicus B. azotofixans B. horneckiae B. thiaminolyticus B. malacitensis 0 n.) B. alkalinitrilicus B. azotoformans B. horti B. thioparans B. mannandyticus o t..) o B. alkalisediminis B. badius B. huizhouensis B. thuringiensis B. marisflavi c,.) .6.
.6.
B. alkalitelluris B. barbaricus B. humi B. tianshenii B. marismortui t.) oe B. altitudinis B. bataviensis B. hwajinpoensis B. bypoxylicola B. marmarensis B. alveayuensis B. betjingensis B. idriensis B. tusciae B. massiliensis B. alvei B. benzoevorans B. indicus B. validus B. megaterium B. amyloliquefaciens B. beringensis B. infantis B. vallismortis B. mesonae B. berkeleyi B. infernus B. vedderi B. methanolicus P
= B.
B. beveridgei B. insolitus B. velezensis B. methylotrophicus , a. subsp. Amyloliquefaciens ...]
1-, b i i B ii Bi i Bil B. ogorenss B. nvctae B. vetnamenss B. mguanus 0 o ..
o =
B. a. subsp. Plantarum r., B. boroniphilus B. iranensis B. vireti B. mojavensis '7 B. borstelensis B. isabeliae B. vulcani B. mucilaginosus 0"
r., B. brevis Migula B. isronensis B. wakoensis B. muralis B. dipsosauri B. butanolivorans B. jeotgali B. weihenstephanensis B. murimartini B. drentensis B. canaveralius B. kaustophilus B. xiamenensis B. myco ides B. edaphicus B. carboniphilus B. kobensis B. xiaoxiensis B. naganoensis B. ehimensis B. cecembensis B. kochii B. zhanjiangensis B. nanhaiensis Iv B. eiseniae n B. cellulosilyticus B. kokeshiiformis B. nanhaiisediminis 1-3 B. enclensis t=1 B. peoriae Iv B. centrosporus B. koreensis B. nealsonii t..) o B. endophyticus n.) B. persepolensis o B. cereus B. korlensis B. neidei 'a B. endoradicis o .6.
t..) t..) un B. farraginis B. chagannorensis B. kribbensis B. persicus B. neizhouensis o tµ.) o B. fastidiosus B. chitinolyticus B. krulwichiae B. pervagus B. niabensis tµ.) o B. fengqiuensis B. chondroitinus B. laevolacticus B. plakortidis B. niacini c,.) .6.
.6.
tµ.) B. firmus B. choshinensis B. larvae B. pocheonensis B. novalis oe B. flexus B. chungangensis B. laterosporus B. polygoni B. oceanisediminis B. foraminis B. cibi B. salexigens B. polymyxa B. odysseyi B. fordii B. circulans B. saliphilus B. popilliae B. okhensis B. formosus B. clarkii B. schlegelii B. pseudalcalophilus B. okuhidensis B. fortis B. clausii B. sediminis B. pseudofirmus B. oleronius P
B. fiimarioli B. coagulans B. selenatarsenatis B. pseudomycoides B. oiyzaecorticis , 1-, B. fiiniculus B. coahuilensis B. selenitireducens B. psychrodurans B. oshimensis o ..
B. fiisiformis B. cohnii B. seohaeanensis B. psychrophilus B. pabuli '7 , B. galactophilus B. compost! B. shacheensis B. psychrosaccharolyticus B. pakistanensis r., B. galactosidilyticus B. curdlanolyticus B. shackletonii B. psychrotolerans B. pallidus B. galliciensis B. cycloheptanicus B. siamensis B. pulvifaciens B. pallidus B. gelatini B. cytotoxicus B. silvestris B. pumilus B. panacisoli B. gibsonii B. daliensis B. simplex B. purgationiresistens B. panaciterrae B. ginsengi B. decisifrondis B. siralis B. pycnus B. pantothenticus Iv n B. ginsengihumi B. decolorationis B. smithii B. qingdaonensis B. parabrevis 1-3 t=1 Iv B. ginsengisoli B. deserti B. soli B. qingshengii B. paraflexus tµ.) o tµ.) B. globisporus (eg, B. B. solimangrovi B. reuszeri B. pasteurii 'a o .6.
tµ.) tµ.) un g. subsp. Globisporus; or B. B. solisalsi B. rhizosphaerae B. patagoniensis c;;) t..) g. subsp. Marinus) B. songklensis B. rigui o t..) o B. sonorensis B. runs c,.) .6.
.6.
n.) B. sphaericus B. safensis oe B. sporothermodurans B. salarius B. stearothermophilus B. stratosphericus B. subterraneus B. subtilis (eg, B.
P
s. subsp. Inaquosorum; or B.

1-, s. subsp. Spizizeni;
or B.
o 2 oe N, s. subsp. Subtilis) '7 , Caenimonas Campylobacter Cardiobacterium Catenuloplanes Curtobacterium Caenimonas koreensis Campylobacter coli Cardiobacterium hominis Catenuloplanes atrovinosus Curtobacterium Campylobacter concisus Catenuloplanes castaneus .. albidum Caldalkalibacillus Carnimonas Campylobacter curvus Catenuloplanes crispus Curtobacterium citreus Caldalkalibacillus uzonensis Carnimonas nigrificans Campylobacter fetus Catenuloplanes indicus Iv Campylobacter gracilis Catenuloplanes japonicus n Caldanaerobacter Carnobacterium Campylobacter helveticus Catenuloplanes nepalensis t=1 Iv Caldanaerobacter subterraneus Carnobacterium t..) Campylobacter hominis Catenuloplanes niger o t..) alterfunditum o 'a Campylobacter hyointestinalis o .6.
t..) t..) un Campylobacter jejuni Carnobacterium divergens o t..) Caldanaerobius Chryseobacterium o Campylobacter lari Carnobacterium funditum t...) o Caldanaerobius fijiensis Campylo Chiyseobacterium bacter mucosalis Carnobacterium gallinarum c...) .6.
.6.
Caldanaerobius balustinum t..) Campylobacter rectus Carnobacterium oe polysaccharolyticus Campylobacter showae maltaromaticum Citrobacter Caldanaerobius zeae Campylobacter sputorum Carnobacterium mobile C. amalonaticus Campylobacter upsaliensis Carnobacterium viridans Caldanaerovirga C. braakii Caldanaerovirga acetigignens C. diversus Capnocytophaga Caryophanon P
Capnocytophaga canimorsus Caiyopha C. farmeri non latum Caldicellulosiruptor C. freundii Capnocytophaga cynodegmi Caiyophanon tenue 1-, ;3' o Caldicellulosiruptor bii o C. gillenii .?.
rup esc Capnocytophaga gingivalis r., Caldicellulosiruptor kristjanssonii Catellatospora Catellat C. koseri , Capnocytophaga granulosa 0"1 Caldicellulosiruptor owensensis Catellatospora citrea C. murliniae ,3"1 Capnocytophaga haemolytica Catellatospora C. pasteuriim Capnocytophaga ochracea methionotrophica C. rodentium Capnocytophaga sputigena C. sedlakii Catenococcus C. werkmanii Catenococcus thiocycli Iv C. youngae n ,-i m ,-o Clostridium t..) o t..) o (see below) 'a cA
.6.
t..) t..) un C
t..) Coccochloris o t..) o Coccochloris elabens .6.
.6.
n.) oe Corynebacterium Coiynebacterium flavescens Coiynebacterium variabile Clostridium Clostridium absonum, Clostridium aceticum, Clostridium ace tireducens, Clostridium acetobutylicum, Clostridium acidisoli, Clostridium aciditolerans, P
Clostridium acidurici, Clostridium aerotolerans, Clostridium aestuarii, Clostridium akagii, Clostridium aldenense, Clostridium aldrichii, Clostridium 1-, algidicami, Clostridium algidixylanolyticum, Clostridium algifaecis, Clostridium algoriphilum, Clostridium alkalicellulosi, Clostridium aminophilum, 1-, .
o r., Clostridium aminovalericum, Clostridium amygdalinum, Clostridium amylolyticum, Clostridium arbusti, Clostridium arcticum, Clostridium argentinense, 2 '7 Clostridium asparagiforme, Clostridium aurantibutyricum, Clostridium autoethanogenum, Clostridium baratii, Clostridium barkeri, Clostridium bartlettii, r., Clostridium betjerinckii, Clostridium bifermentans, Clostridium bolteae, Clostridium bomimense, Clostridium botulinum, Clostridium bowmanii, Clostridium biyantii, Clostridium butyricum, Clostridium cadaveris, Clostridium caenicola, Clostridium cam inithermale, Clostridium carboxidivorans, Clostridium camis, Clostridium cavendishii, Clostridium celatum, Clostridium celerecrescens, Clostridium cellobioparum, Clostridium cellulofermentans, Clostridium cellulolyticum, Clostridium cellulosi, Clostridium cellulovorans, Clostridium chartatabidum, Clostridium chauvoei, Clostridium chromiireducens, Clostridium citroniae, Clostridium clariflavum, Clostridium clostridioforme, Clostridium cocco ides, Clostridium cochlearium, Clostridium colletant, Clostridium colicanis, .0 n Clostridium colinum, Clostridium collagenovorans, Clostridium cylindrosporum, Clostridium difficile, Clostridium diolis, Clostridium disporicum, 1-3 t=1 Clostridium drakei, Clostridium durum, Clostridium estertheticum, Clostridium estertheticum estertheticum, Clostridium estertheticum laramiense, Iv t..) o t..) Clostridium fallax, Clostridium felsineum, Clostridium fervidum, Clostridium fimetarium, Clostridium formicaceticum, Clostridium frigidicamis, Clostridium o 'a o .6.
t..) t..) un frigoris, Clostridium ganghwense, Clostridium gasigenes, Clostridium ghonii, Clostridium glycolicum, Clostridium glycyrrhizinilyticum, Clostridium grantii, n.) o Clostridium haemolyticum, Clostridium halophilum, Clostridium hastiforme, Clostridium hathewayi, Clostridium herbivorous, Clostridium hiranonis, tµ.) o Clostridium histolyticum, Clostridium homopropionicum, Clostridium huakuii, Clostridium hungatei, Clostridium hydrogeniformans, Clostridium w .6.
.6.
tµ.) hydroxybenzoicum, Clostridium hylemonae, Clostridium jejuense, Clostridium indolis, Clostridium innocuum, Clostridium intestinale, Clostridium irregulare, oe Clostridium isatidis, Clostridium josui, Clostridium kluyveri, Clostridium lactatifermentans, Clostridium lacusfiyxellense, Clostridium laramiense, Clostridium lavalense, Clostridium lentocellum, Clostridium lentoputrescens, Clostridium leptum, Clostridium limosum, Clostridium litorale, Clostridium lituseburense, Clostridium ljungdahlii, Clostridium lortetii, Clostridium lundense, Clostridium magnum, Clostridium malenominatum, Clostridium mangenotii, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium methylpentosum, Clostridium neopropionicum, Clostridium nexile, Clostridium nitrophenolicum, Clostridium novyi, Clostridium oceanicum, Clostridium orbiscindens, Clostridium oroticum, Clostridium oxalicum, Clostridium papyrosolvens, Clostridium P
paradoxum, Clostridium paraperfringens (Alias: C. welchii), Clostridium paraputrificum, Clostridium pascui, Clostridium pasteurianum, Clostridium 2 ...]
1-, peptidivorans, Clostridium perenne, Clostridium perfringens, Clostridium pfennigii, Clostridium phytofermentans, Clostridium piliforme, Clostridium 1-, ..
polysaccharolyticum, Clostridium populeti, Clostridium propionicum, Clostridium proteoclasticum, Clostridium proteolyticum, Clostridium psychrophilum, 2 '7 , Clostridium puniceum, Clostridium purinilyticum, Clostridium putrefaciens, Clostridium putrificum, Clostridium quercicolum, Clostridium quinii, Clostridium o r., ramosum, Clostridium rectum, Clostridium roseum, Clostridium saccharobutylicum, Clostridium saccharogumia, Clostridium saccharolyticum, Clostridium saccharoperbutylacetonicum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scatolo genes, Clostridium schirmacherense, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporo genes, Clostridium sporosphaeroides, Clostridium stercorarium, Clostridium stercorarium leptospartum, Clostridium stercorarium stercorarium, Clostridium stercorarium the rmolacticum, Clostridium sticklandii, Clostridium straminisolvens, Clostridium subterminale, Clostridium sufflavum, Clostridium sulfidigenes, Clostridium ,t n symbiosum, Clostridium tagluense, Clostridium tepidiprofundi, Clostridium termitidis, Clostridium tertium, Clostridium tetani, Clostridium tetanomorphum, 1-3 t=1 Iv Clostridium thermaceticum, Clostridium thermautotrophicum, Clostridium thermoalcaliphilum, Clostridium thermobutyricum, Clostridium the rmocellum, t.) o tµ.) o Clostridium thermocopriae, Clostridium thermohydrosulfuricum, Clostridium thermolacticum, Clostridium the rmopalmarium, Clostridium 'a o .6.
tµ.) tµ.) un thermopapyrolyticum, Clostridium thermosaccharolyticum, Clostridium thermosuccinogenes, Clostridium thermosulfurigenes, Clostridium 0 t..) o thiosulfatireducens, Clostridium tyrobutyricum, Clostridium uliginosum, Clostridium ultunense, Clostridium villosum, Clostridium vincentii, Clostridium t...) o viride, Clostridium xylanolyticum, Clostridium xylanovorans c...) .6.
.6.
n.) oe Dactylosporangium Deinococcus Delftia Echinicola Dactylosporangium aurantiacum Deinococcus aerius Delftia acidovorans Echinicola pacifica Dactylosporangium fulvum Deinococcus apachensis Desulfovibrio Echinicola vietnamensis Dactylosporangium matsuzakiense Deinococcus aquaticus Desulfovibrio desulfuricans Dactylosporangium roseum Deinococcus aquatilis Diplococcus Dactylosporangium thailandense Deinococcus caeni Diplococcus pneumoniae P

Dactylosporangium vinaceum Deinococcus radiodurans 1-, t..) Deinococcus radiophilus ..
0"
r., '7 Enterobacter Enterobacter kobei Faecalibacterium Flavobacterium "
E. aero genes E. ludwigii Faecalibacterium prausnitzii Flavobacterium antarctic urn E. amnigenus E. mori Fangia Flavobacterium aquatile E. agglomerans E. nimipressuralis Fangia hongkongensis Flavobacterium E. arachidis E. oiyzae Fastidiosipila aquidurense E. asburiae E. pulveris Fastidiosipila sanguinis Flavobacterium balustinum Iv n E. cancerogenous E. pyrinus Fusobacterium Flavobacterium croceum 1-3 t=1 E. cloacae E. radicincitans Fusobacterium nucleatum Flavobacterium cucumis Iv t..) o t..) E. cowanii E. taylorae Flavobacterium =
'a o .6.
t..) t..) un E. dissolvens E. turicensis daejeonense 0 n.) o E. gergoviae E. sakazakii Enterobacter soli Flavobacterium defluvii r..) o E. helveticus Enterococcus Flavobacterium degerlachei c...) .6.
.6.
t..) E. honnaechei Enterococcus durans Flavobacterium oe E. intermedius Enterococcus faecalis denitrificans Enterococcus faecium Flavobacterium filum Erwinia Flavobacterium flevense Erwinia hapontici Flavobacterium frigidarium Escherichia Flavobacterium mizutaii P
Escherichia coli Flavobacterium 2 -, 1-, okeanokoites 1-, ..
r., N) '7 '8 N) N) Gaetbulibacter Haemophilus Ideonella Janibacter Gaetbulibacter saemankumensis Haemophilus aegyptius Ideonella azotifigens Janibacter anophelis Gallibacterium Haemophilus aphrophilus Idiomarina Janibacter corallicola Gallibacterium anatis Haemophilus felis Idiomarina abyssalis Janibacter limosus Gallicola Haemophilus gallinarum Idiomarina baltica Janibacter melonis Iv n ,-i Gallicola barnesae Haemophilus haemolyticus Idiomarina fontislapidosi Janibacter terrae t=1 Iv Garciella Haemophilus influenzae Idiomarina loihiensis Jannaschia t..) o t..) o Garciella nitratireducens Haemophilus paracuniculus Idiomarina ramblicola Jannaschia cystaugens 'a o .6.
t..) t..) un Geobacillus Haemophilus parahaemolyticus Idiomarina seosinensis Jannaschia helgolandensis r..) o Geobacillus the rmoglucosidasius Haemophilus parainfluenzae Idiomarina zobellii Jannaschia pohangensis r..) o Geobacillus stearothermophilus Haemophilus Ignatzschineria Jannaschia rubra c...) .6.
.6.
t..) Geobacter paraphrohaemolyticus Ignatzschineria larvae oe Geobacter bemidjiensis Haemophilus parasuis Janthinobacterium Geobacter bremensis Haemophilus pittmaniae Ignavigranum Janthinobacterium Geobacter chapellei Hafnia Ignavigranum ruoffiae agaric idamno sum Geobacter grbiciae Hafnia alvei Ilumatobacter Janthinobacterium lividum Geobacter hydrogenophilus Hahella Ilumatobacter fluminis Jejuia P
Geobacter lovleyi Hahella ganghwensis Ilyobacter Jejuia pallidilutea 2 , 1-, Geobacter metallireducens Halalkalibacillus Ilyobacter delafieldii Jeotgalibacillus .
,-, ..
Geobacter pelophilus Halalkalibacillus halophilus Ilyobacter insuetus Jeotgalibacillus 2 '7 Geobacter pickeringii Helicobacter Ilyobacter polytropus alimentarius 0"
r., Geobacter sulfurreducens Helicobacter pylori Ilyobacter tartaricus Jeotgalicoccus Geodermatophilus Jeotgalicoccus halotolerans Geodermatophilus obscurus Gluconacetobacter Gluconacetobacter xylinus Iv n Gordonia m 1-d Gordonia rubripertincta w o t..) o 'a o .6.
t..) t..) un Kaistia Labedella Listeria ivanovii Micrococcus Nesterenkonia o t..) Kaistia adipata Labedella gwakjiensis L. marthii Micrococcus luteus Nesterenkonia holobia o t...) o Kaistia soli Labrenzia L. monocyto genes Micrococcus lylae Nocardia c,.) .6.
.6.
Kangiella Labrenzia aggregata L. newyorkensis Moraxella Nocardia argentinensis t..) oe Kangiella aquimarina Labrenzia alba L. riparia Moraxella bovis Nocardia corallina Kangiella koreensis Labrenzia alexandrii L. rocourtiae Moraxella nonliquefaciens Nocardia Labrenzia marina L. seeligeri Moraxella osloensis otitidiscaviarum Kerstersia Labrys L. weihenstephanensis Nakamurella Kerstersia gyiorum Labiys methylaminiphilus L. welshimeri Nakamurella multipartita P
Kiloniella Labiys miyagiensis Listonella Nannocystis 2 1-, Kiloniella laminariae Labiys monachus Listonella anguillarum Nannocystis pusilla 1-, ..
Klebsiella Labiys okinawensis Macrococcus Natranaerobius 2 '7 , K granulomatis Labiys portucalensis Macrococcus bovicus Natranaerobius 0 r., K oxytoca Marinobacter the rmophilus K pneumoniae Lactobacillus Marinobacter algicola Natranaerobius trueperi K terrigena [see below] Marinobacter biyozoorum Naxibacter K variicola Laceyella Marinobacter flavimaris Naxibacter alkalitolerans Kluyvera Laceyella putida Meiothermus Neisseria 1-d n Kluyvera ascorbata Lechevalieria Meiothermus ruber Neisseria cinerea 1-3 t=1 Iv Lechevalieria aerocolonigenes Neisseria denitrificans w o t..) Legionella Neisseria gonorrhoeae o 'a o .6.
t..) t..) un Kocuria [see below] Methylophilus Neisseria lactamica 0 t..) Kocuria roasea Listeria Methylophilus Neisseria mucosa o r..) o Kocuria varians L. aquatica methylotrophus Neisseria sicca c...) .6.
.6.
Kurthia L. booriae Microbacterium Neisseria subflava t..) oe Kurthia zopfii L. cornellensis Microbacterium Neptunomonas L. fleischmannii ammoniaphilum Neptunomonas japonica L. floridensis Microbacterium arborescens L. grandensis Microbacterium liquefaciens L. grayi Microbacterium oxydans P
L. innocua , -, o r., Lactobacillus , , , ,D
L. acetotolerans L. catenaformis L. mali L. parakefiri L. sakei r., L. acidifarinae L. ceti L. manihotivorans L. paralimentarius L. salivarius L. acidipiscis L. coleohominis L. mindensis L. paraplantarum L. sanfranciscensis L. acidophilus L. collinoides L. mucosae L. pentosus L. satsumensis Lactobacillus agilis L. composti L. murinus L. perolens L. secaliphilus Iv L. algidus L. concavus L. nagelii L. plantarum L. sharpeae n ,-i L. 116amster116116ius L. coryniformis L. namurensis L. pontis L. siliginis t=1 Iv t..) L. amylolyticus L. crispatus L. nantensis L. protectus L. spicheri o t..) o 'a L. amylophilus L. crustorum L. oligofermentans L. psittaci L. suebicus o .6.
t..) t..) un L. amylotrophicus L. curvatus L. oris L. rennini L. thailandensis o r..) L. amylovorus L. delbrueckii subsp. L. panis L. reuteri L. ultunensis o t..) o L. animalis Bulgaricus L. pantheris L. rhamnosus L. vaccinostercus c...) .6.
.6.
L. antri L. delbrueckii subsp. L. parabrevis L. rimae L. vaginalis t..) oe L. 117amster Delbrueckii L. parabuchneri L. rogosae L. versmoldensis L. 117amster117 L. delbrueckii subsp. Lactis L. paracasei L. rossiae L. vini L. bifermentans L. dextrinicus L. paracollinoides L. ruminis L. vitulinus L. brevis L. diolivorans L. parafarraginis L. saerimneri L. zeae L. buchneri L. equi L. homohiochii L. jensenii L. zymae P
L. camelliae L. equigenerosi L. iners L. johnsonii L. gastricus 2 1-, L. casei L. farraginis L. ingluviei L. kalixensis L. ghanensis 1-, ..
L. kitasatonis L. farciminis L. intestinalis L. kefiranofaciens L. graminis 2 '7 L. kunkeei L. fermentum L. fuchuensis L. kefiri L. hammesii , r., L. leichmannii L. fornicalis L. gallinarum L. 117amste L. 117amster L. lindneri L. fructivorans L. gasseri L. helveticus L. harbinensis L. malefermentans L. frumenti L. hilgardii L. hayakitensis Iv n ,-i Legionella t=1 1-d r..) Legionella adelaidensis Legionella drancourtii Candidatus Legionella jeonii Legionella quinlivanii o t..) o 'a Legionella anisa Legionella dresdenensis Legionella jordanis Legionella rowbothamii o .6.
t..) t..) un Legionella beliardensis Legionella drozanskii Legionella lansingensis Legionella rubrilucens 0 t..) o Legionella birminghamensis Legionella dumoffii Legionella londiniensis Legionella sainthelensi t..) o Legionella bozemanae Legionella eiythra Legionella longbeachae Legionella santicrucis c,.) .6.
.6.
t..) Legionella brunensis Legionella fairfieldensis Legionella lytica Legionella shakespearei oe Legionella busanensis Legionella fallonii Legionella maceachernii Legionella spiritensis Legionella cardiaca Legionella feeleii Legionella massiliensis Legionella steelei Legionella cherrii Legionella geestiana Legionella micdadei Legionella steigerwalth Legionella cincinnatiensis Legionella genomospecies Legionella monrovica Legionella taurinensis Legionella clemsonensis Legionella gormanii Legionella moravica Legionella tucsonensis P
Legionella donaldsonii Legionella gratiana Legionella nagasakiensis Legionella tunisiensis 2 1-, Legionella gresilensis Legionella nautarum Legionella wadsworthii 1-, .
Legionella hackeliae Legionella norrlandica Legionella waltersii 2 '7 Legionella impletisoli Legionella oakridgensis Legionella worsleiensis 0"
r., Legionella israelensis Legionella parisiensis Legionella yabuuchiae Legionella jamestowniensis Legionella pittsburghensis Legionella pneumophila Legionella quateirensis Iv n ,-i Oceanibulbus Paenibacillus Prevotella Quadrisphaera m 1-d t..) Oceanibulbus indolifex Paenibacillus thiaminolyticus Prevotella albensis Quadrisphaera granulorum o t..) o 'a Prevotella amnii o .6.
t..) t..) un Oceanicaulis Pantoea Prevotella bergensis Quatrionicoccus 0 n.) o Oceanicaulis alexandrii Pantoea agglomerans Prevotella bivia Quatrionicoccus r..) o Oceanicola Prevotella Prevotella brevis australiensis c,.) .6.
.6.
t..) Oceanicola batsensis Paracoccus Prevotella biyantii oe Oceanicola granulosus Paracoccus alcaliphilus Prevotella buccae Quinella Oceanicola nanhaiensis Paucimonas Prevotella buccalis Quinella ovalis Oceanimonas Paucimonas lemoignei Prevotella copri Oceanimonas baumannii Pectobacterium Prevotella dentalis Ralstonia Oceaniserpentilla Pectobacterium aroidearum Prevotella denticola Ralstonia eutropha P
Oceaniserpentilla haliotis Pectobacterium atrosepticum Prevotella disiens Ralstonia insidiosa 2 ,-, Oceanisphaera Pectobacterium Prevotella histicola Ralstonia mannitolilytica 1-, o r., Oceanisphaera don ghaensis betavasculorum Prevotella intermedia Ralstonia pickettii 2 '7 , Oceanisphaera litoralis Pectobacterium cacticida Prevotella maculosa Ralstonia 0 , Oceanithermus Pectobacterium carnegieana Prevotella marshii pseudosolanacearum Oceanithermus desulfurans Pectobacterium carotovorum Prevotella melaninogenica Ralstonia syzygii Oceanithermus profundus Pectobacterium chiysanthemi Prevotella micans Ralstonia solanacearum Oceanobacillus Pectobacterium cypripedii Prevotella multiformis Ramlibacter Oceanobacillus caeni Pectobacterium rhapontici Prevotella nigrescens Ramlibacter henchirensis Iv n Oceanospirillum Pectobacterium wasabiae Prevotella oralis Ramlibacter tataouinensis 1-3 t=1 Iv Oceanospirillum linum Planococcus Prevotella oris w o t..) o Planococcus citreus Prevotella oulorum 'a o .6.
t..) t..) un Planomicrobium Prevotella pallens Raoultella 0 n.) o Planomicrobium okeanokoites Prevotella salivae Raoultella ornithinolytica t...) o Plesiomonas Prevotella stercorea Raoultella planticola c...) .6.
.6.
t..) Plesiomonas shigelloides Prevotella tannerae Raoultella terrigena oe Proteus Prevotella timonensis Rathayibacter Proteus vulgaris Prevotella veroralis Rathayibacter caricis Providencia Rathayibacter festucae Providencia stuartii Rathayibacter iranicus Pseudomonas Rathayibacter rathayi P
Pseudomonas aeruginosa Rathayibacter toxicus 2 , 1-, Pseudomonas alcaligenes Rathayibacter tritici t..) .
o r., Pseudomonas anguillispetica Rhodobacter '7 , Pseudomonas fluorescens Rhodobacter sphaero ides o r., Pseudoalteromonas Ruegeria haloplanktis Ruegeria gelatinovorans Pseudomonas mendocina Pseudomonas pseudoalcaligenes Iv n Pseudomonas putida t=1 Iv Pseudomonas tutzeri w o t..) o Pseudomonas syringae 'a o .6.
t..) t..) un Psychrobacter o t..) o Psychrobacter faecalis t...) o Psychrobacter c...) .6.
.6.
t..) phenylpyruvicus oe Saccharococcus Sagittula Sanguibacter Stenotrophomonas Tatlockia Saccharococcus the rmophilus Sagittula stellata Sanguibacter keddieii Steno trophomonas Tatlockia maceachernii Sanguibacter suarezii maltophilia Tatlockia micdadei Saccharomonospora Salegentibacter P
Streptococcus Tenacibaculum 2 Saccharomonospora azurea Salegentibacter sale gens Saprospira 1-, Tenacibaculum n . ) 1-, Saccharomonospora cyanea Saprospira grandis r., [also see below]
amylolyticum r.,0 Salimicrobium , Saccharomonospora viridis ' Tenacibaculum discolor '8 Salimicrobium album Sarcina Streptomyces r., Tenacibaculum Saccharophagus Sarcina maxima Streptomyces Salinibacter gallaicum Saccharophagus degradans Sarcina ventriculi achromo genes Tenacibaculum Salinibacter ruber Streptomyces cesalbus Saccharopolyspora Sebaldella lutimaris Salinicoccus Streptomyces cescaepitosus Tenacibaculum Saccharopolyspora eiythraea Sebaldella term itidis Iv Streptomyces cesdiastaticus n Salinicoccus alkaliphilus mesophilum 1-3 Saccharopolyspora gregorii t=1 Streptomyces cesexfoliatus Iv Salinicoccus hispanicus Tenacibaculum Saccharopolyspora hirsuta t..) o Streptomyces fimbriatus t..) Salinicoccus roseus skagerrakense =
Saccharopolyspora hordei 'a Streptomyces fradiae o .6.
t..) t..) un Saccharopolyspora rectivirgula Streptomyces fulvissimus Tepidanaerobacter o t..) Salinispora Serratia o Saccharopolyspora spinosa Streptomyces griseoruber Tepidanaerobacter t...) o Salinispora arenicola Serratia fonticola Saccharopolyspora taberi Streptomyces griseus syntrophicus c...) .6.
.6.
Salinispora tropica Serratia marcescens t..) Streptomyces lavendulae Tepidibacter oe Saccharothrix Streptomyces Tepidibacter Salinivibrio Sphaerotilus Saccharothrix australiensis phaeochromo genes formicigenes Salinivibrio costicola Sphaerotilus natans Saccharothrix coeruleofusca Streptomyces Tepidibacter Saccharothrix espanaensis the rmodiastaticus thalassicus Salmonella Sphingobacterium Saccharothrix longispora Streptomyces tube rcidicus Thermus Salmonella bongori Sphingobacterium multivorum P
Saccharothrix mutabilis Thermus aquaticus Salmonella enterica Saccharothrix syringae -J,-, ermus fiiform Staphylococcus Thlis t..) Salmonella subterranea .i.
t..) Saccharothrix tangerinus r., [see below]
The rmus the Salmonella typhi h hil r.,0 , Saccharothrix texasensis 0"
N) N) Staphylococcus S. arlettae S. microti S. equorum S. schleiferi S. agnetis S. muscae S.
S. sciuri felis S. nepalensis S.
aureus S. fleurettii S. simiae S. auricularis S. pasteuri Iv n S. gallinarum S. simulans 1-3 S. capitis S. petrasii t=1 S. haemolyticus S. stepanovicii Iv t..) S. caprae S. pettenkoferi o t..) S. hominis S. succinus o S. carnosus S. piscifermentans 'a o .6.
t..) t..) un S. caseolyticus S. hyicus S. pseudintermedius S. vitulinus 0 t..) o S. chromo genes S. intermedius S. pseudolugdunensis S. warneri t...) o S. cohnii S. kloosii S. pulvereri S. xylosus c...) .6.
.6.
S. condimenti S. leei S. rostri t..) oe S. delphini S. lentus S. saccharolyticus S. devriesei S. lugdunensis S. saprophyticus S. epidermidis S. lutrae S. lyticans S. massiliensis P
Streptococcus , , t..) .
Streptococcus agalactiae Streptococcus infantarius Streptococcus orisratti Streptococcus the rmophilus ..
N, Streptococcus anginosus Streptococcus iniae Streptococcus parasanguinis Streptococcus sanguinis , , , ,D
, Streptococcus bovis Streptococcus intermedius Streptococcus peroris Streptococcus sobrinus "
N, Streptococcus canis Streptococcus lactarius Streptococcus pneumoniae Streptococcus suis Streptococcus constellatus Streptococcus milleri Streptococcus Streptococcus uberis Streptococcus downei Streptococcus mitis pseudopneumoniae Streptococcus vestibularis Streptococcus dysgalactiae Streptococcus mutans Streptococcus pyo genes Streptococcus viridans Iv Streptococcus equines Streptococcus oralis Streptococcus ratti Streptococcus n ,-i Streptococcus faecalis Streptococcus tigurinus Streptococcus salivariu zooepidemicus t=1 Iv t..) o Streptococcus ferus t..) o 'a o .6.
t..) t..) un C
t..) o t..) o Uliginosibacterium Vagococcus Vibrio Virgibacillus Xanthobacter c,.) .6.
.6.
t..) Vagococcus carniphilus Vibrio aero genes Virgibacillus Xanthobacter agilis oe Uliginosibacterium gangwonense Vagococcus elongatus Vibrio aestuarianus halodenitrificans Xanthobacter Ulvibacter Vagococcus fessus Vibrio albensis Virgibacillus aminoxidans Ulvibacter litoralis Vagococcus fluvialis Vibrio alginolyticus pantothenticus Xanthobacter Umezawaea Vagococcus lutrae Vibrio campbellii autotrophicus Weissella Umezawaea tan gerina Vagococcus salmoninarum Vibrio cholerae Xanthobacter flavus P
Weissella cibaria Undibacterium Vibrio cincinnatiensis Xanthobacter tagetidis 2 ,-, Variovorax Weissella confusa ;3' t..) Undibacterium pigrum Vibrio coralliilyticus Xanthobacter viscosus 2 .6.
r., Variovorax boronicumulans Weissella halotolerans Ureaplasma Vibrio cyclitrophicus Xanthomonas 2' ' 7 Variovorax dokdonensis Weissella hellenica 0"
Ureaplasma urealyticum Vibrio diazotrophicus Xanthomonas , ,3"
Variovorax paradoxus Vibrio fluvialis Weissella kandleri albilineans Variovorax soli Weissella koreensis Ureibacillus Vibrio furnissii Xanthomonas alfalfae Weissella minor Ureibacillus composti Vibrio gazo genes Xanthomonas Veillonella Weissella Ureibacillus suwonensis Vibrio halioticoli arboricola Veillonella atypica aramesenteroides Ureibacillus terrenus Vibrio harveyi p Xanthomonas Iv Veillonella caviae n Weissella soli Ureibacillus the rmophilus Vibrio ichthyoenteri axonopodis t=1 Veillonella criceti Iv Weissella thailandensis t..) Ureibacillus the rmosphaericus Vibrio mediterranei Xanthomonas o Veillonella dispar t..) Weissella viridescens o Vibrio metschnikovii campestris 'a Veillonella montpellierensis o .6.
t..) t..) un Veillonella parvula Vibrio mytili Xanthomonas citri o Williamsia t..) o Veillonella ratti Vibrio natriegens Xanthomonas codiaei r..) o Williamsia marianensis Veillonella rodentium Vibrio navarrensis Xanthomonas c...) .6.
.6.
Williamsia mans t...) Vibrio nereis cucurbitae oe Venenivibrio Williamsia serinedens Vibrio nigripulchritudo Xanthomonas Venenivibrio stagnispumantis Vibrio ordalii euvesicatoria Winogradskyella Vibrio orientalis Xanthomonas fragariae Wino gradskyella Vibrio parahaemolyticus Xanthomonas fuscans thalassocola Verminephrobacter Vibrio pectenicida Xanthomonas gardneri P
Vibrio penaeicida Xanthomonas hortorum Verminephrobacter eiseniae Wolbachia 2 1-, Vibrio proteolyticus Xanthomonas hyacinthi ? ;3' t..) Wolbachia persica ..
un r., Vibrio shilonii Xanthomonas perforans 2 '7 , Vibrio splendidus Xanthomonas phaseoli 0 N) Verrucomicrobium r., Vibrio tubiashii Xanthomonas pisi Verrucomicrobium spinosum Wolinella Vibrio vulnificus Xanthomonas populi Wolinella succino genes Xanthomonas theicola Xanthomonas translucens Iv n ,-i Zobellia m 1-d Zobellia galactanivorans Xanthomonas w o t..) o Zobellia uliginosa vesicatoria 'a o .6.
t..) t..) un C
Zoogloea Xylella t..) o t..) o Zoo gloea ramigera Xylella fastidiosa .6.
Zoo gloea resiniphila Xylophilus .6.
t...) oe Xylophilus ampelinus Xenophilus Yangia Yersinia mollaretii Zooshikella Zobellella Xenophilus azovorans Yangia pacifica Yersinia philomiragia Zooshikella ganghwensis Zobellella denitrificans Xenorhabdus Yersinia pestis Zobellella taiwanensis P
Yaniella Zunongwangia Xenorhabdus beddingii Yersinia pseudotuberculosis 2 , 1-, Yaniella flava Zunongwangia profunda .3 t..) Xenorhabdus bovienii Yersinia rohdei .2 o r., Yaniella halotolerans Xenorhabdus cabanillasii Yersinia ruckeri 2' , Zymobacter Zeaxanthinibacter Xenorhabdus doucetiae , Yeosuana Yokenella Zymobacter palmae Zeaxanthinibacter r.,"
Xenorhabdus griffiniae Yeosuana aromativorans enoshimensis eiensburg Xenorhabdus hominickii Yokenella reg Zymomonas Xenorhabdus koppenhoeferi Yersinia Zymomonas mobilis Zhihengliuella Yonghaparkia Xenorhabdus nematophila Yersinia aldovae Zhihengliuella Yonghaparkia alkaliphila Xenorhabdus poinarii Zymophilus 1-d Yersinia bercovieri halotolerans n ,-i Xylanibacter Zymophilus paucivorans Yersinia enterocolitica Zavarzinia Xylanibacterium t=1 1-d Xylanibacter oiyzae Zymophilus raffinosivorans t..) Yersinia entomophaga Zavarzinia compransoris Xylanibacterium ulmi o t..) o 'a Yersinia frederiksenii o .6.
t..) t..) un .,.., '.-"E3 e) ,'12.; = ,t2 4-=
= ,..4 ,-.=
= 4t = 4t = ,4 = ,4 4.1 4.1 }... }...

TABLE 2: SaPIs Element Staphylococcal genome Baba* Lindsay and Size Inducing Holden' (kb) phages SaP14 S. aureus str. MRSA252 NA SaP14 15.1 Endogenous prophage SaP11028 S. aureus str. NY940 NA NA 15.6 Endogenous prophage SaPlbovl S. aureus str. RF122 vSa2 NA 15.8 91 1 and 80a SaPlbov2 S. aureus str. V329 NA NA 27 80a SaP1m4 S. aureus str. mu50 v5a3 NA 14.4 Endogenous type I prophage SaPlmw2 S. aureus str. mw2 v5a3 SaP13 14.4 Endogenous type II prophage SeP11 S. aureus str. FRI909 NA NA 9.9 Not known ShP12 S. haemolyticus v5h2 NA 16.6 Not known SaP11 S. aureus str. RN4282 vSal NA 15.2 80a and 913 SaP13 S. (WIWI'S' str. COL vSal SaP11 15.6 Not known SaP15 S. aureus str. USA300 NA Na 14.0 Not known SaP1n1 and S. aureus str. n315 and S. v5a4 SaP12 15 80a SaP1m1 aureus str. mu50, respectively type 1 SaP12 S. aureus str. RN3984 NA NA 14.7 80 and 80a SaRlfusB S. aureus European fusidic NA NA 20.7 Not known acid-resistant impetigo clone SaP1122 S. aureus str. RF122 NA NA 17.9 Endogenous prophage SaP16.L S. aureus strains 8325, COL, v5a4 NA 3.14 Not known USA300, M55A476, type II
Newman and mw2 SsP115305 S. saprophyticus str. 15305 vSs15305 NA 16.7 Not known [N/A, not applicable; *Nomenclature proposed by Baba et al; # Nomenclature proposed by Lindsey &
Holden et all Table 3: Titers of SA100 packaged CGVs after production SA100 Native phage strain TFU/mL PFU/mL
EMG2 1.4E+10 n.d C-la 2.2E+10 <20 C-1792 5.2E+09 <20 SUBSTITUTE SHEET (RULE 26) Table 4: Groups (Example 5) Group Preparation (per 500 I) vehicle 139 ul 9% Sod.carb + 361 I H20 SA100 139 ul 9% Sod.carb + 330 I p114 + 31 I H20 SA100 + induction of CRISPR system 170 I Inducers+Sod.carb + 330 I p114 +

Table 5: Clinical Scores Score 0 healthy Score 1 Minor clinical signs of infection (slower movements, light piloerection in the skin) Score 2 Moderate signs of infection (slower movements, slightly pinched eyes, lack of curiosity or changed activity) Score 3 Severe signs of infection (reduced movements, pinched eyes, changed body posture) Score 4 Severe signs of infection (stiff movements, forced ventilation, pinched eyes) Sacrificed.
Score 5 The mouse does not move, is cold, lying on the side. Sacrificed Score 6 The mouse is dead Table 6: Sequences w o SEQ ID NO: DESCRIPTION SEQUENCE
w o i-J
4,.
1 terS nucleotide AATTGGCAGTAAAGTGGCAGTTTTTGATACCTAAAATGAGATATTATGATAGTGTAGGATAT
w oe sequence TGACTATCTTACTGCGTTTCCCTTATCGCAATTAGGAATAAAGGATCTATGTGGGTTGGCTG
ATTATAGCCAATCCTTTTTTAATTTTAAAAAGCGTATAGCGCGAGAGTTGGTGGTAAATGAA
ATGAACGAAAAACAAAAGAGATTCGCAGATGAATATATAATGAATGGATGTAATGGTAAAAA
AG CAG CAATTTCAG CAG GTTATAGTAAGAAAACAG CAGAGTCTTTAG CAAGTCGATTGTTAA
P
GAAATGTTAATGTTTCGGAATATATTAAAGAACGATTAGAACAGATACAAGAAGAGCGTTTA
, , 1¨
.3 =
ATGAGCATTACAGAAGCTTTAGCGTTATCTGCTTCTATTGCTAGAGGAGAACCTCAAGAGGC
, , TTACAGTAAGAAATATGACCATTTAAACGATGAAGTGGAAAAAGAGGTTACTTACACAATCA
, , ,, ,, CACCAACTTTTGAAGAGCGTCAGAGATCTATTGACCACATACTAAAAGTTCATGGTGCGTAT
ATCGACAAAAAAGAAATTACTCAGAAGAATATTGAGATTAATATTGGTGAGTACGATGACGA
AAGTTAAATTAAACTTTAACAAACCATCTAATGTTTTCAACAG
2 Packaging signal GCATGCGTTTTCCTGCCTCATTTTCTGCAAACCGCGCCATTCCCGGCGCGGTCTGAGCGTGTCAGTGCAACTGCATTAA
AACCG 1-d n sequence CCCCGCAAAGCGGGCGGGCGAGGCGGGGAAAGCACCGCGCGCAAACCCAGAAGTTAGTTAATTATTTGTGTAGTCAAAG

t=1 CTTGACTACATACCTCGTTAATACATTG GAG CATAATGAAGAAAATCTATG G CCTATG
GTCCAAAACTGTCTTTTTTGATG G CAC 1-d w o TATCCTGAAAAATATGCAAAAAATAGATTGATGTAAGGTGGTTCTTGTCAGTGTCGCAAGATCCTTAAGAATTC
w o 'a o 4,.
w w vi 3 Cos packaging signal GCATGCGTTTTCCTGCCTCATTTTCTGCAAACCGCGCCATTCCCGGCGCGGTCTGAGCGTGTCAGTGCAACTGCATTAA
AACCG

sequence CCCCGCAAAGCGGGCGGGCGAGGCGGGGAAAGCACCGCGCGCAAACCGACAAGTTAGTTAATTATTTGTGTAGTCAAAG
TGC w o w CTTCAGTACATACCTCGTTAATACATTGGAGCATAATGAAGAAAATCTATGGCCTATGGTC
i-J
4,.
4,.
4 P2/P4 packaging TGCATTAAAACCGCCCCGCAAAGCGGGCGGGCGAGGCGGGGAAAGCACCGCGCGC
w oe signal sequence sid ATGTCTGACCACACTATCCCTGAATATCTGCAACCCGCACTGGCACAACTGGAAAAGGCCAGAGCCGCCCATCTTGAGA
ACGC
CCGCCTGATGGATGAGACCGTCACGGCCATTGAACGGGCAGAGCAGGAAAAAAATGCGCTGGCGCAGGCCGACGGAAAC
GA
CGCTGACGACTGGCGCACGGCCTTTCGTGCAGCCGGTGGTGTCCTGAGCGACGAGCTGAAACAGCGCCACATTGAGCGC
GTG
GCACGCCGGGAGCTGGTACAGGAATATGACAATCTGGCCGTGGTGCTGAATTTCGAACGTGAACGCCTGAAAGGGGCGT
GTG
ACAGCACGGCCACCGCCTACCGGAAGGCACATCATCACCTTCTGAGTCTGTATGCAGAGCATGAGCTGGAACACGCCCT
GAAT P
GAAACCTGTGAGGCGCTTGTCCGGGCAATGCATCTGAGCATTCTGGTACAGGAAAATCCGCTCGCCAACACCACCGGCC
ATCA , 1¨
GGGCTACGTCGCACCGGAAAAGGCTGTCATGCAGCAGGTGAAATCATCGCTGGAACAGAAAATTAAACAGATGCAAATC
AGC , 1¨
CTCACCGGCGAGCCGGTTCTCCGGCTGACCGGACTGTCAGCGGCAACACTCCCGCACATGGATTATGAGGTGGCAGGCA
CACC

GGCACAGCGCAAGGTGTGGCAGGACAAAATAGACCAGCAGGGAGCAGAGCTTAAGGCCAGAGGGCTGCTGTCATGA
, , , , ,, 6 psu ATGGAAAGCACAGCCTTACAGCAGGCCTTTGACACCTGTCAGAATAACAAAGCAGCATGGCTGCAACGCAAAAATGAGC
TGG ,, CAGCGGCCGAACAGGAATATCTGCGGCTTCTGTCAGGAGAAGGCAGAAACGTCAGTCGCCTGGACGAATTACGCAATAT
TATC
GAAGTCAGAAAATGGCAGGTGAATCAGGCCGCCGGTCGTTATATTCGTTCGCATGAAGCCGTTCAGCACATCAGCATCC
GCGA
CCGGCTGAATGATTTTATGCAGCAGCACGGCACAGCACTGGCGGCCGCACTGGCACCGGAGCTGATGGGCTACAGTGAG
CTG
ACGGCCATTGCCCGAAACTGTGCCATACAGCGTGCCACAGATGCCCTGCGTGAAGCCCTTCTGTCCTGGCTTGCGAAGG
GTGA
AAAAATTAATTATTCCGCACAGGATAGCGACATTTTAACGACCATCGGATTCAGGCCTGACGTGGCTTCGGTGGATGAC
AGCC 1-d GTGAAAAATTCACCCCTGCGCAGAACATGATTTTTTCGCGTAAAAGTGCGCAACTGGCATCACGTCAGTCAGTGTAA
n ,-i m ,-o 7 Delta ATGATTTACTGTCCGTCGTGTGGACATGTTGCTCACACCCGTCGCGCACATTTCATGGACGATGGCACCAAGATAATGA
TTGCA w o w CAGTGCCGGAATATTTATTGCTCTGCGACATTTGAAGCGAGTGAAAGCTTTTTCTCTGACAGTAAAGATTCAGGAATGG
AATAC
'a o ATTTCAGGCAAACAGAGATACCGCGATTCACTGACGTCAGCCTCCTGCGGTATGAAACGCCCGAAAAGAATGCTTGTTA
CCGG
w w vi GTGCACGGATCCGGGCTGTGGTCTGGTGTTTAAAACGCTTCAGACCATCAACCGCTTCATTGTCCGCCCGGTCACGCCG
GACG r..) o r..) AACTGGCAGAACGCCTGCATGAAAAACAGGAACTGCCGCCAGTACGGTTAAAAACACAATCATATTCGCTGCGTCTGGA
ATGA
i-J
.6.
.6.
8 Q through S of P2 TCAGTCGTTGTCAGTGTCCAGTGAGTAGTTTTTAAAGCGGATGACCTCCTGACCGAGCCAGCCGTTTATCTCGCGGATC
CTGTC r..) oe CTGTAACGGGATAAGCTCATTGCGGACAAAGACCTTTGCCACTTTCTCAATATCACCCAGCGACCCGACGTTCTCCGGC
TTGCC
ACCCATCAACTGAAAGGGGATGCGGTGCGCGTCCAGCAGGTCAGCGGCGCTGGCTTTTTTGATATTAAAAAAATCGTCC
TTCG
TCGCCACTTCACTGAGGGGGATAATTTTAATGCCGTCGGCTTTCCCCTGTGGGGCATAGAGAAACAGGTTTTTAAAGTT
GTTGC
GGCCTTTCGACTTGACCATGTTTTCGCGAAGCATTTCGATATCGTTGCGATCCTGCACGGCATCGGTGACATACATGAT
GTATCC
GGCATGTGCGCCATTTTCGTAATACTTGCGGCGGAACAACGTGGCCGACTCATTCAGCCAGGCAGAGTTAAGGGCGCTG
AGAT
ATTCCGGCAGGCCGTACAGCTCCTGATTAATATCCGGCTCCAGCAGGTGAAACACGGAGCCGGGCGCGAAGGCTGTCGG
CTC
GTTGAAGGACGGCACCCACCAGTAAACATCCTCTTCCACGCCACGGCGGGTATATTTTGCCGGTGAGGTTTCCAGTCTG
ATGAC P
CTTACCGGTGGTGCTGTAACGCTTTTCCAGAAACGCATTACCGAACACCAGAAAATCCAGCACAAAGCGGCTGAAATCC
TGCT L.
, L.
, 1¨
GGGAAAGCCATGGATGCGGGATAAATGTCGAGGCCAGAATATTGCGTTTGACGTAAATCGGCGAGCTGTGATGCACGGC
AGC .3 .
r..) CCGCAGGCTTTTTGCCAGACCGGTAAAGCTGACCGGTGGCTCATACCATCTGCCGTTACTGATGCACTCGACGTAATCC
AGAAT

, GTCACGGCGGTCGAGTACCGGCACCGGCTCACCAAAGGTGAATGCCTCCATTTTCGGGCCGCTGGCGGTCATTGTTTTT
GCCG , , CAGGTTGCGGTGTTTTCCCTTTTTTCTTGCTCATCAGTAAAACTCCAGAATGGTGGATGTCAGCGGGGTGCTGATACCG
GCGGT
GAGTGGCTCATTTAACAGGGCGTGCATGGTCGCCCAGGCGAGGTCGGCGTGGCTGGCTTCCTCGCTGCGGCTGGCCTCA
TAG
GTGGCGCTGCGTCCGCTGCTGGTCATGGTCTTGCGGATAGCCATAAACGAGCTGGTGATGTCGGTGGCGCTGACGTCGT
ATTC
CAGACAGCCACGGCGGATAACGTCTTTTGCCTTGAGCACCATTGCGGTTTTCATTTCCGGCGTGTAGCGGATATCACGC
GCGGC
GGGATAGAACGAGCGCACGAGCTGGAACACGCCGACACCGAGGCCGGTGGCATCAATACCGATGTATTCGACGTTGTAT
TTT
TCGGTGAGTTTGCGGATGGATTCCGCCTGGGTGGCAAAGTCCATGCCTTTCCACTGGTGACGCTCAAGTATTCTGAATT
TGCCA
1-d CCGGCCACCACCGGCGGTGCCAGTACCACGCATCCGGCGCTGTCGCCACGGTGTGACGGGTCGTAACCAATCCATACCG
GGC n GGGAGCCGAACGGATTGGCGGCAAACGGCGCATAGTCTTCCCATTCTTCCAGCGTGTCGACCATGCAGCGTTGCAGCTC
CTCG t=1 1-d AACGGGAACACCGATGCCTTGTCGTCAACAAATTCACACATGAACAGGTTTTTAAAATCGTCGGCGCTGTTTTCGCGTT
TGAGC r..) o r..) TGCTCAATGTCGAACAGCGTGCAGCCGCCTTTCAGGGCGTCCTCAATGGTGACAATCTGTCGCCACTGGCCGTCCGCAC
AGAG =
'a AAGCCCACCGGCAAGGGCGTTATGACTGACGTCGATTTCCACGCGTTCGGCGGCGCTGGCGCGTCCCCGGTTAAACAGT
TCAC o .6.
r..) r..) un CCGACCAGAACGGGTAGGCGTCGTGCGCCAGCGTGGACGGGGTGGAGAAATAGGTCGAGCGCAGGTGACTCTGTGAGGC
CA

TACCTGATGCCACCTTACGCAGTACCTGAAAATTCGGGATCCAGAAAATCTCATCGACGTACAGGTCGCCGTTATGACT
CTGCG n.) o n.) CGGTGTTGGAGTTGGTGCCGAGAAAAATCAGTTTTGCGCCGTTATTGCCCAGGACAATCGGGTCACCGGTCAGGTCAAC
GTCA
i-J
ACCAGACGGGCAAAGGCGATGATGTATTCGCGGAACACATACGCCTGTGTTTTACTGGCCGACAGAAAAATCTGGTTAT
GACC .6.
.6.
n.) GGTTTTCAGGGCACGCAGCAGCGCCTCGCGGGAAAAATAAAACGTCGCGCCAATCTGGCGGGATTTCAGGATATCGCGG
ATG oe CGGTGCTCAAGCCCGGCACGATACCAGTGCAGCTGATAGTCGAAAGACTGCTCAAAGAAAATCTGCTCCAGCTTTTCGA
TGGC
CTCGTCACTGAAAAAATTCTTTTTCGGTTTGCGCCGTCCGCCTTTGTTACGGTTAGCGACGTTCGGATTAAGGTCTGCC
TCGTTG
CCGGTCTGGCTGTAGCGGTTGACCCGTGCCAGTCGTTCAATCTGGCGTCCCAGCAGGTCAATTTCCTTGAAGTCACCGC
CGGTT
TTCTGTGGTTTGATGATGAGCTGGGTCAGCCGCGCTTCCAGACTCATTTCGACACGGCTGATGGGGGCAACGCTGTCCC
AGCC
GTCGCGCTGTTTCCAGCTCTGCACTGTCGGGCGTTTCATCTGCAACATGGCGGCAATCTGCGGCACGGAAAACCCCTGC
CAGTA
CAGCAGCGCCGCCTGACGACGCGGGTCGTGTAAAAGAGTGGTGTCTGTGGTGATGGTCATGAATACCTCGCCGTGATGA
ATA
P
CACGGCAAGGCTACTGAGTCGCGCCCCGCGATTCGCTAAGGTGCTGTTGTGTCAGTGATAAGCCATCCGGGACTGATGG
CGG

AGGATGCGCATCGTCGGGAAACTGATGCCGACATGTGACTCCTCTAATCACTATTCAGGACTCCTGACAATGGCAAAAA
AAGT
CTCAAAATTCTTTCGTATCGGCGTTGAGGGTGACACCTGTGACGGGCGTGTCATCAGTGCGCAGGATATTCAGGAAATG

.3 AAACCTTTGACCCGCGTGTCTATGGTTGCCGCATTAACCTGGAACATCTGCGCGGCATCCTGCCTGACGGTATTTTTAA

ATGGCGATGTGGCCGAACTGAAGGCCGAAAAGATTGACGATGATTCGGCGCTGAAAGGCAAATGGGCGCTGTTTGCGAA
AAT
.31 .3 CACCCCGACCGATGACCTTATCGCGATGAACAAGGCCGCGCAGAAGGTCTACACCTCAATGGAAATTCAGCCGAACTTT
GCCA
ACACCGGCAAATGTTATCTGGTGGGTCTGGCCGTCACCGATGACCCGGCAAGCCTCGGCACGGAATACCTGGAATTCTG
CCGC
ACGGCAAAACACAACCCCCTGAACCGCTTCAAATTAAGCCCTGAAAACCTGATTTCAGTGGCAACGCCTGTTGAGCTGG
AATTT
GAAGACCTGCCTGAAACCGTGTTCACCGCCCTGACCGAAAAGGTGAAGTCCATTTTTGGCCGCAAACAGGCCAGCGATG
ATGC
CCGTCTGAATGACGTGCATGAAGCGGTGACCGCTGTTGCTGAACATGTGCAGGAAAAACTGAGCGCCACTGAGCAGCGC
CTC
GCTGAGATGGAAACCGCCTTTTCTGCACTTAAGCAGGAGGTGACTGACAGGGCGGATGAAACCAGCCAGGCATTCACCC
GCC
IV
TGAAAAACAGTCTCGACCACACCGAAAGTCTGACCCAGCAGCGCCGCAGCAAAGCCACCGGCGGTGGCGGTGACGCCCT
GAT n GACGAACTGCTGACCGGCGTCAGTCAGTCCGGGAAAACCTTCACGATTAACCCTTAATTTCAGGAAAAACTATGCGCCA
GGAA t=1 tl ACCCGCTTTAAATTTAATGCCTACCTGTCCCGTGTTGCCGAACTGAACGGCATCGACGCCGGTGATGTGTCGAAAAAAT

o GTTGAACCGTCGGTCACCCAGACCCTGATGAACACCATGCAGGAGTCCTCTGACTTTCTGACCCGCATCAATATTGTGC
CGGTC 'a o AGCGAAATGAAAGGGGAAAAAATTGGTATCGGTGTCACCGGCTCCATCGCCAGCACTACCGACACTGCCGGTGGTACCG
AGC tt n.) un GTCAGCCGAAGGACTTCTCGAAGCTGGCGTCAAACAAGTACGAATGCGACCAGATTAACTTCGATTTTTATATCCGCTA
CAAAA

CGCTGGACCTGTGGGCGCGTTATCAGGATTTCCAGCTCCGTATCCGTAACGCCATTATCAAACGCCAGTCCCTTGATTT
CATCAT n.) o n.) GGCCGGTTTTAACGGCGTGAAGCGTGCCGAAACCTCTGACCGCAGCAGCAATCCGATGTTGCAGGATGTGGCGGTCGGC
TGG
i-J
CTGCAGAAATACCGCAATGAAGCACCGGCGCGCGTGATGAGCAAGGTCACTGACGAGGAAGGCCGCACCACCTCTGAGG
TTA .6.
.6.
n.) TCCGCGTGGGTAAGGGCGGTGATTATGCCAGCCTTGATGCACTGGTGATGGATGCGACCAACAACCTGATTGAACCGTG
GTAT oe CAGGAAGACCCTGACCTTGTGGTGATTGTGGGGCGTCAGCTACTGGCGGACAAGTATTTCCCCATCGTCAACAAGGAGC
AGG
ACAACAGCGAAATGCTGGCCGCTGACGTCATCATCAGCCAGAAACGCATCGGTAACCTACCAGCGGTACGCGTCCCGTA
CTTC
CCGGCGGATGCGATGCTCATCACGAAGCTGGAAAACCTGTCCATCTACTACATGGATGACAGCCATCGCCGCGTGATTG
AGGA
AAACCCGAAACTCGACCGCGTGGAGAACTACGAGTCAATGAACATTGATTACGTGGTGGAAGACTACGCCGCCGGTTGT
CTG
GTGGAAAAAATCAAGGTCGGTGACTTCTCCACACCGGCTAAGGCGACCGCAGAGCCGGGAGCGTAACCGATGACGAGTC
CCG
CACAGCGCCACATGATGCGGGTCTCGGCAGCGATGACCGCGCAGCGGGAAGCCGCCCCGCTGCGACATGCAACTGTCTA
TGA
P
GCAGATGCTGGTTAAGCTCGCCGCAGACCAGCGCACACTGAAAGCGATTTACTCAAAAGAGCTGAAGGCCGCAAAAAAA
CGC

GAACTGCTGCCGTTCTGGTTGCCGTGGGTGAACGGCGTGCTGGAGCTGGGCAAAGGTGCACAGGATGACATTCTGATGA
CGG
TCATGCTGTGGCGTCTGGATACCGGCGATATTGCCGGTGCGCTGGAGATTGCCCGTTATGCCCTGAAGTACGGTCTGAC

.6.
.3 CCGGGTAAACACCGCCGTACCCCGCCGTACATGTTCACCGAGGAGGTAGCGCTTGCGGCCATGCGCGCTCACGCTGCCG

GTCTGTGGATACCCGCCTGCTGACGGAGACCCTTGAACTGACCGCCACGGCTGACATGCCTGATGAAGTGCGCGCAAAG
CTGC
.31 .3 ACAAAATCACCGGTCTGTTTCTGCGTGACGGTGGTGATGCCGCCGGTGCGCTGGCGCACCTGCAACGTGCGACACAGCT
CGAC
TGTCAGGCAGGCGTCAAAAAAGAGATTGAACGACTGGAGCGGGAGCTGAAACCGAAGCCGGAGCCGCAGCCCAAAGCGG
CC
ACCCGCGCCCCGCGTAAGACCCGGAGCGTGACACCGGCAAAACGTGGACGCCCGAAAAAGAAAGCCAGTTAACAACCGA
ATG
CGCCCCGCGCCAGGGCGGCACGCCGGTCAGTGACGGTGAATCACCTGACACTGCACCGGCGTCCACCGCCCGACTTTTC
AGAG
GTAGTCATGATGACGCTGATTATTCCGCGAAAGGAGGCTCCCGTGTCCGGTGAGGGTACGGTGGTCATCCCGCAACCGG
CAG
GCGACGAGCCGGTGATTAAAAACACGTTCTTTTTTCCCGATATCGACCCGAAGCGCGTCCGGGAACGTATGCGCCTTGA
GCAG
IV
ACCGTCGCCCCCGCCCGTCTGCGTGAGGCCATCAAGTCAGGCATGGCTGAAACGAATGCGGAGCTGTACGAGTACCGCG
AAC n AGAAAATTGCCGCCGGTTTTACGCGTCTGGCTGACGTCCCGGCGGACGATATCGACGGTGAAAGCATCAAGGTTTTTTA
CTAC t=1 tl GAGCGCGCCGTGTGTGCGATGGCGACCGCGTCGCTTTATGAGCGTTATCGCGGTGTGGATGCCAGTGCGAAAGGCGACA

o AGGCTGACAGCATTGACAGCACCATTGATGAGCTGTGGCGGGATATGCGCTGGGCGGTGGCGCGCATCCAGGGCAAGCC
GC 'a o GCTGCATCGTGAGTCAAATCTGATGAAGACCTTTGCGCTACAGGGCGACACGCTCGACGCCATTTGTGTCCGCTATTAC
GGGC tt n.) un GCACTGAGGGCGTGGTTGAGACCGTGCTCGCCGCAAATCCGGGACTGGCTGAACTGGGGGCGGTGCTGCCACACGGCAC
CG

CCGTCGAACTGCCCGACGTTCAGACCGCGCCCGTGGCTGAAACTGTCAATCTGTGGGAGTAACGCATGACAGCAGAAGA
AAA n.) o n.) AAGCGTCCTGTCGCTTTTCATGATTGGGGTGCTGATTGTTGTCGGCAAGGTGCTTGCCGGTGGTGAACCTATCACCCCG
CGTCT
i-J
GTTTATCGGGCGCATGTTGCTCGGTGGTTTTGTCTCGATGGTTGCCGGTGTTGTTCTGGTGCAGTTTCCTGACCTGTCA
CTGCCA .6.
.6.
n.) GCGGTGTGCGGCATCGGCTCCATGCTGGGTATCGCCGGTTATCAGGTGATTGAGATTGCCATTCAGCGCCGCTTTAAGG
GCAG oe GGGGAAACAGTAATGCCGGTAATTAACACGCATCAGAATATCGCCGCCTTTCTCGACATGCTGGCCGTGTCCGAAGGGA
CGGC
GAATCATCCACTGACGAAAAACCGGGGCTATGACGTGATAGTCACCGGACTGGACGGGAAGCCGGAAATTTTCACCGAC
TAC
AGTGACCACCCGTTCGCACATGGCCGACCGGCGAAGGTGTTTAACCGTCGCGGTGAAAAATCCACGGCCTCCGGTCGCT
ATCA
GCAGCTTTACCTGTTCTGGCCGCATTACCGCAAACAGCTTGCCCTGCCGGATTTCAGTCCGTTGTCACAGGACAGACTC
GCCAT
TCAGTTGATCCGCGAACGCGGAGCACTGGATGACATCCGGGCGGGACGCATTGAGCGCGCCATTTCACGCTGTCGCAAT
ATCT
GGGCGTCCCTGCCGGGTGCCGGTTACGGTCAGCGTGAGCATTCACTGGAAAAACTGGTCACCGTCTGGCGTACCGCTGG
CGG
P
CGTACCGGCTTAAACGGAGTAAATACCATGAAGAAATTATCCCTTTCACTGATGCTGAACGTGTCGCTGGCGCTGATGC
TGGC

ACTGTCCCTGATTTACCCGCAGAGCGTGGCCGTCAATTTTGTCGCTGCCTGGGCGATTCTGGCGACGGTTATCTGTGTG
GTTGC
CGGTGGTGTGGGCGTGTATGCCACTGAGTATGTGCTGGAACGCTACGGGCGGGAGCTGCCGCCGGAATCGCTGGCCGTG

un .3 GATTGTCACGTCGCTGTTTTTGCAGCCGGTGCCGTGGCGCAGACGGGCGGCGGCTCTGGTAGTGGTGGTGGCGACGTTT

CGCTGGTCGCTGCCGGGTGGATTTTTACCGCGCTGATTTATCTTGTGGTGTCGCTGTTTTTCCGGCTGATACGTAAAGC
CTGTC
.31 .3 GTCAGCGTCTTGAGGGGCGGGAACCATGTCAAGGCTGATGATTGTGCTGGTCGTGTTGTTATCGCTGGCGGTGGCCGGT
CTGT
TTCTGGTGAAACACAAAAATGCCAGCCTGCGCGCCTCGCTGGACAGGGCGAACAACGTCGCCAGCGGTCAGCAGACGAC
CAT
CACCATGCTGAAAAATCAGCTTCATGTTGCGCTCACCAGGGCAGATAAAAACGAGCTGGCGCAGGTGGCACTGCGTCAG
GAA
CTGGAGAACGCCGCGAAACGTGAAGCACAGCGCGAGAAAACCATCACGAGGTTACTTAATGAGAACGAAGATTTTCGCC
GCT
GGTACGGTGCTGACCTGCCTGATGCTGTGCGCCGGTTGCACCAGCGCCCCGCCTGCACCGACGCCAGTGATTGTCCCCA
ACGC
ATGCCCGAAAGTGAGCCTTTGCCCGATGCCGGGCAGTGACCCGCAGACGAACGGCGATTTAAGTGCCGATATCCGGCAG
CTT
IV
GAGAACGCGCTGGCACGCTGTGCCAGCCAGGTAAAAATGATTAAACACTGTCAGGACGAAAACGATGCTCAAACCCGAC
AGC n CTGCGCAGGGCGCTGACTGATGCCGTCACGGTGCTGAAAACTAACCCCGATATGCTGCGGATATTCGTGGATAACGGGA
GTAT t=1 tl TGCCTCCACACTGGCGGCGTCGCTGTCATTCGAAAAGCGTTACACGCTCAATGTGATTGTGACCGACTTTACCGGTGAT

o CCTGCTCATTGTGCCGGTGCTGGCGTGGCTGCGGGAAAATCAGCCCGACATCATGACCACCGACGAAGGCCAGAAAAAG
GGC 'a o TTCACGTTTTATGCAGACATCAACAATGACAGCAGCTTTGATATCAGTATCAGCCTGATGCTGACCGAGCGCACGCTGG
TCAGT tt n.) un GAGGTGGACGGCGCACTGCATGTGAAGAATATCTCGGAACCCCCGCCGCCGGAGCCGGTCACCCGCCCGATGGAGCTGT
ATA

TCAATGGCGAACTGGTGAGTAAGTGGGATGAATGAGTTTAAGCGTTTTGAAGACCGGCTGACCGGACTGATTGAATCGC
TGTC n.) o n.) ACCGTCAGGGCGTCGGCGACTGAGTGCCGAACTGGCGAAACGTCTGCGGCAGAGTCAGCAGCGTCGGGTGATGGCACAG
AA
AGCCCCGGACGGCACACCCTACGCGCCACGCCAGCAGCAGAGCGTCAGAAAAAAGACCGGTCGCGTTAAGCGAAAAATG
TTT .6.
.6.
n.) GCGAAACTTATTACCAGTCGTTTTTTGCATATCCGTGCCAGCCCGGAGCAGGCATCAATGGAATTTTACGGCGGGAAGT
CGCC oe GAAAATCGCCAGTGTGCATCAGTTTGGTCTGTCGGAAGAAAACCGGAAAGACGGTAAGAAAATTGATTATCCGGCGCGT
CCCC
TGCTCGGCTTTACCGGTGAGGATGTGCAGATGATTGAAGAGATTATCCTGGCTCACCTTGAGCGTTAG
9 V through G of P2 ATGAACACTCTCGCAAATATTCAGGAACTCGCGCGCGCACTGCGCAACATGATTCGCACTGGCATTATCGTCGAAACCG
ACCTT
AACGCCGGTCGCTGCCGCGTGCAGACCGGCGGCATGTGCACCGACTGGCTTCAGTGGCTGACCCATCGCGCAGGACGTT
CGC
GCACATGGTGGGCACCTTCCGTGGGGGAACAGGTGCTGATTCTGGCCGTGGGTGGTGAACTCGACACGGCGTTCGTTCT
GCC
GGGGATTTATTCCGGCGATAACCCCTCGCCGTCTGTGTCGGCGGATGCCCTGCATATCCGTTTCCCTGACGGGGCGGTG
ATTG P
AATATGAACCCGAAACCAGTGCACTCACGGTAAGCGGAATTAAAACGGCCAGCGTGACGGCTTCCGGTTCTGTTACTGC
CACG L.
, L.
, 1-, GTGCCGGTGGTCATGGTGAAAGCATCAACCCGCGTCACCCTGGACACCCCGGAGGTGGTCTGCACCAACAGGCTGATTA
CCG .3 .
eA
GCACGCTGGAAGTGCAGAAAGGCGGGACGATGCGCGGCAACATTGAACACACCGGCGGTGAACTCTCATCAAACGGTAA
GG

, TACTGCATACCCATAAACACCCCGGCGACAGCGGCGGCACAACCGGGAGTCCTTTATGACAGCGCGTTATCTCGGAATG
AATC , , GCAGTGATGGCCTGACTGTCACTGACCTTGAGCATATCAGCCAGAGTATCGGCGATATCCTGCGCACACCGGTCGGCTC
ACGG
GTGATGCGTCGTGATTACGGCTCGTTGCTGGCGTCAATGATTGACCAGCCGCAGACCCCGGCGCTTGAGTTGCAGATTA
AAGT
CGCCTGTTACATGGCAGTGCTGAAATGGGAACCCCGCGTCACCCTGTCATCCGTCACCACGGCGCGCAGTTTTGACGGG
CGAA
TGACGGTCACGTTAACCGGCCAGCACAACGACACCGGCCAGCCACTTTCATTAACCATCCCTGTGAGTTGAAACCATGC
CGATT
ATCGACCTGAACCAGCTACCCGCACCGGATGTGGTCGAGGAGCTGGACTTTGAAAGCATTCTCGCTGAACGCAAGGCGA
CACT
GATTTCCCTTTACCCGGAAGATCAGCAGGAGGCGGTCGCCCGTACCCTGACACTGGAATCTGAGCCTCTCGTCAAACTG
CTGG
Iv AAGAAAATGCTTATCGTGAGCTTATCTGGCGTCAGCGTGTGAATGAGGCCGCACGGGCGGTGATGCTGGCCTGTGCCGC
CGG n TAATGACCTTGATGTGATTGGTGCCAATTACAACACCACGCGCCTGACTATCACCCCGGCAGATGATTCGACCATCCCG
CCGAC t=1 Iv ACCGGCAGTGATGGAATCTGACACCGATTATCGTCTGCGTATTCAGCAGGCTTTTGAGGGCTTAAGCGTCGCCGGGTCA
GTGG n.) o n.) GAGCCTATCAGTATCATGGTCGCAGTGCTGACGGGCGTGTCGCGGATATTTCTGTCACCAGTCCGTCTCCGGCCTGTGT
CACCA
'a TCTCTGTGCTGTCACGTGAAAATAACGGCGTCGCATCCGAAGACCTGCTGGCTGTGGTGCGTAACGCCCTTAATGGCGA
GGAC eA
.6.
n.) n.) un GTCAGGCCGGTGGCCGACCGCGTGACCGTGCAGTCTGCCGCCATCGTTGAATACCAGATAAACGCCACGCTTTACCTTT
ACCCT

GGTCCCGAAAGCGAACCCATCCGCGCTGCCGCTGTGAAAAAGCTGGAAGCGTATATCACGGCACAGCACCGGCTGGGGC
GCG n.) o n.) ACATCCGTCTGTCTGCCATTTATGCCGCTTTGCATGTGGAAGGTGTGCAGCGTGTCGAACTGGCTGCACCACTGGCCGA
CATCG
i-J
TGCTCAACAGTACGCAGGCGTCTTTCTGTACCGAATACCGCGTCGTGACCGGAGGCTCGGATGAGTGATTCGCGACTGC
TGCC .6.
.6.
n.) GACCGGCTCATCACCGCTTGAGGTCGCCGCCGCAAAAGCCTGTGCGGAAATTGAAAAAACGCCGGTCAGTATTCGTGAA
CTGT oe GGAACCCGGACACCTGTCCGGCAAATCTGCTGCCGTGGCTGGCGTGGGCGTTTTCGGTCGACAGGTGGGATGAAAAGTG
GCC
GGAAGCGACAAAACGCGCCGTTATCCGCGATGCCTATTTCATCCACTGTCATAAGGGCACGATAGGTGCAATCCGGCGT
GTGG
TGGAGCCGCTCGGCTATCTCATCAACGTGACGGAGTGGTGGGAAAACAGTGACCCGCCCGGCACCTTCCGGCTTGATAT
TGGT
GTACTGGAAAGCGGTATCACAGAGGCAATGTATCAGGAAATGGAACGGCTGATTGCTGATGCCAAACCTGCAAGCCGTC
ACC
TTATTGGCCTGAACATTACCCGGGACATTCCCGGCTATCTGTTCGCCGGTGGTGTGGCTTACGACGGCGATGTAATTAC
GGTTT
ACCCCGGATAAGTGAGGAATAATGAGCATAAAATTCAGAACCGTTATCACCACTGCCGGTGCAGCAAAGCTGGCAGCGG
CAA
P
CCGCGCCGGGAAGGCGGAAGGTCGGCATTACCACGATGGCCGTCGGGGATGGCGGTGGTAAATTGCCTGTCCCGGATGC
CG

GACAGACCGGGCTTATCCATGAAGTCTGGCGACATGCGCTGAACAAAATCAGCCAGGACAAACGAAACAGTAATTATAT
TATC
GCCGAGCTGGTTATTCCGCCGGAGGTGGGCGGTTTCTGGATGCGTGAGCTTGGCCTGTACGATGATGCGGGAACGTTAA

--.1 .3 CCGTGGCGAACATGGCCGAAAGCTATAAGCCAGCCCTTGCCGAAGGCTCAGGACGTTGGCAGACCTGTCGCATGGTCAT

GTCAGCAGTGTGGCCTCAGTGGAGCTGACCATTGACACCACAACGGTGATGGCGACGCAGGATTACGTTGATGACAAAA
TTG
.31 .3 CAGAGCACGAACAGTCACGACGTCACCCGGACGCCTCGCTGACAGCAAAAGGTTTTACTCAGTTAAGCAGTGCGACCAA
CAGC
ACGTCTGAAACACTGGCCGCAACGCCGAAAGCGGTAAAGGCCGCGTATGACCTGGCTAACGGGAAATATACCGCACAGG
ACG
CCACCACAGCGCGAAAAGGCCTTGTCCAGCTTAGTAGCGCCACCAACAGCACGTCTGAAACGCTCGCCGCAACACCAAA
AGCC
GTTAAGACGGTAATGGATGAAACGAACAAAAAAGCGCCATTAAACAGCCCTGCACTGACCGGAACGCCAACGACGCCAA
CTG
CGCGACAGGGAACGAATAATACTCAGATCGCAAACACGGCTTTCGTTATGGCCGCGATTGCCGCCCTTGTAGACTCGTC
GCCT
GACGCACTGAATACGCTGAACGAGCTGGCGGCGGCGCTGGGCAATGACCCGAATTTTGCTACCACCATGACTAATGCGC
TTGC
IV
GGGTAAGCAACCGAAAGATGCTACCCTGACGGCGCTGGCGGGGCTTGCTACTGCGGCAGACAGGTTTCCGTATTTTACG
GGG n AATGATGTTGCCAGCCTGGCGACCCTGACAAAAGTCGGGCGGGATATTCTGGCTAAATCGACCGTTGCCGCCGTTATCG
AATA t=1 tl TCTCGGTTTACAGGAAACGGTAAACCGAGCCGGGAACGCCGTGCAAAAAAATGGCGATACCTTGTCCGGTGGACTTACT

o AAAACGACTCAATCCTTGCCTGGATTCGAAATACTGACTGGGCGAAGATTGGATTTAAAAATGATGCCGATGGTGACAC
TGAT 'a o TCATACATGTGGTTTGAAACGGGGGATAACGGCAATGAATATTTCAAATGGAGAAGCCGCCAGAGTACCACAACAAAAG
ACC tt n.) un TGATGACGTTGAAATGGGATGCACTAAATATTCTTGTTAATGCCGTCATTAATGGCTGTTTTGGAGTTGGTACGACGAA
TGCAC

TAGGTGGTAGCTCTATTGTTCTTGGTGATAATGATACCGGATTTAAACAGAATGGAGACGGTATTCTTGATGTTTATGC
TAACA tµ.) o tµ.) GTCAGCGTGTATTCCGTTTTCAGAATGGAGTGGCTATTGCTTTTAAAAATATTCAGGCAGGTGATAGTAAAAAGTTCTC
GCTAT
i-J
CCAGCTCTAATACATCCACGAAGAATATTACCTTTAATTTATGGGGTGCTTCCACCCGTCCAGTGGTTGCAGAGTTAGG
CGATG .6.
.6.
tµ.) AGGCCGGATGGCATTTCTATAGCCAGCGAAATACAGATAACTCGGTAATATTTGCTGTTAACGGTCAGATGCAACCCAG
CAAC oe TGGGGAAATTTTGATTCCCGCTATGTGAAAGATGTTCGCCTGGGTACGCGAGTTGTTCAATTGATGGCGCGAGGTGGTC
GTTA
TGAAAAAGCCGGACACACGATTACCGGATTAAGAATCATTGGTGAAGTAGATGGCGATGATGAAGCCATCTTCAGGCCG
ATA
CAAAAATACATCAATGGCACATGGTATAACGTTGCGCAGGTGTAAGTTATGCAGCATTTAAAGAACATTAAGTCAGGTA
ATCC
AAAAACAAAAGAGCAATATCAGCTAACAAAGAATTTTGATGTTATCTGGTTATGGTCCGAAGACGGAAAAAACTGGTAT
GAGG
AAGTGAAGAACTTTCAGCCAGACACAATAAAGATTGTTTACGATGAAAATAATATTATTGTCGCTATCACCAGAGATGC
TTCAA
CGCTTAATCCTGAAGGTTTTAGCGTTGTTGAGGTTCCTGATATTACCTCCAACCGACGTGCTGACGACTCAGGTAAATG
GATGT
P
TTAAGGATGGTGCTGTGGTTAAACGGATTTATACGGCAGATGAACAGCAACAACAGGCAGAATCACAAAAGGCCGCGTT
ACTT .
, TCCGAAGCGGAAAACGTTATTCAGCCACTGGAACGCGCTGTCAGGCTGAATATGGCGACGGATGAGGAACGTGCACGAC
TGG
...]
1¨
.3 AGTCATGGGAACGTTACAGCGTTCTGGTCAGCCGTGTGGATCCTGCAAATCCTGAATGGCCGGAAATGCCGCAATAA
.
oe , , Fl through ogr of P2 ATGAGTGACTATCATCACGGCGTGCAGGTGCTGGAGATTAACGAGGGCACCCGCGTCATTTCCACCGTATCCACGGCCA
TTGT , , CGGCATGGTCTGCACGGCCAGCGATGCAGATGCGGAAACCTTCCCCCTCAATAAACCTGTGCTGATTACCAATGTGCAG
AGCG
CAATTTCAAAGGCCGGTAAAAAAGGCACGCTGGCGGCATCGTTGCAGGCCATCGCTGACCAGTCAAAACCGGTCACCGT
TGTC
ATGCGCGTGGAAGACGGCACCGGTGATGACGAGGAAACGAAACTCGCGCAGACCGTTTCCAATATCATCGGCACCACCG
ATG
AAAACGGTCAGTACACCGGACTAAAAGCCATGCTGGCGGCGGAGTCGGTAACCGGTGTTAAACCGCGTATTCTCGGCGT
GCC
GGGACTGGATACCAAAGAGGTGGCTGTTGCACTGGCATCAGTCTGTCAGAAGCTGCGTGCTTTCGGGTATATCAGCGCA
TGG
GGCTGTAAAACCATTTCCGAGGTGAAAGCCTATCGTCAGAATTTCAGCCAGCGTGAGCTGATGGTCATCTGGCCGGATT
TCCTC
1-d GCATGGGATACGGTCACCAGTACCACCGCCACCGCGTATGCCACCGCCCGTGCGCTGGGGCTGCGCGCTAAAATCGACC
AGG n ,-i AGCAGGGCTGGCATAAAACGCTGTCCAATGTCGGGGTGAACGGTGTTACCGGCATCAGCGCATCTGTATTCTGGGATTT
GCAG t=1 1-d GAGTCCGGCACCGATGCTGACCTGCTTAACGAGTCAGGCGTCACTACGCTGATTCGCCGCGACGGTTTCCGCTTCTGGG
GTAA tµ.) o tµ.) CCGTACCTGCTCTGATGACCCGCTGTTCCTCTTTGAAAACTACACCCGCACCGCGCAGGTCGTGGCCGACACGATGGCT
GAGGC =
'a GCACATGTGGGCGGTGGACAAGCCCATCACTGCAACGCTGATTCGCGACATCGTTGACGGCATCAATGCCAAATTCCGT
GAGC
.6.
tµ.) tµ.) un TGAAAACAAACGGCTATATCGTGGATGCGACCTGCTGGTTCAGCGAAGAATCCAACGATGCGGAAACCCTCAAGGCCGG
AAA

ACTGTATATCGACTACGACTATACACCGGTGCCTCCTCTCGAAAACCTGACCCTGCGCCAGCGTATTACCGATAAATAC
CTGGC n.) o n.) AAATCTGGTCACCTCGGTTAACAGCAATTAAGGAGCCTGACCGATGGCAATGCCGCGCAAACTCAAGTTAATGAACGTC
TTTCT
i-J
GAACGGCTACAGCTATCAGGGCGTTGCAAAGTCCGTCACGCTGCCAAAACTGACCCGTAAGCTCGAAAACTATCGCGGT
GCGG .6.
.6.
n.) GGATGAACGGCAGCGCACCGGTAGACCTCGGCCTTGATGACGATGCGCTGTCAATGGAGTGGTCGCTCGGTGGCTTCCC
GGA oe TTCGGTTATCTGGGAGCTTTACGCCGCAACCGGTGTGGATGCCGTGCCGATTCGTTTTGCAGGCTCTTACCAGCGCGAC
GATAC
CGGCGAAACGGTGGCCGTCGAAGTGGTCATGCGTGGACGTCAGAAAGAAATCGACACCGGCGAGGGTAAACAGGGAGAA
G
ACACTGAGTCGAAAATCTCCGTGGTCTGCACCTATTTCCGGCTGACGATGGACGGTAAGGAGCTGGTCGAAATTGACAC
CATC
AACATGATTGAGAAGGTGAACGGCGTCGATCGGCTGGAGCAACACCGCCGCAATATCGGCCTGTGATTTTCATCCGGTC
AGCC
TGGCTGGCCGGTTAACCCTGATTCAGAAGTGAGAAAACCATGAACAAAGAAAATGTCATTACCCTGGACAATCCGGTCA
AACG
TGGTGAGCAGGTTATCGAACAGGTCACGCTGATGAAACCCAGTGCCGGGACGCTACGCGGTGTCAGTCTGGCTGCGGTT
GCA
P
AACTCCGAAGTCGATGCACTGATTAAGGTGCTGCCGCGCATGACGGCACCGATGCTGACCGAGCAGGAAGTCGCCGCGC
TGG

AACTGCCTGACCTTGTGGCGCTGGCCGGTAAGGTGGTCGGTTTTTTGTCGCCGAACTCGGTGCAGTGACGTTTCCGAAA
AATC
TCTCGGTCGATGACCTGATGGCGGATGTGGCAGTGATATTTCACTGGCCGCCATCAGAACTGTATCCCATGAGCCTGAC

o .3 CTCATCACATGGCGCGAAAAGGCGCTCCGGCGAAGCGGAAACACGAATGAGTAACAATGTAAAATTACAGGTATTGCTC

GCTGTTGACCAGGCATCCCGCCCGTTTAAATCCATCCGCACAGCGAGCAAGTCGCTGTCGGGGGATATCCGGGAAACAC
AAAA
.31 .3 ATCACTGCGCGAGCTGAACGGTCACGCATCCCGTATTGAGGGATTCCGCAAGACCAGTGCACAGCTCGCCGTGACTGGT
CATG
CACTTGAAAAGGCACGGCAGGAGGCCGAAGCCCTTGCCACACAGTTTAAAAACACCGAACGTCCGACCCGTGCTCAGGC
GAA
AGTCCTGGAATCCGCAAAGCGTGCGGCGGAGGACTTACAGGCGAAATATAACCGCCTGACAGATTCCGTTAAACGCCAG
CAG
CGGGAACTGGCCGCTGTGGGAATTAATACCCGCAATCTTGCACATGATGAGCAGGGACTGAAAAACCGTATCAGTGAAA
CCA
CCGCACAGCTTAACCGTCAGCGTGATGCGCTGGTGCGTGTCAGTGCGCAACAGGCAAAACTTAACGCAGTAAAACAGCG
TTAT
CAGGCCGGAAAGGAACTGGCCGGAAATATGGCCTCAGTGGGCGCTGCCGGTGTGGGGATTGCGGCGGCGGGAACGATGG
C
IV
CGGTGTTAAGCTACTGATGCCCGGTTATGAGTTTGCGCAGAAAAACTCAGAATTACAGGCTGTGATCGGAGTGGCAAAA
GACT n CCGCCGAAATGGCCGCACTCCGCAAGCAGGCGCGCCAGCTCGGCGACAATACCGCCGCCTCGGCAGATGATGCAGCCGG
TGC t=1 tl GCAGATTATTATTGCGAAAGCCGGTGGGGATGTTGATGCCATTCAGGCGGCAACGCCGGTCACGCTGAACATGGCGCTG

o AACCGTCGCACAATGGAAGAAAACGCCGCCCTGCTGATGGGGATGAAATCCGCCTTTCAGCTTTCAAACGATAAGGTCG
CTCA 'a o TATCGGGGATGTTCTCTCCATGACGATGAACAAAACCGCCGCCGATTTTGACGGCATGAGCGATGCGCTGACCTATGCC
GCAC tt n.) un CTGTGGCAAAAAATGCCGGTGTCAGCATTGAAGAAACCGCCGCAATGGTCGGGGCGCTGCATGATGCAAAAATCACAGG
CTC

AATGGCGGGGACGGGAAGCCGTGCCGTGTTAAGCCGCCTGCAGGCACCGACGGGAAAAGCATGGGATGCACTCAAAGAG
CT n.) o n.) TGGAGTGAAAACCTCAGACAGCAAAGGAAACACCCGGCCAATATTTACCATTCTGAAAGAAATGCAGGCCAGTTTTGAG
AAAA
i-J
ACCGGCTCGGTACTGCCCAGCAGGCTGAATACATGAAAACTATTTTCGGGGAGGAGGCCAGCTCAGCCGCTGCCGTGCT
GAT .6.
.6.
n.) GACTGCCGCCTCAACCGGAAAGCTGGACAAACTGACCGCTGCGTTTAAAGCCTCAGACGGGAAGACCGCCGAGCTGGTA
AAT oe ATCATGCAGGACAACCTAGGCGGTGACTTTAAAGCGTTTCAGTCCGCTTATGAGGCGGTGGGGACTGACCTGTTTGACC
AGCA
GGAAGGCGCGCTGCGTAAGCTCACGCAGACGGCCACAAAGTATGTGTTAAAACTCGACGGCTGGATACAGAAAAACAAA
TCA
CTGGCGTCAACCATCGGCATCATTGCCGGCGGTGCACTGGCGCTTACTGGCATCATCGGTGCCATTGGCCTCGTAGCCT
GGCC
GGTTATCACCGGCATCAATGCCATCATCGCGGCAGCAGGCGCAATGGGGGCAGTCTTCACGACGGTTGGCAGTGCTGTT
ATGA
CCGCCATCGGGGCTATTAGCTGGCCGGTTGTGGCCGTGGTGGCTGCCATTGTCGCCGGTGCGTTGCTTATCCGTAAATA
CTGG
GAGCCTGTCAGCGCATTCTTTGGTGGTGTGGTTGAAGGGCTGAAAGCGGCATTTGCGCCGGTGGGGGAACTGTTCACGC
CAC
P
TTAAACCGGTTTTTGACTGGCTGGGCGAAAAGTTACAGGCCGCGTGGCAGTGGTTTAAAAACCTGATTGCCCCGGTCAA
AGCC

ACCCAGGACACCCTGAACCGTTGCCGTGACACGGGCGTCATGTTCGGGCAGGCACTGGCTGACGCGTTGATGCTGCCGC
TTAA
.6.
TGCGTTCAACAAACTGCGCAGTGGTATTGACTGGGTACTGGAAAAACTCGGTGTTATCAACAAAGAGTCAGACACACTT

o .3 AGACCGCCGCCAGAACTCATACCGCCACGTATGGTACCGGTGACTATATTCCGGCGACCAGCTCTTATGCAGGCTATCA

ATCAGCCGGTCACGGCACCGGCTGGCCGCTCTTATGTAGACCAGAGTAAAAACGAATATCACATCAGCCTGACGGGGGG
GAC
.31 .3 TGCGCCGGGGACACAGCTTGACCGCCAGTTACAGGATGCGCTCGAAAAATACGAGCGGGATAAACGTGCGCGCGCCCGT
GCC
AGCATGATGCATGACGGTTAAGGAGGTGACGAAAAATGATGCTCGCGTTAGGTATGTTTGTTTTTATGCGCCAGACGCT
GCCA
CACCAGACCATGCAGCGTGAATCAGATTATCGCTGGCCGTCAAATTCCCGTATCGGTAAACGGGATGCCTTTCAGTTTC
TCGGT
GTGGGTGAGGAAAACATCACGCTGGCCGGTGTGCTTTATCCCGAACTGACCGGCGGCAAGCTGACGATGACCACGCTCA
GGC
TGATGGCAGAGGAGGGGCGGGCGTGGCCGTTGCTGGATGGCACCGGCATGATTTACGGCATGTATGTCATCAGCAGGGT
GA
GTGAAACAGGGAGTATTTTCTTTGCAGACGGCACACCCCGGAAAATTGATTTTACGCTGTCACTCACCCGCGTTGATGA
ATCAC
IV
TGGCCGCGCTTTATGGCGATATCGGTAAACAGGCGGAATCGCTCATCGGTAAGGCCGGCAGTATGGCGACCAGATTCAC
AGG n TATGACGGGGGCGGGATAATGCTGGATGCGCTGACATTTGATGCAGGCAGTACGCTGACGCCGGATTACATGCTGATGC
TCG t=1 tl ACAGCAGGGATATTACCGGCAATATCAGCGACCGTCTGATGAGCATGACCCTGACGGATAACCGGGGCTTTGAGGCTGA

o GCTTGATATTGAACTGAACGATGCCGACGGGCAGGTCGGGCTGCCGGTTCGTGGCGCTGTCCTGACGGTGTATATCGGC
TGG 'a o AAAGGTTTTGCCCTGGTATGCAAAGGGAAATTTACCGTTGATGAGGTTGAACACCGGGGCGCACCGGATGTAGTCACCA
TCCG tt n.) un CGCCCGGAGTGCAGATTTTCGCGGGACGCTCAATTCCCGCCGGGAAGGCTCCTGGCATGACACCACGCTCGGTGCGATT
GTTA

AGGCGATAGCCACCCGTAACAGGCTGGAAGCCAGTGTCGCTCCGTCACTGGCCGGAATAAAAATTCCACACATCGACCA
GTCG t,.) o CAGGAGTCTGATGCGAAATTCCTGACCCGTCTTGCAGAACGCAACGGCGGTGAGGTGTCGGTAAAAATGGGAAAACTGT
TGT
i-J
TTCTCAAAGCGGGGCAGGGAGTGACGGCCAGCGGTAAAAAAATCCCGCAGGTCACCATAACCCGCAGCGACGGCGACCG
CCA
4,.
TCATTTTGCGATTGCTGACCGTGGAGCCTACACCGGTGTAACGGCAAAATGGCTACACACTAAAGACCCGAAGCCGCAA
AAGC oe AGAAGGTAAAACTGAAACGCAAAAAGAAAGAGAAACACCTGCGCGCACTGGAGCACCCGAAAGCGAAACCGGTCAGGCA
GA
AGAAAGCGCCTAAAGTACCGGAAGCGCGTGAAGGTGAATACATGGCCGGTGAGGCTGACAACGTTTTTGCCCTGACCAC
GGT
ATATGCCACGAAAGCGCAGGCCATGCGCGCCGCTCAGGCGAAGTGGGATAAACTGCAACGGGGCGTTGCGGAGTTCTCT
ATC
AGCCTGGCTACCGGTCGGGCAGATATTTACACGGAAACACCGGTCAAAGTGTCTGGCTTTAAGCGCGTCATAGACGAGC
AGG
ACTGGACAATCACTAAGGTGACACATTTTCTGAATAATAGCGGCTTCACGACGTCCTTAGAGCTTGAGGTCAGGCTTTC
TGATG
TGGAGTACGAAACAGAAGATGATGAGTGATGTTTTTGTTTTATCTGTTTGTTTTGTAAGGATAAATTAACTAAAATGGC
ACCAT
P
CAACAAAACCGGAAGAGGTGCTCGCGATGTTTCATTGTCCTTTATGCCAGCATGCCGCACATGCGCGTACAAGTCGCTA
TATCA .
, CTGACACGACAAAAGAGCGTTATCATCAGTGCCAGAACGTGAATTGCAGCGCCACGTTCATCACTTATGAGTCGGTACA
GCGA
_.]
1¨
.3 4,.
TACATCGTGAAGCCGGGAGAAGTCCACGCCGTAAGGCCGCACCCGTTGCCATCAGGGCAGCAAATTATGTGGATGTAA
.
..
1¨
, , 11 S. aureus phi11 ANGATTTANTCC
, , packaging signal 12 cpmA M KTESYF KEYNQFVLDQH KAI QE LEQE R NALESKI
KLDKSTYKQLIM DGQDD KADN LYQATDAD E KKLKALN KR LETKKSVSKEVKY
QKTI E LLKH QS ELSSLYESE KQSAI EKLKKAVDAYN EIIDEIEDIND RYE D EHQQYASVYSQEQLYDD
KEARKALNG H FKE N I FTSFINGN
DLPYEHNNKLFLKC
13 cpmB M KTKYELN NTKKVANAFCLN EE DTN LLI NAVD LD I
KN N M QE ISS E LQQAEQSKQKQYGTTLQN LAKQN RIIK 1-d n 14 ptiA
MDKQQIKDFVCDYHERTRSDVLIDDDINTDEFFSIADENSNEWMADDNIDDHIVKNHLEMIVDRVANDKEFYIFDSLIQ
GRSYQDIS t=1 1-d GVLDCSEQSVRFWYETLLD KIVEVI E
t,.) o o 'a o 4,.
vi 15 ptiB
MESIAEKETYHLPTEHLQVFNVIKNTSNKYITKTKILNQLGYEYNSSNERWLRRVINSLVYDYGYPIGCSYKPSERGYV
IITTEQEKQQA

MRSIKKLADGSMKRYEALKRIEV
w o w o 16 ptiM
MIAYPIRVGSVYRGEQMKLLKTKNCLYYRNGDNKLSEYQLLTQFNPTFINKKIRMCEFQIESMYHMSASTTTCDEMMGV
VSVSYPIE

.6.
.6.
KLVIKIIETKARLQNYKNRSISNMVLLKTVLNHYTEKEQKKVVKYMRSNGRYKPYNVIERLQVDLYQASIKQRSERQKQ
RNIAIENSKIA w oe RVNAYHQSSYVKVV
17 gene #29 (terS) [00119]
through gene #53 atgaacgaaaaacaaaagagattcgcagatgaatatataatgaatggatgtaatggtaaaaaagcagcaattacagcag gttatagtaagaaaacagc (lysin) of phi11 agagtctttagcaagtcgattgttaagaaatgttaatgtttcggaatatattaaagaacgattagaacagatacaagaa gagcgtttaatgagtattacagaagcttta gcgttatctgcttctattgctagaggagaacctcaagaggcttacagtaagaaatatgaccatttaaacgatgaagtgg aaaaagaggttacttacacaatcacacca acttttgaagagcgtcagagatctattgaccacatactaaaagtacatggtgcgtatatcgataaaaaagaaattactc agaagaatattgagattaatattggtgagt P
acgatgacgaaagttaaattaaactttaacaaaccgtctaatgttttcaatagaaacatattcgaaatactaaccaatt acgataacttcactgaagtacattacggtg .
, 1¨
gaggttcgagcggtaagtctcacggcgttatacaaaaagttgtactcaaagcattgcaagattggaaatatcctaggcg tatactgtggcttagaaaagtacaatcaa , .3 .6.
.
w caattaaagatagtttgttcgaagatgttaaagattgtttgataaactttggtatttgggacatgtgcctttggaataa gactgataacaaagttgaattgccaaacggc r., gcagtttttttgtttaaaggattagataacccagagaaaataaagtcgataaaaggcatatcagacatagtcatggaag aagcgtctgaattcacactaaatgattac , , , , acgcaattaacgttgcgtttgagggagcgtaaacacgtgaataagcaaatatttttgatgtttaacccagtatctaaac tgaattgggtttataagtatttctttgaacat r., ggtgaaccaatggaaaatgtcatgattagacaatctagttatcgagataataagtttcttgatgaaatgacacgacaaa acttagagttgttagcaaatcgtaatcca gcatattacaaaatttatgcgttaggtgaatttgctacactagacaaattggttttccctaagtatgaaaaacgtttaa taaataaagatgagttaagacatttaccttct tattttggattggactttggctacgttaatgatcctagtgcttttatacattctaaaatagatgtaaagaaaaagaagt tatacatcattgaagagtatgttaaacaaggt atgctgaatgatgaaatagctaatgtcataaagcaacttggttatgctaaagaagaaattacagcagatagtgcagaac aaaaaagtatagctgaattaaggaatct agggcttaaaaggattttaccaaccaaaaaagggaagggctcggttgtacaagggttacaattcttaatgcaatttgaa atcattgttgatgaacgttgtttcaagact attgaagagtttgacaactacacatggcaaaaggacaaagatacaggtgaatataccaatgaaccagtagatacataca atcattgtatcgattcgttgcgttattca 1-d n gtggaacgattctacagaccggttagaaaacgcacaaatctcagttcgaaagttgacacaataaaatctctaggattat aggagggaacaaatgttaaaagtaaacg 1-3 t=1 aatttgaaacagatacagatctacggggaaacataaattacttatttaatgatgaagccaatgttgtttacacatatga cgggacggaatccgatttattacaaaacgt 1-d w o taatgaagtaagtaaatacattgaacatcacatggattaccaacgacctagattgaaagtgttaagtgattattacgaa ggtaaaactaagaacttagttgagttaac w o -a-, acgacgcaaagaagagtacatggcagataaccgtgtagcgcatgattacgcatcttatattagcgattttatcaacggc tatttcttgggtaatccaattcaatatcaa o .6.
w w vi gatgatgacaaagatgtattagaagttattgaggcgttcaatgatttaaatgatgttgagtcacacaatagatctttag gattagatttgtcaatttatggcaaagcttat gagttaatgattagaaaccaagatgatgaaacgcgtttatacaagagtgatgcaatgagtacttttgtcatatacgaca atacaattgaacgtaatagtatcgcaggc n.) o n.) gttagatatttaagaactaaaccaatagacaagactgacgaagatgaagtgtttacagttgatttattcacttcacacg gtgtttatagatatcttaccagtagaacaa o atggattgaagctcacaccacgtgaaaacggttttgaatcacactctttcgaacgtatgcctattacagaatttagcaa caacgaaagaagaaaaggggattatgag .6.
.6.
n.) aaagtaatcactttaattgatttgtatgataatgctgaatcagatactgctaactatatgagtgatttaaatgacgcta tgttacttattaaaggtaatttaaatttagatc oe ctgtagaagttagaaaacaaaaggaagctaacgtgttgtttttagaaccgactgtttatgctgatagcgaaggtagaga aacagaaggctctgttgatggtggttatat ttataagcaatacgatgtacaaggtaccgaagcttataaagaccgtttaaacagtgatatacacatgtttaccaacacg cctaacatgaaagatgataactttagcgg cactcaatcgggcgaggcaatgaaatacaaattatttggattggaacaacgtactaaaactaaagaaggattgtttact aaagggttaagacgtcgtgctaagttgtt agagacaatacttaaaaatacatggtcgattgacgctaacaaagatttcaatactgttagatacgtatacaacagaaac ttacctaaatcattgattgaagaattaaa agcttatattgattctggtgggaagattagccaaacaactttaatgtctctattctcgttcttccaagaccctgaatta gaagttaagaaaatcgaagaagatgagaaa gaatctattaaaaaagctcaaaaaggtatttataaagaccctagagacatcaatgatgacgaacaagatgatgatacaa aagatactgttgataaaaaggaatgatt P
gtaattgcctaacaaaaacactcaagaatattgggaagaacgcggacgcaaagcaatcgagaatgagttgaagcgtgat aaaactaaagctgaagaaatagaacg .
, tatattgaatatgatgattaagcgcattgaaaaagagatcaatgcgtttattgtcaagtacggagattttgcaggcgtt acattacaagaagcacaaaagattattgat ,, , 1¨
.3 .6.
.
c,.) gagttcgatgtaaaagcgtttcaagaagaagcaaaaagattggtcgaaaacaaggagtttagcgatagagcaaatgaag aattaaagaagtataacacgaaaatg .
r., tatgtatctagagaacagatgttaaagattcaaatagaattcttaattgcttatgcaacagctcaaacagaattatcga tgagggaatatttcgaatcaacagcttatc "
, , , gtgtgttcagtgatcaagcgggtattttaggtgaaggtgtacaagtagctaaagaagttatagatacaatcgttgatac acaatttcatggtgtcgtttggtcagagcga 0 , N, N, ttatggactaataccgaagcaatgaaacaagaagtagaagaaataattgctaatgtagttattagaggtcgacatccta atgaatatgttaaagatatgcgcaagca cttaaataaattcgaaggcacagcacgacaaaagaccgcagcaattaaatcattgctttatacggaatcggcacgtgtt cacgcacaatcaagcattgacagcatga aagaaatttcaccggaaggatattatatgtatattgcaaaaatcgataatagaacaactaaagtatgcaaagggcttaa tggagaaatattcaaagttaaagacgct aaaattggtgttaatttctatcctatgcatatcaattgtcgttcagattgcgctttactacctaaatctatgtggccga aaaaaccaagcaagaaacgaaaaacaaaat acttcggagggaaagtgaaaagcggtgattgatttaaaagtgaagttttttaaaggcaagttagttttgtatgacagta aattaaatgtttggaggatactaatatgag taatactgacaaataccttagagacatagcaagagaattaaaaggtatacgtaaagagttacaaaagcgaaacgaaaca gttattattgatgcaaacttagacagtt 1-0 n taaggtcggcagtattagccgataaagaaaaatcgaaatataatgaacctctcttttaatagctagcacttaattgtgt tggctattttttatgtccaaaacgtgctgatg 1-3 acataaaaagcacgcatggaaaaacagtcgacagactataaatggaggtatatctcatggaagaaaataaacttaagtt taatttgcaattttttgcagaccaatcag t=1 n.) atgatccggacgaaccaggcggagatggtaaaaaaggaaatcctgataagaaagaaaatgacgaaggtactgaaataac tttcacgccagagcaacaaaagaaa =
n.) o gttgatgaaatacttgaacgtcgtgtagcccacgaaaagaaaaaagctgatgagtatgcaaaagaaaaagcagcagaag ctgctaaagaagctgctaaattagcg -a-, c, .6.
aaaatgaacaaggatcaaaaagatgaatatgaacgcgaacaaatggaaaaagaactggaacaattacgttcagaaaaac aattaaacgaaatgcgttcagaagc n.) n.) vi OP OA , (0 OA r0 0 r0 0 OP OA
OD OD ,sr, ,(;)n 0 CLO (0 2 OA tj t,' tc; -u M MI
-uM-our04-,0,0(00(0c0cOutj-- 0 r0 03 (0 -I-' (0 (0 co 4-, (.0 " (13 OD U FO, .iL-' (0 (0 OA 03 co 4_, 4_, (i, 4_, rOtjr0r0rotj4-,(0cou,,?;oroOOrOrOrauSu3r0BOrOut,' -1,- (0 U (0 (13 4-, (0 (0 co 2 U a-, co (0 t; (0 co (0 , -1e,., (0 co op (10 4., (13 4 (0 (0 (0 (0 4-, r0 (0 03 0 0,0 4-, 4-, (0 (.0 (0 CO CO (0s4.4., 4-, r0 03 0 r0 0 (0 4- +., 4-' -' (0 (0 4-, co (0 ,(45, OP co 0 (13Z-r,E; 2 oo (-.
co 0 .(,-9, 2, r ( = 0 -' o(0- 4-,' 2 (.0 (0 4(2 0 4_, 0 3 n, 0, (-0 , 0 00 (.0 2 0,0^ tv ro +, ro 0 u ro r (0 OA 2 (ao (0 a ttotE; u (0 CO 0 U 4-' (0 r.: "M 4- 2 õro' tE; CO
ere CIO -'rir (0 CLO ti' 6) U
,.., .... (xi op 0.0 _ -_ .1-, 4_,-uu-o03c0 =L''(0 w:' 010 co CIO
-u -o (J4- co OP (0 co co -o M OLO M 0-0 co 0 t , ^ .1- ; 00 op 0 (0 ,_,,., .1-; 541 (0 4_, (13 ,(7.),., OP n3 0 0 (0 0 0 OP (.0 co 03 co r0 n3 4.-; OA -3 OD 4-, 4_, ro 0 OD '4. .1-, 4-1 CO 4-, 4-' U
(0 03 0 (0 -1-J .1-; OD 0 (13 hn c0 ,...,(13 U c0 4- M M, (0 ro 0 t,,o ro .0 rr, H r (0 b' OA OA c.71 (0 61 M OD 2 Vo (0 ST MI
0,0 cOr0(0(00.04-,c0-u- m 0,13-aro4-, Ms (0-u .E1 2 : I 2 t Lou .t , BO - u( . 0 c or 0 3 Lo a . 5 , ( . 0 .. , r 0 (. or 0 .. t : , , 4-J( . 0 mu 0 , 00 , 0 . 0( 0 (Z, 0 cjr BO t:), 0 r 0 t :,(13 (31 4-, 4-, 4-, U U CO _ 4_, 03 03 op (i3 u !,7, 4.., n3 CD U 4-1 a-, (0 0,0 0,13 CD 4-',..,0.0op op U 4-, (10 00 03 03 03 co 4_, 03 4_, 4_, _ n3 sam 00 .1-; 4-, OD (xi 4_, 4_, (0 ,,,, 4-, ttO .1_=
(0 4-'õ CO 0 4 (0 CO 013 CO 4-, 4-, U CO '' CO (0 (-0 (-0 OD `(.6" r0 03 03 0 , -..:, 03 .1-; 0 (13 U U 0 0 ,., U .1-; .1-; (0 ,r, U 4-' (0 0 t' `:,' co co co 0 iii "(13 ((3.30 ((.73) (% 03 (a, õ(13 BO
(0 _, (0 4-, (.0 ,,,:, 4_, l:113 c0 U 4-' 0 u CD CO +-, U OP
Ur0C0c0(0(13.t,' M r0+,(0r0a-'n32 2 t,' tE;
(00c0030.0(00(0+-,(04-,c0(0ZrOopur00-uut0'23,,r0(00 0 ---, co 03 03 (a0 4-, op -I-' 0 -u a, 4-, 0 -.It r0 --Ir-:: OW (13 a (0 (0 (13 -u (0 Ms 4-t 0 o mro Ms 0 0 mro 0 4- -, CD ro ...,- CD +., 0 -, ro 0.
CO CD

(13 (13 U ' ' (13 CO .1_= -I-, 0 U 0,0 4-, :i CO
.1-, 0 .1_= 0 0,0 0 j3 if U 4-' CO
0 CO CO 0 0 op op 0 j (0 0 CD (0 (13 4-, (13 (13 (0 0 03 0 Op õ,, 0 (0 co 03 co 4_, t5 03 ti r0 0 r0 r0 OA 0,0 tnn (:(0 OP OLO -o u-' (0 MI co -o co 0 h,-,(13 ,T, co OP 0 s's 4-(-0 ' (0 4--, Op 4-, 03 co 03 03 ..õ.,-, 0- OP 03 0 r0 Ms OA co 0,0 +., (0 MI r0 -u M M 0.0 4t1 0.0 OD ro 0-0 - r0 OP co OO
0 ^ -o co , ss-s" OP -o OP m co 03 0 0 op BO -,O,, co +., +_, (0 co 0.0 op T.0-' , 0 (0 ts, õ, (0 _ .... 4_, hn _ -' OP µ6. MI U (70- ((.,, SIP, (a) 2 0.0 2 "(-0 0,0 0 0.0 (,,, ro (0 4-, t.,, 0 -,t,' s,$).0 t,' co 03 r0 03 (0 -o tr, 4-, (0 (0 0 co (0 0 u .2 co (0 03 +., OP (0 op OP 0 co OP 0 , 0 4_, co , OP , r0 r0 0 op -.1,:,' OW 2 (.0 .1-, 1.T3' (0 (-0 447.,, (:113 CO 0,13 CO t: 0.0 CO (13 OD OD (0 U (a U ,(513 2 ro -(0, op a 0 OA to op OP
(0 0 M
0 -u Ms 0,0 -r; -_,t; co -o 0 0 co S U -^-' , c0 03 OP U op uUr0r0r0 00,0UCCI -(13(13(130,00,0 00(00,0U
(00,00a-,+,+.,+,(13c0UUWCOM cot ''U Ur0r00.0+' (0 U 0 j3 U 0 j3 (X1 (0 CO 03 (0 U .o .o .1.-= 4_, (13 (.0 0 4_, , . 2 0,0 0 4-, +., Ms MI (0 co (0 Ms r0 OA co 'um h n 4- ^ U -' -' OP hn Ms m u -o OP 0-0 0 -o OP (0 4-, }I
0 4- '' OP -o 03 - - r0 OP (0 r0OD 0 OP a-, OP op -Jif (0 (0 a-, 03 OP t.r3 2 ro , (0O 4-, 4-, ,., 0 0 4-, U
,., '' MI MI ' M M (a CD (0 (a0 ta' 2 oo on 0.0 0.0 0 (0 0 .E19 4t: (),., .t,' U U
.F., U (13 (.0 CO (a CO +-= 4=- "jõ.., CO .F. , CO 4- 0 tp r o 4 -J 0 co co co r 0 õ . ,,, s , t; - oõ.., Ms -o 0 03 - r0 co 0 4-, OP s sss 0 03 03 -1--, op ,T, 4-, CO (0 .1-, (0 (-0 CO " s . 4-, 0coo-uhnromo (0- Op4-, 4-, +.,opc0+,0c0 CD 0 4-, (0 co "u 0,0 Op co tf, hn- 03 .':, MI +., hnc 0 MI 2 (ao 2 OP co ro -u (0 0.0 U 4-, +.' (0 (0 2 (-0 (-0 417 +.1 op all u 4,4, 03 OP 4-, OP 4-, 03 a-, CD 8 oo -, 4-, 03 co 03 OP (0 Si? 29 Op 03 1_2 (AO (30 .1-, S19, S. 2 _it, 22:1, (0 t' -(1;r3 (If a t : U ( a 0 ( 0 6' rj (al -r. : µ( . ki 4 - ' B 2 0 (0 (0 2, t,'u 4u ro Lao 0 ,r,(ao -_,t ,,0 n3 eto (0 , ..".õ0 ro (.0 0 (.0 4_, 0 (.0 4-, (0 -, ro - 4-,, (.0 0 A OLO r 0 CD 1 0 O t 0 Z - ,u 0 i jp' r 0 op C ti;
%CI
(0 (0 OA (13 (0 U 4-, (13 0 (13 (13 ,., (13 (0 (13 (13 U OD
(13OP OP 0 op aT,"
4-',..,õ (.0 (.0 0 00 s sa -10, (.0 0 U 0 4-' 03 .1-; a-, (0 03 M tp CtO -u M 0 m OA CO 4-' -' (0 W3 (0 ^ M MI (0 W3 (a r (0 (a CO M
OD U MI (a M U OD (a CO MI r MI OD (0 OD U CO 0 CO rocOopMour0+,03-u-r%0t3u03(003-ur00-000 (0 r0 _,_,4-' OA co OA M r0 (a -u co (a MI co 2 (0 (ao 4-, r 4-, .-t--,' (0 r (ao r "ro (0 0 2 -a -0.13-rorotitirotto0+-,4-Jurou'ro+-' CO 4-, OP
00 4_, 4_, 03 -4-, OP (0 (0 0 0 03 op (0 01:1 4-' 01:1 4-, (0 OP 00 0,0 op .I.J U 4_, (0 r0 (0 r0 hi3 OP 4-, CO 4-j (13 4-, op 4_, 0 OP (0 OP co OP OP 0 , op -1--, OP op (0 4-, 0 0,..õ,(04-,courOuro,,+-,00,0c04-,4-,c00,0000,,c0c0+,00A-u CO CO ro (0 4--' (.0 op op -.1-,-;õ if a, co dA S U (0 4..; u a, all U -,-',.., (0 4.7,j,, 3 '(-0 rom 4-'r 4-'r .).) t --Fi I?, 2 B 4-' co mr(E', Wt.,' tE; (0 r (-0 co 2 (a13 b' 3 (,0-- ro Bo 1-. 4.7_, co (0 su; 4-, 4- 0.0 0 t; 3 03 op 4- op 0 U ST r-: 2 0 (70) OD OD
0.0 MI T^ 010 .1.'.1 ti; (0 õi, it?... t..2 4.5, (0 (0 õ .1-; OP (0 03 (.0 03 0 03 OP 0 r0 4-' OP t;
op op 4-, (.0 op µ(.6-' ',,-u 6uzi (0 .(i2, (0 -,-, (0 (0 ro 0 ttO (.0 4-, 4-' .1-, CO (:)J3 (0 (13 4-, CO U (13 0 U 4_, ,...," _,_, rr, 4.,, (0013 0 4-',,,, õ., t', (13 03 OP 0 co OP
03 OP (0 03 r0 4-, ,..,.," Tip iii ' . +-, OLO co (0 r0 Op _Ls.,, õ..,- õ..,- -F-0 co 0 4-, op r0 n3 -i.i3 OP r0 (10 op ,47', OP ,__,,,s, 0.0 (.0 2 233 3 li 17;j3 -ILI co op srifi O.
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MI (13 4-, OA (0 u CO +,, OD 0 co 0 U u 00 4-' t,' CO CO t,' 2 CD 0 r 0 r 0 4- , r o 0 r 0 r 0 o r 0 4 - (- 0 r o r 0 r 0 0.0-+t + - , r 0 .._,- (..) ,,9 0 ro ro 0 -1--, ti-'3 4-, ro ro U U ro (.0 3 (-0 u -,-, (-0 4-, ro -'-' U "rill OP n, 4-, U u OD 4-' .,,, co 4-; U 1.7',.. CO 0 U i. 03 _ 4_, 4_, CO 4-' 0 (13 4-;
ro OA co CO ¨
COCO0,04-,(0`"'OPCOctur0 -co`(.00.0t,'( 0,04-'CO(0-04-'('' ti' (0 U r r COMUMMUCOCOMRICOM
4_, ro , 0 " Op 0 j3 4-1 2 4,,, 4(3 U 4., (.0 ozi 0.0 rp 4-1 , , U ,., n'w CO 0 4-, OP (13 4-, ro 0.0 ro ro ro ro ro ro co 4-' .,, 4-, ro ,-; op ro 0,0 t a ro a, co (.0 u op -' co on +., 0 +, u t,' ro co u 4-, co ozi op , CO 4.5, 0D 4.5, H
0 ro OP 4-',õ 4-, 4-; op 0 CO 4-, .F., (.0 (:)J3 4-; 4-, tk., 4-, 4-; ,..¶., _ ro ro u 4_, ro ro4-' ,,, ro 4_, 4-, co.
4,7.: 2, r0 m 4-, c ro ro -' an on ro '4" ,U ro ro an (0 __co tto (0 Fa' 0.0 an ro 70' 61, 4u an ro L'D ro U 4-; (:113 U U 4-, 4-, 4-; CO OP 4--' op U -,:,-, CO t.-.,:, ,,3 0 4-; (13 4-' 4-' ro 4-, ro 4-, 4-' U U 013 tIZI U 4-, 4-, OD 4-' "" 4-' `-µ,, CO U 4-, U 0.04-, (.0 4-, 0,0 0,0 0,0 4-, 4_, OP 0 4-, CD U OP OP 03 ro 4-, ro 0 4-, ro ro ro ro ro ro 0 4-, OP 4-, a-, a-. CO a-, CO a-, CO CO tll3 CO CO a-, a-. CO tll3 U U
4-, CD CD 0 4-,a-,ouur000 (0 ro 4-, oo .1-; 4_, 03 (0 m r0 r0 oo u , u ro (0 CO 00 ro o co "" co -'' 00 ro co 0.0 ro 0,0 (''' i.3 co co ro ro an (49 3 (0 - t' 0 (0 tin (0 (0 U 4-, u ro 4-' ro a-, MI '' 0 ro -' on ro 2 ro 4-, r 0 u if r 0 .o U (0 4.1: on (0 4-, 4-, ( ) On 'I.- U -' '' (-0 0.0 (0 0.0 (10 (0 (0 CO (11:10.13(13 MI 0.0(0 -' CO 0 MI - (..õ., 4-, U on ro 4-, U On W3 C0 (0.2 te:PrO (0 (13"+-, mtl 76 0,0 r0 te ro 0 00 (0,,, õ.,0 ,õ,(0 r0 B ro (0 2 A On hcM". (0 r U5 4-' (0 U 4-= (0 CO r ". ". "' 4-' ro on (0 u (0 4-, (.0 (0 u u 0 i.i:, ,,.(- (.0 c,- (33 44-, 4_, 4-, (0 CD 03 (:(j3 (:(. On 1.T; U
CO OA 4-' (0 ro u ljz, (0 r0 4F0' oto Fo" ro on r0 ro ro ((.1', o 1.-0' 4-'õ., ro ,4:',.. 01:1 1F .1-; 2 OA ri3 4-' co 00 u on r0 ro ro +, -' on -u an r.,' ro 2 ro µr,Y., r, a t, 0 r 0 +-;
4_, 4_, .1-; (10 ere .1-, 4-, CO u OA m co oo u rou4-, (0 0,00,04-,,,-(0 ooro u (0 ro 4-' 4-' on o o ro - (0 (0 4-, õ., 4-, (0 0 U+' 4- W3r0 (0 U W3(0 0.1:1CW.,, 0M 0%0 p. 0 0 , 0U t : ( MI UM .. t-J+ ' 00.0 3 ro +.,-- -ro Bo (0 ,,,,,r0 ,.0 on (-,, tE; u U 4-, ro .1-; ro o 03 4_, ro u ro 44f, 03 ro a004-, ro monro co, r04-,ro (pro on.F,uro mroc,0-(3'.2("3-(%.t,'ro.F, r0 B 00 I'S u -c; (0 u an (0 0.0 (0 co u (0 4,74 (0 2, t-_,, en , (:to 2 +, 4-= .C,:i U
(0 (10 0.1:1 (0 U "MI -(0' t,en 3 ro on .s,. 2, ro +, 0 u 00 m CO ro m u 4-' r0 4-, .1-=
MI (13 0 on 3 õ.,(13 4-5' 2 (134--10 hrio,orl3U-4-,(00.0(00-13c0 co OA , U
ro op 4-1 ======= ro ro u r0 OA u 0 u 4-, u 4-' oo a, t 41(00.0u6',0 2 3 0 0 0 + , r 0 r 0 r o r o o n + , r o r o ,,,0 r o t: .t,' , . . , r 0 CD, on (0 u (0 on oo t-,, rot, u+,+, onro um .,, 0,13-42-0, 0 on FO. (0 (.0 0 (3,3 o (0 ro u 4-' OA (0 tX, ro 4-' 4-, 4-, (0 u rj 00 4-' +' 4-' MI ro 4_, oo OA ro 4-, hn 4-, U '' CD -,-, m ro (0 m .0 .0 03 (34 CO 03 on o on 4-, ro (0 (0 (I,- (0 , u u ro CO
I. 0 ' s' U U .1-; (0 (0 on (0 t, (13 (13 (0 OA (0 00 ro 4-; ,,., OA (0 :-., ro ro (0 u 4_, 4-, 4-, -u0.0rorouror0robow:,(0(.0ro00`(.'o'roro4Z+,rororo u op ro 4-, U (0 013 (-0 (0 4.5, u .,, 0 j0 (.0 .i., U (-0 t.: CO U On 4-, u ro -' 4-, , (0 2 on on on +-, u ro on ro OA m (0 U 2, ro µ(.6-= t, OA r0 44f, r0 (34 00 on 03 ri3 on -' 4-, OA on 4-, -(% +,4-= CO 4-, (0 on t, -(% ro Z ro a onro U ro 00 -u (0 on 4--, romBO,,u0Orom4-,0,0-ourorOo,ror0 rOro co oprou on u 4_, ro ro 0.0 ro -u -u r0 4-, 4-' (0 MI MI -' 0,0 an 4_, (.0 m op (:,,:, 4_, ,:,,3 õo 4_, ro oo (0 OA ro on 4., "ro 0 2 tr., (0 on o on 4_, u (.0 s_. on 4_, 4_, u u (.0 ro 4_, -+, u (0 on u 0.0 ( 0 co r 0 r o r 0 oo u Ms (0 4-1 on co m 4.5, u 03 -(13js -8J-3, 4-, 03 ro 03 4-, 4.,., ri3 ((.3.. 4-' ri3 ro (0 OA 4- (0 CO
OA
u MI -' ur 4- CO CIO on 00 U OA MU 0 t) CO (73) (0 On (0 a .t,' U CO
r CO CO 4-' CO (0 - CO .1-, .1-; - ro ro O ,õ 4-, 4.,O ro 03 r0 .1-; CO on 4- .F., ., CO ,z, (.0 .,., (0 +
+., ro ro u 0 .,A OA ro 81, CD+, ro 03 CD CD CD CDCD ro 4-, (0 (.0 u U -' (0 .1- = .1-; U
+., ro r0 +, on on co m (0 CDan (0 co (0 OA OA op u ro (0 4-, CDCD(0 (.0 4_, 4_, 4_, õ= 4-, CD(.0 (0 OA co 0 .1-; 4-; 01:1 CO U (0 .t: an -(0 }-!, (0 co 0 (10 00 e, r, 00 +., ro hn 4-, u (0 u " 2 co õr -"r0 BC) ro (0 ''' (1 t,' ' t.') (0 r r to Cr to 4-, OA (0 (4 (0 s ' ro OA m- OA 4-, 4-' .1-; CO co , 0 co on CO 0 4_, 0 on u on oo on 4-, on (0 4_, u , -,_ u ro 00 Ms r0 Ms romr 0.0 'o ,,,,(1 0 (I 0-0 0 2 Ms a, 4-, (0 0 (0 ro ro , .
rOrOror00.0r0-u --rororOuu 4-' on r0 CO 4-, ro an on ro u ro p.,r,r0 u ' 0 -' (0 - -u u u (3-13 u ro on ro u ro ro on OA (0 u u OA 0 on 0 ro 4_, 0 ro ro onro ononro -u hn _ 00 (13 On }I ,,., (13 .II .., 0 (13 0 0j0 On (0 4-' CO (0 On mr r ..6 mW (0 0 (0 tig3 s(1.; tj (0 (1 Z:54 (1301_30(.00 (0 co -I-, OA 4-, (13 (-0 ... ro 03 - 4_, ro 0 03 (34 on ro .1-; CO -1-' (0 ,,,(10 oo .1-; CO (-0 4-, (0 (0 U U 0 (0 u ro 4-, on 4-, -1-' _,_,4-' ' w 0,0 .1-; 0 -L) U On U On (0 on U CO on ',,,.. 4-' OA +., a o 4, :0- r 0 r 0 r o r 0 : -, :, 4- , (0 + , r 0 oo +a r, r, ro +, 4-' u on -' on 4-, +, +, (0 4-, , OS 4-, (-0 (.0 ,,, co ,:u3 (.0 ,z, s. .1-; U (0 on on n3 on 4-= 4-, ,z, r,,(:u3 4-, 0 4-, - -' on (..) 4-, U 4-, (13 4., a-, U co ro ro CO 4., 0 CO (0 4_, 0.0 0.0 ro .(i2 (.0 ro on on 4 d 2 , i i ,- op 0,0 on u ro (0 0 on OA OA 4-, -81 4d 2 233 .-t: ,, (0130 ( 0 .t, v 2 CO (0 (T, u u on ro -' -' ro .,, , ro 4- CO
4- U (0 (0 0 +., on U '' (13 W3 (11:1 U '' 0 0 0 4..-...' (.0 U t u 1-,, OA
,(7,, rill. (4 .t,' CO 00 00 oo ro (0 0.0 (0 4-, (0 Ms U (0 (0 4-' 2 - ro (0 r 0 tE; z (0 (.'; -..-5 z.v 4- ,,, ro ro 0 r 0 4- - t; -J, õ, y. ,, +, on +, -8;3 u , (0 E (0 OA r0 _ 4_, (0 oo 4-, 4-, - m 174' if on OA -653 op - ,,,-, 03 3 (0 on ro 4-, 4-, .1-, (.0 oo oto row mr0 mu 0.0 -' (0 (0 ro 4-, õ., 4-, (0 .1-, OA 4-, 4--' õro -ti ri3 OA m (0 00 (0 r0 4-, ro 1-.,E,' -6' r0 4-, CO 4-' 4-, ".. ".. ,,,u onn3 (0 u- on,rorou 4-, 4_, on ro to +.,ro wro ,(5.),0 -0,0 (29 uu 3 mu mro +.,m, (.0 uro (pro 1_3(0 4-(0 +.,m, mon mu .t' wro '3 .t.6, -ro --' t' 4,õ -(i.T,; 4(9 (0 .,_= (0 (0 u on 3 (0 .(,2 õc, on (.0 u (0 2 OA ro ,:,j3 -(1.T3' _ET (.0 4 = ' + , 70 t . i -I, ro r o On 2 r 0 t; tj -5' ro u 0 4_, on 4_, 4_, u (0 u , (34 ri3 (.0 4, .tian, a-, ro (.0 03 on CO OA on on u r0 0 co u OA m 4-, CO 4-' 'I.' On CO 4-, On 4., ro ro CO On 4., hn OA 44f hn U co (10 CO (0 (0 U co U 0.0 +-' op (0 00 ro :C.," Ms ro -' -+t,' ro ..04 Ms m 4,"' (0 ro +., 4-, ,,..,,r0 u +., on r0 4-, (0 co On õ OA 4-, (0 4-, on (0 03 r 0 u u (3,3 r 0 2, 4 - ' -4t f, 3 - -r% ti;
u (0 ano an r o r o 0,0 co -.,t,' r 0 0.0 4-, 00 mum 0 mumroro,r0(.0(.0u0.0 4-, ro .,., ro 4-' 00 ro an (1 co ("3 u ro 00 -u ro 00 ("3 on ro (1' +, 0.0 u on 4_, (.0 44 ro u (.0 OA (0 r0 m m 4-, (13 ,...., (0 4r2( On u 4_, U on OA
4_, on ro (0 on 44 u on OA ro BO co u ro .(,-9 'rt.,'t; OA r0 ro u Ms 0,0 4-, (0 CO on u ..,u on ro ro (.0 ao ro ro (0 ro .2 t,' 4-, on 00 B ro 4-, 1-21 (0 U ,,,, +.; on ro on +, +, 4-, (0 u an t,' 2 ..), a 2 -,,,' hnr0 a BO ko) r0 mr0 t:,' (0 BO t C to' r0 8 4-m Bo (0 .2 CO
(0 -' co (10 ili (al 2 ro t, ro u (0 +, Ec, ro (0 u an u -u 4- u u 00 4-, CO 4-, r0 u , ro 4-, ro OS ro r0 u 4-, u 4-, +.1 on On u ro ro ro (0 4-1 (0 (13 (0 0 4-, (0 (0 ro CO OS OA
OA ro on ro 0 OA (0 .(,2 ro 0 ,:,z, co oo (0 4-, .1-; 0,0 U 4-' OS (0 (0 OS
OA -I-' (0 (0 -' u ro U (0 223, 2 1::: 233 (0 r0 -6, co t4 2 ro ro if U u ro um ro ro ro ro 4_, 0.0w 4-, ro On 0 (13 (3 on 0 on 4-, ro 2 ro ro (0 OA u ro co on OA 4-, (0 u -' .,., +, ro r0 ro 03 , on 4-, gjo , +, U (0 (.0 4(.T:,' (0 .1-; a-, u On 4-1 4-' 4t1 4-, (13 (0 CO CO (.0 (0 (0 CO oo 4-, r0 On 00 u ro ri3 ro ro 0,0(0-u ro+, co u 0.0+-= onro o 0+,4-, u ro-u o t,'1-2, on r,(113 (13 U -' 4_, on n3 ro (.0 4-, u õ., n3 u ,,., 4_, on .... 4-, u OA . n (13 CO ,,. .1_= u .iL-' CO
co (13 on ro t,' (0 ro (0 (0 ro ro ' an, , r, .L'2, on ro ro '-'a õflon ro µc, (0 (.0 (.0 on on '10 ro ro on 2 00 BO E.0, 0 2 (ojo -E, 0 4., rO co u (0 6.. 44 (.0 ,z, , 3 oo (0 4-, ro ,õ0 2 a 3 1-3 ro (0 oo u 0-0 , u (0 on (,:, ro op +., oo 00 '-"' r0 ' w _ (.0 (.0 õ,., 4.5, (.0 U (-0 U
UtnUr0r0(000(1303(13MU4-'M CO 4- '4,' U CO MI -' (0 .i.7 (0 (0 (0 u ro 4-'_ 4_, u "(13 (0 o 4-, ro ro_ 4-, on u ro 4-, 4-',.r, õ(13 4-'õ., ro (.0 ,z, (0 (0 ,z, .,. 41.1 (0 On .iLl, .,. U
(13 (13 l 1.1 (13 ro on on (0 - - -.,-==, U u co u u 4-, (-0 .iti 4-, 4-, ; 4 .1--, ro ro CD 4-' 4' .-; u (0 4- , (0 ro ro ro CD.,., +, OA r0 r0 +., +., op u +, co op , on CD CD CD CDu 4- on (0 u i.0 4-CO OA rill OA OA OA rill U U rill rill U U U 4-, U U OA U 4-, OA rill 4-, CO 4-, U ro atatcaaaacgaaagaattgttatttaaacgtcgcatcgatataggcggtgtgaataacaactttaaaggagatttcca agaggctgagggtctagatatgtattacg atctagaaacaggacgtaaagcacttctaatcggggtaactattggacctggtaacaacagacatcattcaatttattc tatcggtcaaagaggtgtaaaccaattctt n.) o n.) gaaaaacatcgcacctcaagtatcaatgactgattcaggcggacgtgttaaaccgttaccaatacagaacccagcatat ctaagtgatattacggaagttggtcatta o ctatatctatacgcaagacacacaaaatgcattagatttcccgttaccgaaagcgtttagagatgcagggtggttcttg gatgtactgcctggacactataatggtgctc .6.
.6.
n.) taagacaagtacttaccagaaacagcacaggtagaaatatgcttaaattcgaacgtgtcattgacattttcaataagaa aaacaacggagcatggaatttctgcccgc oe aaaacgccggttattgggaacatatccctaagagtattacaaaattatcagatttaaaaatcgttggtttagatttcta tatcactactgaagaatcaaaccgatttact gattttcctaaagactttaaaggtattgcaggttggatattagaagtaaaatcgaatacaccaggtaatacaacacaag tattaagacgtaataacttcccgtctgcac atcaatttttagttagaaactttggtactggtggcgttggtaaatggagtttattcgaaggaaaggtggttgaataatg gtagtagataatttttcgaaagatgataactt aatcgagttacaaacaacatcacaatataatccggttattgacacaaacatcagtttctatgaatcagatagaggaact ggtgttttaaattttgcagtaactaagaat aacagacccttatctataagttctgaacatgttaaaacatctatcgtgttaaaaaccgatgattataacgtagatagag gcgcttatatttcagacgaattaacgatagt agacgcaattaatgggcgtttgcagtatgtgataccgaatgaatttttaaaacattcaggcaaggtgcatgctcaggca ttctttacacaaaacgggagtaataatgtt P
gttgttgaacgtcaatttagcttcaatattgaaaatgatttagttagtgggtttgatggtataacaaagcttgtttata tcaaatctattcaagatactatcgaagctgtcg .
, gtaaagattttaaccaattaaagcaaaatatggctgatacacaaacgttaatagcaaaagtgaatgatagtgcgacaaa aggcattcaacaaatcgaaatcaagca ,, , 1¨
.3 .6.
.
oe aaacgaagctatacaagctattactgcgacgcaaactagtgcaacacaagctgttacagctgaattcgataaaatagtt gaaaaagagcaagcgatttttgaacgtg .
r., ttaacgaagttgaacaacaaatcaatggcgctgaccttgttaaaggtaattcaacaacgaattggcaaaagtctaaact tacagatgattacggtaaagcaattgaat "
, , , cgtatgagcagtccatagatagcgttttaagcgcagttaacacatctaggattattcatattactaatgcaacagatgc gccagaaaagacggatataggcacgttag 0 , N, N, agaagcctggacaagatggtgttgatgacggttcttcgttcgatgaatcaacttatacatcaagcaaatctggtgtgtt agttgtttatgttgttgataataatactgctc gtgcaacatggtacccagacgattcaaacgatgagtacacaaaatacaaaatctacggcacatggtacccgttttataa aaagaatgatggaaacttaactaagcaa tttgttgaagaaacgtctaacaacgctttaaatcaagctaagcagtatgtagatgataaattcggaacaacgagctggc aacaacataagatgacagaggcgaatgg tcaatcaattcaagttaacttaaataatgcgcaaggcgatttgggatatttaactgctggtaattactatgcaacaaga gtgccggatttaccaggtagcgttgaaagtt atgagggttatttatcggtattcgttaaagatgatacaaacaagctatttaacttcacaccttataactctaaaaagat ttacacacgatcaatcacaaacggcagactt gagcaacagtggacagttcctaatgaacataaatcaacggtattgttcgacggtggcgcaaatggtgtaggtacaacaa tcaatctaactgaaccgtacacaaactat 1-0 n tctattttgttggtaagtggaacttatccaggtggcgttattgagggattcggactaaccgcattacctaacgcgattc aattgagtaaagcgaatgtagttgactcaga 1-3 cggcaacggtggcggtatttatgagtgcttactatccaaaacaagtagcactactttaagaatagataacgatgtgtac tttgatttaggtaaaacatcaggttctgga t=1 n.) gcgaatgccaacaaagttactataactaaaattatggggtggaaataatgaaaatcacagtaaacgataaaaacgaagt tatcggattcgttaatactggcggtttac =
n.) o gcaatagtttagatgtagatgataacaatgtgcctattaaatttaaagaagagttcgaacctagaaagtttgttttcac taacggcgaaattaaatacaatagcaatttc -a-, c, .6.
gaaaaagaagacgtaccgaatgcatcaaaccaacaaagtgcgtcagatttaagtgatgaggaacttcgcggaatggttg cgagtatgcaaatgcaggtggcacaag n.) n.) vi -' u ro 4-, u tw_mtwro (0, m 2 n , n (.0 4-, 0 u m m 4-, 2 op oz, 0 ^ `-µ,' op 03 ro 4-, (0 OP 0 ,,-, au u (II
(0 ' ro o ,õ u r or o 2 . o4 - , or 0 + -=,r 0 t :Thu mop wrill t: 2 .,(0 60 (00A .,co wro -r.103 0D ad + . ,r 0 roro coop row roro co- .,03 0-13 to op U 03 ozi op 03 co -1-' OA oo OA 4-, -' OP OP
r0 co co 4-, OP 03 03 4_, ro CO U CO (-0 CO oo oz, ro ro 4-, U co OP 4-, OP OP 0 OP -' 4-, -' -I-' 4., (-0 M CO CO (-0 4-, CO
Mr C00.00.00.000CCIM'w4-'(-0--.F., ""r 4-' 0,13+,0(13- (0 CO ro ro ro ro ao u co on ,snu r u tto ,..,(1' BO ro ((r o) ,_,(1' õnu -u u ro ao ro 1-3 ro , oz, op ro 03 co ro 4-_,w (1-3.- 3 ( (,0, 4-; 4-, co 44 co u a 3 (0 (0 0 4_, co U
no M co 4-, OD 4-; r0 OP co u .2 :0 m ro tto 0,0 3 oo o ro to V.1 ro ro 0 tE; ro ro t'-', +-, ro ro u -u u (0 u +.,- 0 t OA u Bo t,' co -(0' ro -0 õnnõ4.-iirourorow,u 4-, 233 ',nal, op,c0'-wro,uttOuurr,030 ^ CO co op - õ, 4_, 0D ro co ro u ro 03 (1 r0 tp OP ro ez, op -I-' OP OP op ro '', 4-, u ro (13 OP ro op ro ro OP 4-, ro u ro a, m, (0 u ao 4- on 0,0 +, (.0 ro ao ao õ, (0 +-, OA otO -um oto m 0 u co co 4_, ro on u 4_,urowro- 4_, (0 4-, - ro - 4-, 0 OP o^ p 4_, CO ro u ro 4_, oz, U OA U ro 4-' OA ro ro OA OD OD CIA all U a-, 0 a-, '4.. ro ro co U ro ,.., U U
op ,õ CO op 0 ( 0 op 4., (00,1:1(0rOUCOCCICOCCI.2(10(00.00+.1 -(3"drob'-'sr6uroa.o0.0(0-6' OP ro 03 co U _it, CO a-a (13 tx, t:ll3 4-' u U ty0 0 ro .(i2, ro (0 ro ta ro ro 4.5, co tw 4-, U (0 MI (1 (1 CO MI (113 +' -' MI MI 1-.,) r l:IA CO d.13 CO MI CO
co U 4-' (13 ro ,,c, . 4,74 4(2, 4-' ,,,.,(13 2 (ao n, on n, n, 0 , ro tax) 4., on u 4-, ro m , ro (0 +' 44=,' s . no 111 4`-_,'.. co .,(2, 4_, U u co 4-, OJO b) -1-' 4-, CO 4-' CO CD s ' 4-' U (0 CO nn ro ro 0.0 otO (II (II r 0-0 ro L,:v3 ro (%ro' ro ro ro (0 Lto ro ro ((i,' Oo ro u (0 , u rr,- u ro ror000mmooBorprorouromromm+-,coro+-,0.0,corom-ro, (0 u co 0 u co - õõ Op ro ro on 4-, 4- ro ro ro ro ra m - , a, 0 m u ro ,,,, 4-, 0 CO 4-, 4_, CO OA P. 4_, r CO hnr 'WM 03 4-' U
(0 ro co ro ro (0 4 ao u oo +-, ro 0.13-a CO 0.04-' ..."' ro ''' ao ro -e, u ,r +'ro +-'op ra 0 ra ,õ +., OP OP co co t 4-, U (-0 CO 4_, 4-, CO CO
co ro (13 u oz, 223, 03 co a-, ,,-, OP 4-, u ro ro 4-, co '' +., (113 u U (.
r, U CO
CD on 4., 4-1 co CO 1::: (0 ^ Op 0 0 0 op 0D r0 OP r0 ozi op op 4-, ro OP - +., op U 4., 4--' -I-, 4-, 4_, co OP co (13 ro ro opOPrOc0 4-'rOur0O.PrOcOu Ms op t r 0 2 r, 4-, co+-' MU co+, co CO ClIlt,' TIE; all U+,4-' 2 , + - , r 0 + -, r 0 CO all 0,0 ro rõ, co 0 ri, 2 on CO 4_, on 0 ro ro 0 ro ro CO 4-, OD 4. op 0 op -'(00.0Mco4-'CLCiourOMIcOc0(00-0r0(0+-,(0 0 +., ro OP OP 0 CO op -1-' -' +-' +, U 0 r CD-CD
co ro .i.-; ozi all 4_,z, U CO 1T3' tu3 (.0 4_, no ,.., co 4-, co 4_, U 4_, no 4_, MI 03 4- +, (13 4-' (0 (0 0,0 ca 0 ro ro 0 .,,,,, (.0 ao ro ro OP ro co (.0 , 03 03 u a-a (13 4t: U CO BO 4-' ro 0,0 a, , all ro op OP 4-1 CO ojo (-0 MU (11:1-j CO+, Mr 0,13(0+' (113(0 OD+, CO CO+, COBOU all (044 , op 4-, OP op op OP 4-' op OP ro ro 0 co mu ro (0 co op co OP
" ro ro IT tp co U
U ro ro t 4-, 4-, U U +., OD 4-' +./ MI ' . OD co OP OP t:'- - ro u u -I-, 4--' U
CO 0,0 CO , CO U .t, (10 ro +., CO (-0 co (-0 01:1 44 ,,,, 4_, ojo co co 4_, ,,, oo u CO 0 tn U OD r 0 1-3 CO CA t: (10 (0 co 'cLY., U U OD CO OD (0 2 '44 4-,õ u ao ro =z.,.- ro ro (0 tto ro ro ,-.4, u -' -r; ro t', , õ, OP co +., u op co u OP _,_,=-= OP
-' Ms 0,0 OP ca OD 4-, +., 0,0 tj 0 MI (0 r0 ' OP 'v7 u u ra -' u u OP OP 'El Ms 0 (0 (10 co OA MI OP Ms OA ro ro '10 -0 oo 4-' CO +./ MI (1 -(Lo' MI CO OD U
CO 0 (10 CO ro (0 ro ro ro ro 4-, oz, tx, u ro ro op OP 03 4_, ro ro 2 rõ, (-õ, 03 4_, a-a -!--; U CO ozi op u all a-a (0 CO 4-, ro ro ro 0,0 (0 r, C00.0,...,''4.7,',tf,+4-'01:1C111+,4-JUUCOU0 0c000(0000 0,0 Ms 0 OP ttO .L'f, '-'-'4 op Ms +., (10 r0 MS OP ca -' 0,0 Ms Ms MI OA
OP co (13 CO
ro co ro+,+,4-,ro.,,r0 0 u4-,r0 uttoro-u8r04-,ro , ro u u 0,0 co co 4-, OP oz, co OP co U ro U 2 ,õ ro ro õ OP WI
roc003cOuur00,003-60,0cOur0+,:,;0,0rOurocourorProrou (0 4t, (-0 CO CO OA CO ,..,., OD 0 DOACOUCCI(13(00.t,'+,U(OUUCCIrat' U OD CO MI (10 (0 -,t: 4j CO 0D (0 (13 -(0' -(0j OP 0,0 r0 op (0 OA (o co u U -' co fo 03 oz, 03 0,0 U -I-' oz, (0 4-, _ u 1--,, 4_, 4_, .(1.3, oo 4.2 co u 4_, co 4_, 4_, u co tw I.
U op 0,0 (0 K1 3 , 0 (- 0 t: : 4 i - = , 0 z ; + -=, , 0 0 OP w 4-, Ms ca O
00U 2 -' ,T, 4-, ro ro OP 4-' ro - +., u OP OP 0 - ro ro 4-, 4-, ro OP rill -r, u õ..,"' co a-, (13 4-; CO Cll:1 OD OD (13 4-, OJO co U µ s (13 co ro ro t,o ro CIA (:113 CO /1 a-, 4-' OA ro co 4-, ro a-, op - ^ 4_, 03 4_, 4_, 4_, 4_, (.0 u - õõ co co 4., 4-, co 4_, (-0 4., u OP 4-, OA OD (-0 ro (0 4-, ro m (0 co ''' 4-' u OP OP 03 4-, ro ro 03 OP -4 r0 0,00,0 co 4-, (0 OA, u co - OA U r0 MI OA
on +.1 ClAttOroUoproCCIroro+.1 Uotnr0 op 1-3 U3 OA (10 +.,(0 .000 ror0 (6,0 +-,(0 rou ror %.1:1 Zm ro(10 +-,(0 co 0DM 0,000 _our uU cora (00-0 tn,..,, (00.0 al hj, .6,' ro õ9 ti u44 u 0,0(0,4-,õ,(04:5 um u n, ro ro 4-, OP CO
co tx, (13 u OP -5' ,õ(0 op 1-2, co co 0,0 0,0 mr0 ,T,U ro LI 4t r or 13 r ou 0 3( 1 0 3( 1 4j LW mall (al,(13 ro to .t, Lto u 45.
u , ro -u ro ro - -roun,rorouro 03 OP 03 co ,,,, 0 0 OP co -' +., co OP ,44-' rõR co CO u 4-, ro ro (13 on , 0j3 4-, ro u - ro en (61 (.0 u (.0 ro (0 -0 co 4., ,_,U (0 ro OP 03 ll, -1-',_ 8 4-, 4-',,, 4-, CIIIMU-+.,UU 0,0 0j3 co .1=, tto op u 4_, oz, , . 4_, s . ro U r0 u (10 4-' -' (õ,-,1 op u 0 op co 0 ^ op , (.0 r0 co ro (.0 co (.0 u CD 4_, 4_, U 4-, CO µ...4 co 4_, co ojo u 4_, co 4_, co 4_, 4_, u no hn on õ, co U a-, ro 4-'ro 2 6a0 rpm 4-'u .I.-,t' U U 4-',., 4-'µ , CO 4_,4-' OD 4õ7,' OD OA U
CO CO C13--. CD 0 -0 6' t S 4 - 'a 13 0 u no 4_, co (a rub 3 4C2, 4t,. õ.,`-' tiO co 4-' ,_,"-' CO (:113 -I-' ro U
ro ,..õ r0 0.0 4_, 4_, 4-, 4-, ".. ro ro - 4-' u ro a-, co a-, 'w CO 4., (-0 U 4-' U (0 CO '' OD (1 OD U 4-' CO -' r U
OD oto ro m ro c'D t:,' ro 8 ( , :, ,, um - 6 ,:,õ` 1 2 2 3 s r orc' ,4 - , 3( = 0 s g2 - , .0', , ( . 0 n , n , 0 . 0 4-; OP U ...,, (13 ,,,, , (-0 ro 4-, , , r0 tto µ..,_ u ro 4_, (.0 4_, 0 4_, _ 4-,r0rOWZro+attOurO`' 4-, ,,-, -' MI op 3 0 . 0 t E ; u r 0 , 0 , 0 4 -,c) .2 2 "( = 0 , ow or 0 mu 0 .
om ot, 0 -t:', z s . õ- ' r or 0 + - ',. 0 ,` ' CO a-, CO a-, (0 co .2 0.0 W:Th r ti'o OD U Fcr to 4-' fa (0 CO r ti U (0 CO CO
-6' ro V , ro ro (.0 , TIE; 4., U u (53' ojo ro (-0 oo Op 0 L,^ u OP (13 4-, U 2 U 3 4-, r (13 U OP to op R? F33 2 U - (1 T1; BO 83 If a-, 2 2 0.0 -, u u u oto 0 ro 0 0 on u ro ro ro CO CO (:113 (10 -1--' U
CO U U U U ro oz, CO 4., 4-' m CO (13 u 4-, r CO CO op ro 0j3 (0 CO OP OP u CD 0 c0-(0'corOc uu 4-'CLOMIro U u 4-, CD CD4-, 4-; ...,--(-0 4., (0 l:ll3 Cll:1 U U w CO CO BO u 0 CD CD co co u u 03 0,0 op 0,0 OA u -+t,' u cor0c0u4-,4-,+-,c0OPc0(0c0(00,0000,0urPrOcouut,'-ur0 .,., +., , co r0 -' 4-' r CO OA
(1 on On CO ''" U '' CO r 0 m CO MI
4_, ro 4.5, ,S, ,y 0 OD CO ()J3 (0 ((ro) 4-, op (cil , ro ro z., 4-, 2 - i -=, u .,, ro u r 4-' a (You .,,:i t, -+t: (:Lo -r.: (0 ro co u -m (.2 t 5 a t2, +
- , ( .' 73' "r 0 u r, u +., ca 0.0 ro -.t:,' on 4-, on ti 4- m- u -',õ on Lao t ro - ro (ao (ao oz, co (0 4-' CO (-0 4-' dA 4-, 4-, 4-' 4.D
(-0 , . 4-, - 4-; r (-0 4-, OA
co CO co ro ro 4-, a-, 4., CO -1-' 4-, 4-' -1-' (-0 (-0 ro , a, u 4-, OA 2 0 ro rõ, co OD +./ t,' CO CO CO U CO CO U t: CO (0 +, OP OP .,., OP OP OP .,., u ca +., OP CO
03 rill rill a-, CO CO toiO U U OA CO 03 OP a-, CO CO 4-, (0 t1J3 OA CO a-, a-, co ro a-, U
a-, CO CO CO CO CO OA CO CO CO OA a-, CO CO CO OA CO a-, U OA CO CO CO U 4-, CO CO

0 rn ro (.0 , ro ro u oo oo oo ho ro u 0.0 +, oto B ((:, )3 tj U CO r U CO
2 (II (a ro ro ro OA -' '' OLO -' U CO u (0 r MI r +., U 0 0 I.
0.0 on (0 (0 (.0 oz, moo 2 (0 on u (0 0,3 (0 (ao (ao (ao 0 l5Aro 0+, Fo-ar0 44 0.0(0(000 inn U ¨ ¨ 0 ro bn CO MI ''( ) (0 OA OA +-, CO (0 rn m cO
4-, ty3 oo 4-, -' ¨ hn tto OD a, ro 0 u ,,,¨ m 0 .4¨ ro 4-, u u ro oo ro +, ro u-' - (13 +, CO OA U U it., 0.13 Z.:w (73 (13 Zj co ro Op .1-, ro 0 (0 CO (0 _ 4_, U u , , OA
+-,(00.0(0 -Ot: 1 ( O.O.O.00.0Mr w3 4-'c'0' 3 ?.., (-L0) ti (.0 , (.0 (4 u , 0 (.0 4., 03 0,0 0,0 CO .it', CO 4-, (.0 ,,,, (.0 co (0 (0 ro (0 (0 (0 0.0 (-0 a. 0 (0 (0_ r 0 tE; ti ro 'µ,' 4-, 0 ro oo oo oo u ro co co U MI - La0 OD OD (5.0 (0 U " (0 (5,0 U (0 (5.0 U (0 (0 13;3 ((.11.), 03 co 4(:), 4(:), T.0 ro ro cc, 44 4(2( 0 (.0 (.0 (00 0.0 ro (.0 (.0 0 ro 0 OP
U OP 0 tE; U U U U CO CO t-.,' to +-, (a op (0 4-, (0 CO OP 4t,' 83 OP (0 U 0 CO
.1-; 03 CID oz, 4-, (13 4-, r0 P OA O CO
4-, CO U 03 u 4-, if U u oyo ozi 03 .!..! U 0D co CD m to U co CO
CO U co co 4-, tto ttO , (0 OA 4_, OA .1-; rn _ 4_, -r,r, co .1-; 0 4-, = "' 0,13r CO 0 CO (10(0 +-,.F., tnr0 (0 U U 0.00,13(0 CO U '',....4-' f, -r, +-, , ro sc, op ro ro (-0 4-, oo -u ro u 4, op 1-', U 4-' -' oyo +-' co +-, " CO
CO " 4-, ro u m ro ro .--,:, u ro (13 4-, `-' Op CO OA 03 OA op CO tj OP co oz, 4-' U
U%-",U-to-0003opc0 +.,Opouto ODopOP CO4-, OA
4-,(000.0+.,( C0+.,C0t0LWrOrOopCOOPrOUouUroto(a U
CO . CO m r OD 4-' 4-' U OD (0 r CO 4=- (0 r "..: (-13 CO .2 U CO p.09 hriCd3 OD
t) t.,' (0 '' OD U MI OP -' MI OA CO (0 U CO (13 '' r CO OP 0 U '' (0 L. co OA -r.r, U .,(a 4_,(13 tj 4-= 4-' 2., U 2 - ro t,,o ro 4_, , ro 0.0 ro ¨ ro 4-, = ,-, 0.0 4_, , 0,3 ¨ 4-, õ., .1_=
.1-; l:113 U ,W ro 4t, 1-,. op 4t1 ri3 2 ro tri (13 0 1-', ri3 4-, (13 C1:1 +11 .1., 4-, OD CO U
U 76 di3 CO U(F0) 2 -(,-,-3, co OP (F0) 4-, op OP 4-, 0 -6' 03 CD
2 ao c'ci ro -'((:0' (.0 ti, 0 ro a , as Bo 3 2 (y, w3 (.0 2 ;0.0 murorOm ro so U u C1:1 (.5 t' 4(3 C1:1 T.E; (V .(i2, 44:: 03 (13 (%/3 4-, TIE; 4-' U
C1:1 1553' u ro a 4-, 1.T3' U CO MI (a 2 (:(.0 (:(.0 -' -813 CO OA OP (0 U (V co 2 ao 3 g2 cd:' (:(.0 Bo -(;E; .0 (V (.0 (o:d3 0.0 mr Bo (.0 co -(0' ac9 4-' co -(t:; u -(0' t' u (0 (.0 s.,,o ro .0 (To (0 co (0 u 4-, 0 2 -(.6' (.0 .2 (4 (0 "(0 3 ro+,N32.2+,,,,r0 ro (0, u, 0.0^ 0.0 0.0 4-, ,,,, 0.0 (0 (0 (0 (0 -,,õ _51,0 .L,OO tj 8.0" 0 2 SG (-V
L'; 4r2' 4(2 (0 C(E)1 1-.,) .1-, U OD (13 , C1:1 4_, (0j0 ro r0 rn ro +, -r, 4-' 0 (0 (0 .L'_I -63 '' CO
ro 0 .-t-', OP OA co to OA CO OA OP OA
U CO 03 ro OP (.0 1.T3' 0 CO CO oyo op (.0 2 ,(:),., (0 , -!-,, oz, (.0 õ,:, (0 2 (0 .i.,:,, ti 2 -,,,, (.0 (0 (0 (.0 ,,,, (.0 t 2 (u40.2sz-i2(04-,-.t:: oti_4-,(0 CO OP r0 t, 4_, (-0 -r.,, 0 (6,3, ,,, 0 (.0 0 (.0 .0 (.0 CO 0 ro ro 4(2, co u 2 (.0 ro (.0 (.0 (4 4_, (.0 (13 CO
rOCOOLOCOUM.2 4-'-'+.,d.00ON4-'+-'(-"130.0,,MIZ (-000mU.F.,-rnUt,' CO CO OPOPoprOor00.0000h¨n4-'0OMO.0`"COOprOiirOii (.0 4-, ro 4-, , 4-, ¨ co ro 0 0 .0 OA _ 4-, u +-,OD
CO U +-, 4-' r CO OD OD OD 4-' (0 ". u 3 tn iii3) I CO ti (.0(aci +.,4-' (.0(. .(,..i ro 3 rou u ro ro oo e., õas:, , ,O.0 ro rn to ,,,,O.0 t:,' 1-2, +., cr 0D mCO õ.0 0 (0 (.0 (ao co (0 co co tto 013 (0 .1-; ...A., -.. U co ro -..., 0 3 (4 (.0 4_, a. (0 u (.0 (0 u ri3 (0 CIA CO MI co (0 ..r.: -' 4-, U-',+./.,,..0 (0 W3 OD 0 -' CO U r '' (0 MI ro MI CO
.:,) '' OD `5 '= 0 I. 'thj.' 0 4-' .F., O .0 . = 3 03(-0,,,,r003(.0roro+,ro.0ro.,,(00(04-,,u 4-,ror000,+, = co OD (5.1:1 (11:1 COM 0,0 (0 CO t,'COCCI(5,000tt or 03 (0 03 !TRIMS U fl, 4-, OA co (0 U OA co ro .1-; ,T, n5 .1-; CO OP to ro OP 4-'._ ,., U OA ro co (0 CO U r CO sCj '' " +., CO CO .A.' (0 U
(13 .1-; OP 4-, ¨4-, CO OP -' CO CO
CO
MI 4-' ro CO r0 U Z.,. r0 (0 4-' r0 -u OD OD MI u OD ,(.7?r, u '' op MI u 4-1 +, a-, ro ro op 1:,, 03 U 4-1 U CO Op u ,õ--4 ro op , 0 .0 u oz, OA
CD
CD +., +., op +-, ro _ 0 .,, 0 (13 .t, ro ro u op -F0 U +, OD (13 U C1:1 op ' . CO
op U .1-, co OD (13 "-'.,., 4-, OA 0 co (0 4., 0 ro ro OP .1.., ro 4-' 4-, ro oo 0 ro (13 OA
03 Op to , op 0,0 um co Op +., 0j0 u co +, u rn 4_, .,., 4-, ro ro ao +, 4-, 0 OA op op op oyo 4., OP 4..1 OD (-0 4_, 4-' 4., OD .1-; OD (.0 CO u a-a (1:1 4-' ^ ro CO 4-, (-0 u ro ro ro u 0j3 (13 a-, CO OP 4-, op op co 4-' U 4-, (-0 CO (,.,-. 4-, (13 , OA 4-, U co u OA -I-' co .it', op TIE; .it', CO 03 4., (13 ro u 0.0 OA OA s w ro u (:113 CO oyo OP 44f 44 co u 03 _ 4_, u CO `,.. 0j0 4-, 00 U U (15 0.0 MI CO 4-' +., U +.; CO +., U 0 "m 4-2, C 0 MI, , MI q p t U .,(11 Z 2., , Op ro OD .1-; OD 4-, ,T, 4-, (.., .1_= ¨ ¨ ro =-= to' 4_, 0,0 u 4_, 4-, U C1:1 ri3 CIA (513 U C1:1 +11 C1:1 .;:r (0 (0 OD ro U õ., (13 4., (13 co CO
CO op to 03 0 = 0 OA 4-' op U 4-' OP OP ro ro s- 4_, ro 0 u u "¨ u (.0 4_, 0j0 (:(o ro CO (a .1-; ro ro ro ro 0D 4-,, ro ¨ 4_, (.0 ro õ., 4_, co (.0 co OP co co CO44=,' ro ttO 0.0 ¨, ro OP (13 ro u 4-, .iL' ro -,-, u (13 (13 CO (0 r r CO Z:r ro r rt:' r 2 -' 4-= Cd3 U +-' (a 4-' 4- U rnM 11(7)n r (.7 CO
OP 03 op ro U op (o 0,0 +., CO CO CO +., .t, U CO +., +.1 4-, +., t' U ¨ ¨ -' a..! ,.,., 03 03 op .1-; u ,.õ( 03 OD (0 U .1-; 03 C1:1 C1:1 2 a-, (a) U
ro U CO -1--, U
(µ5'i3 '0'5 03 .1- i B) 0 j3 ( . 0 '0'5 1 f BO (13 BO 4t.1 - = 4- (%) 0 j3 a 4 _, C1:1 4, co (i13,.0 3 ro ti-3' (6,13) (13 0 a, op 0 3 (13 (r-)0 OP OA "t1-8 r (V
w3r0+,t,'4-'0.04-,w3tiouu000.oumu .2 co co 010 u r t co -' co r ro r u OA 4-' ro u ro ro .0 0 +, ro u tto OP o U CO +-, tt, (.0 (0 0,0 (.0 +-, 0,0 t,' (%.0 00 ^ 0,0 t 03 U (13 (5j3 CO (13 C1:1 U 0.0 'a t:: OP -(. a a CO OP (01 CO U
co to CO u ro U 0 03 op (0 ro Li (017'0 gi2 44:: (a 2 29, õc= o 2 0 (.0 0,0 r 0 ti õ0 -(0' w:, (0 (-0 a -(.0' ("3 c'D .0 u r u u co ro ro 4t,' (0 ¨ ,,, 4-, tIA .1-, C1:1 ¨ .1-; 4-, 0.0-rr,+.,(1:1(0MCLOCLOUCOCO
t.1 r U OLO r 4- 0 2 U (0 -' MI -2 a to w3 613 U CO -,..,r, (a0 r0 u ro m (loopt::(.0Urp.00(.00.0(0,0,00,0S,COok7,COUCOroC06-pt) COmr0U-mtor131.2,4-'rou'C'04-'`wr rOrOUO.04-'CO
CD co 03 u OP co op 4-, CO ._,., OP oi3 1T3' 4-, .1-; 0,0 4-, OD U ro u op CO ro 4-, µ6, (0 u (.0 Bo co oo u ro 1,...,.. tto 00 OP ((.1), 2 0 4-, 0 1.T3' CO (0 (0 oto u ro u (0 03 , 03 4_, 03 4t, OA co 41.1 -I-, . 2 (-0 4_, , OA OP CO 0 4-, (4 u 03 ty3 U ro rn u 0.0 oo ro 0 ro r (0 u r op ro .1t .' (a B .(.% r op td3 CO r0 u u ro OD
(.0 op t (0 .1..1 ro oi3 fO, CO op 2 (.0 (.0 4_, u ro 4., (.0 ,(rõ) 4., co (13 -1--' 4-, OP U CO
U CO sowp OP -6,-.0 "(0' CO Z.; 1-2, "(0' +., U Op OP 2, CO OP 0 `(.'6 S co CO
2 2 O. BO CO
-(1-3-3' to ro OP 03 oi3 U U co 4_, CO 4-, .1-,4_, 0 (.0 (0 4_, 0 j3 op 4_, co +., +-, CO MI OA op MI to' '' (0 MI .2 tE; 0 0D ro an tE; U RI r (13 M
.I-; U n3 C1:1 U
UU
4., u CO 0 CO .1.-= U (.0 u (.0 UroM U WCOCOOD
OA dA 4-, CDu (0 0 4-1 1.T3' On (13 03 u CDCD03 CDu 1-3 4-, OA t t.,1 4-, U CD
CDop 4-, (0 (-0 U OA oz, co .1-; 4t,' 03 co 03 4., dA dA 0 4., 4- u o a-o A U U D p U 4-, U CO
co 1-3 op co CO ri3 4t', 4-, 03 fl5 OP 0 j3 op u 0 oi3 4-1 03 OD OA OD 03 0,0 OD CD-(1.T0' 0 rornroror04-,(0Z,,ro(.0+,roo(.01-3(0b) tzl t:: U OA 2 3 (- 0 Bo CO ri, s ' .1_, 4-' 4-' 4-, (0 ,...., CO O CO op o co 0 ro ., CDoo rn CD ii (0., +, 0.0 013 (0 CO, 0+, ''' C1:1 O C1:1 4-, 4-, (13 (13 CO , CO .1-; d A
U d A 4-, ro o CD ro h CDao CD 4-, ^ a-, CD CO OA CD OA CD a-, a-, CD CO CD U U CD OA tll3 CO OA tll3 CO CD
tll3 a-, a-. OA

on w3 co an - co 2, +, an Bo 2 0 (.0 .p, 0 < 0 H u < 0 0 0 u <
ro OA co OA 1-T; -1-' co < 0 < H H H
0.0 -04-'0Opla 03(Or C000Ur 4-' O op MI
OA - '' OAot La 0 < 0 0 (D L9 < H
4-, (0 r on ro ro on 1_3 3 .-,t, (0 4t, (V (0 ro CD m co rill ,õ '=..... U 0 U , õI- (-9 CD
OD OA CO OA -I-' 4-, 0 ro rm. - L7 < U H U L7 L7 H
4_, (0 (-0 tn M U .1-, µ(.6 CO ro .1.t,' CO CIA 65 0 ro -,1.74 on ro ro no H < U < <
01:1 U U CDU CO CD C0 1-3 `6' u g2 < W 0 u W W H < 0 < 0 (0 ro ro 2 4-, ^ 0 OA 4-; tE; ps,-,C4 .1-, tl CO mall (-90uUL-9(-9U<U
4-, , , on rou corn .t., 2 .2 .,-, to on to, .ro. UL7Ho<<L71-uL75 (.1:' O
(0 (10 U r U o ro - 4-' CD H U H < (-CD CD0,0 co ro 4-, ro co U L7 U 0 U y +., < L9 (-9 <
<UULDH 8 <
(-0 OA OD CO ro 4-, all all U ro ro 4-, all op 1(2 8 * u 0 -',..., op õ., ,-, OA co 4-, ^ +-' ro ,,, 0 ,,, - U Q
H < U < W W
2 2 2 .,r 0 'r 0 r ou .t,' flII. t4 0 ro (-0 tton3 4(2, 076, 0.0 2 n, H (D EH < UU LDIL2 (D
(0 CO CO 2 U CO roOA +-, 4-, -0.0 CO OA CO .F., -õ.., U r, 0 , u 44 44 .-.1 < < < (-90,õ(-90<(DE
u op ro U CDs == CO 4-, all OA (13 co a-, co 0 U C-) 0 (-9 U W U
CD co op (13 4_, 4-, CD-I-, OD 4-1 OA co HI-0 00<0 U ' (0 0.0 ' ,-rs -0 CO OA tf, r -',õ, u u u < HI-< H 0 CD4-, 4-, 4-, , ,4-, CO 4-; . s' U 4-' (0 4-' ro (13 U M U
4-, ''' co ro tto ro on 0 H
- 4= 4- < U U
4-, M 4-, U 4-' 4-; u CD0 tto < co 0 0 U U 0 OH <- 0 4; -; (13 0.13 on 0 ttO ro U (13 CO CO U (0 CO CD4-, CO 0 (-0 on 4-, CDCO
OD OD (0 OD -' U U M 4-, U 4- +-'rn OD M U 0 u U U U (-9 - U
ro .1-, M co 4t, (00 OD all a-, On ro ,.õ.,--õH U L7 U u < (-7 I-- 4-; M on M M CO 0 CO CO (0 0U 0 < H W
,_ - U .1= (-0 -, 4-; 4-'õ
O
o (0 pow ro 4-, 4_, t u on ro 4-, 0 -' Op ro L -' ro on u 4= +-, CO -' +-, m (.0 s ''',,.. (D U

OA 4_, .1= CO i.-, .1-, ,-,.. 0 op 4-, (.0 co tu 0 co 0 0 m co j0 .' 0 ro 0 ro OD (.0 +-, ro OD 0 4-, OA
O (%0 2 õc, -(iT,; 4(3 12 T ,,:, if (a tiej a op 1-0, 2 H t)UL7õ<01-UH<Uuu ,1-<U`"Uerl-ro 4-, ro 4-, ro ro o 4-, co 4-, 4_, 4_, 0 (0 H U 0 , r, , ,.., H u - u -.
0 j0 ro 0 0 all U ro 0 0 0 dA U ro MI tiO MI
(13 4-, 4-, 4-; 4_, U 4-, U (-0 .F., (0 (0 ,47',., (0 M
4-; 4-, co co U 0 4-, I-! C0 tto U
u ono roror,ro70$194-,(04-,0 ro f ri Uf)I-ro on u u =-.., < --- (.5 H , .-, (0 (0 U RI OD p , < 5 u 0 H u H u < H
mu 42 2 2 0 0 t i , 2 2 - u u < u < (.9 '9 o < u 0 u 0 < 0 H <
1.,) (.0 _(-0 u tE;
4 - ., (13 ro CO
-(0'CCIa0O.t,'.t:COCO(.000rod.0 Ur0 < ILD(D<L7L9uUUHU<
oHU(D(DUu(D 0. U an4-,+-' 2 0 (0 _ _ . , u u u u , < c,--:, < =-= ,, <
ro (13 (13 ro U all U , U ro H
((7, )3 :,ti CO tf, = U U 4- ' U (10 8 (0 0 u u (49 0 u u < H
CD< op dA 4-, CO
RI r CD ro ro CD
4^ F ' co co co co OA 4-, L. 4-, u (.0 4_, co (0 u t.,-?, ro ro u , 0 u u ro t ro 0 U <
2 4- U U U < (11 U 0 < U U

(.0 , - (.0 ro ro ro U u on OA 0 <000 Lc:c)Q0QL<_S(D<
Qui 4-' U rj U (13 .1-, CO 4-' CO 4-, ro 0 j0 CO co u co CIII U 4-' (:Lo 0 0 -.,,, ,L0 4-; (0 (.0 ro 0 ro U < < L9 "L9 U < <
OD M CO W3 OD (113 +., 2 co op Bo 4t, .1f, 44:: ro õ, -Id 44 .f2 r(.1k8r,.õ,(5,(2u0(.1 (0 .0 u CDu tt0_,_ õ1:10 u ro ttO u 1::: co 4-, 4_, U ro ro u ro tal., .i...-, ro op C0 4.., 4-, ro (13 D -1-' < 0 < < < H 0 U H < U
U U CO O 4_, (0 u 4-0 j0 ; (0 U M 4_, u CD
< I- < L7 < ( D < ( D U ( D <
CO U U CO 013 CO .1-, .1-, CO U CO M a-, U CO
CU
rq u .(0 w .+7 u ,1-0_ ( = 0 0_ 0 ..._... 4_, 013 (11 U CO SD- CO

,-I

ACAGTAAAGATTCAGGAATGGAATACATTTCAGGCAAACAGAGATACCGCGATTCACTGACGTCAGCCTCCTGCGGTAT
GAAA

CGCCCGAAAAGAATGCTTGTTACCGGATATTGTTGTCGGAGATGTAAAGGCCTTGCACTGTCAAGAACATCGCGGCGTC
TGTC w o w TCAGGAAGTCACCGAGCGTTTTTATGTGTGCACGGATCCGGGCTGTGGTCTGGTGTTTAAAACGCTTCAGACCATCAAC
CGCTT
i-J
CATTGTCCGCCCGGTCACGCCGGACGAACTGGCAGAACGCCTGCATGAAAAACAGGAACTGCCGCCAGTACGGTTAAAA
ACA
4,.
w CAATCATATTCGCTGCGTCTGGAATGAGGGCTGCCGGTTAACACCGGCCGTCGCCGCACACCGTATTTTTATTCTTCAG
CATGA oe TGAGAAAGAGATAACGATGGAAAGCACAGCCTTACAGCAGGCCTTTGACACCTGTCAGAATAACAAAGCAGCATGGCTG
CAA
CGCAAAAATGAGCTGGCAGCGGCCGAACAGGAATATCTGCGGCTTCTGTCAGGAGAAGGCAGAAACGTCAGTCGCCTGG
ACG
AATTACGCAATATTATCGAAGTCAGAAAATGGCAGGTGAATCAGGCCGCCGGTCGTTATATTCGTTCGCATGAAGCCGT
TCAG
CACATCAGCATCCGCGACCGGCTGAATGATTTTATGCAGCAGCACGGCACAGCACTGGCGGCCGCACTGGCACCGGAGC
TGA
TGGGCTACAGTGAGCTGACGGCCATTGCCCGAAACTGTGCCATACAGCGTGCCACAGATGCCCTGCGTGAAGCCCTTCT
GTCC
TGGCTTGCGAAGGGTGAAAAAATTAATTATTCCGCACAGGATAGCGACATTTTAACGACCATCGGATTCAGGCCTGACG
TGGC
P
TTCGGTGGATGACAGCCGTGAAAAATTCACCCCTGCGCAGAACATGATTTTTTCGCGTAAAAGTGCGCAACTGGCATCA
CGTC .
, AGTCAGTGTAAAATTCCCCGAAAATCCGCCCGTTTTTACTGAAAAAAGCCATGCATCGATAAGGTGCATGGCTTTGCAT
GCGTT
, 1¨
.3 vi TTCCTGCCTCATTTTCTGCAAACCGCGCCATTCCCGGCGCGGTCTGAGCGTGTCAGTGCAACTGCATTAAAACCGCCCC
GCAAA .
w GCGGGCGGGCGAGGCGGGGAAAGCACCGCGCGCAAACCGACAAGTTAGTTAATTATTTGTGTAGTCAAAGTGCCTTCAG
TAC "
, , , ATACCTCGTTAATACATTGGAGCATAATGAAGAAAATCTATGGCCTATGGTCCAAAACTGTCTTTTTTGATGGCACTAT

, N, N, AAATATGCAAAAAATAGATTGATGTAAGGTGGTTCTTGTCAGTGTCGCAAGATC
19 Native P2 (acc nr:
GGCGAGGCGGGGAAAGCACTGCGCGCTGACGGTGGTGCTGATTGTATTTTTTCAGCGTCTCAGCGCGTCGTGACGGCAC
TTA
NC_001895) GTCTGCCCGTTGAGGCGTTGTGTGTCTGCGGGGTGTTTTGTGCGGTGGTGAGCGTGTGAGGGGGGATGACGGGGTGTAA
AA
AAGCCGCCCGCAGGCGGCGATGTTCAGTCGTTGTCAGTGTCCAGTGAGTAGTTTTTAAAGCGGATGACCTCCTGACCGA
GCCA
GCCGTTTATCTCGCGGATCCTGTCCTGTAACGGGATAAGCTCATTGCGGACAAAGACCTTTGCCACTTTCTCAATATCA
CCCAGC
1-d GACCCGACGTTCTCCGGCTTGCCACCCATCAACTGAAAGGGGATGCGGTGCGCGTCCAGCAGGTCAGCGGCGCTGGCTT
TTTT n GATATTAAAAAAATCGTCCTTCGTCGCCACTTCACTGAGGGGGATAATTTTAATGCCGTCGGCTTTCCCCTGTGGGGCA
TAGAG t=1 1-d AAACAGGTTTTTAAAGTTGTTGCGGCCTTTCGACTTGACCATGTTTTCGCGAAGCATTTCGATATCGTTGCGATCCTGC
ACGGCA w o w TCGGTGACATACATGATGTATCCGGCATGTGCGCCATTTTCGTAATACTTGCGGCGGAACAACGTGGCCGACTCATTCA
GCCAG =
'a GCAGAGTTAAGGGCGCTGAGATATTCCGGCAGGCCGTACAGCTCCTGATTAATATCCGGCTCCAGCAGGTGAAACACGG
AGC o 4,.
w w vi CGGGCGCGAAGGCTGTCGGCTCGTTGAAGGACGGCACCCACCAGTAAACATCCTCTTCCACGCCACGGCGGGTATATTT
TGCC

GGTGAGGTTTCCAGTCTGATGACCTTACCGGTGGTGCTGTAACGCTTTTCCAGAAACGCATTACCGAACACCAGAAAAT
CCAGC n.) o n.) ACAAAGCGGCTGAAATCCTGCTGGGAAAGCCATGGATGCGGGATAAATGTCGAGGCCAGAATATTGCGTTTGACGTAAA
TCG
i-J
GCGAGCTGTGATGCACGGCAGCCCGCAGGCTTTTTGCCAGACCGGTAAAGCTGACCGGTGGCTCATACCATCTGCCGTT
ACTG .6.
.6.
n.) ATGCACTCGACGTAATCCAGAATGTCACGGCGGTCGAGTACCGGCACCGGCTCACCAAAGGTGAATGCCTCCATTTTCG
GGCC oe GCTGGCGGTCATTGTTTTTGCCGCAGGTTGCGGTGTTTTCCCTTTTTTCTTGCTCATCAGTAAAACTCCAGAATGGTGG
ATGTCA
GCGGGGTGCTGATACCGGCGGTGAGTGGCTCATTTAACAGGGCGTGCATGGTCGCCCAGGCGAGGTCGGCGTGGCTGGC
TTC
CTCGCTGCGGCTGGCCTCATAGGTGGCGCTGCGTCCGCTGCTGGTCATGGTCTTGCGGATAGCCATAAACGAGCTGGTG
ATGT
CGGTGGCGCTGACGTCGTATTCCAGACAGCCACGGCGGATAACGTCTTTTGCCTTGAGCACCATTGCGGTTTTCATTTC
CGGCG
TGTAGCGGATATCACGCGCGGCGGGATAGAACGAGCGCACGAGCTGGAACACGCCGACACCGAGGCCGGTGGCATCAAT
AC
CGATGTATTCGACGTTGTATTTTTCGGTGAGTTTGCGGATGGATTCCGCCTGGGTGGCAAAGTCCATGCCTTTCCACTG
GTGAC
P
GCTCAAGTATTCTGAATTTGCCACCGGCCACCACCGGCGGTGCCAGTACCACGCATCCGGCGCTGTCGCCACGGTGTGA
CGGG

TCGTAACCAATCCATACCGGGCGGGAGCCGAACGGATTGGCGGCAAACGGCGCATAGTCTTCCCATTCTTCCAGCGTGT
CGAC
un CATGCAGCGTTGCAGCTCCTCGAACGGGAACACCGATGCCTTGTCGTCAACAAATTCACACATGAACAGGTTTTTAAAA

.3 GGCGCTGTTTTCGCGTTTGAGCTGCTCAATGTCGAACAGCGTGCAGCCGCCTTTCAGGGCGTCCTCAATGGTGACAATC

CCACTGGCCGTCCGCACAGAGAAGCCCACCGGCAAGGGCGTTATGACTGACGTCGATTTCCACGCGTTCGGCGGCGCTG
GCG
.31 .3 CGTCCCCGGTTAAACAGTTCACCCGACCAGAACGGGTAGGCGTCGTGCGCCAGCGTGGACGGGGTGGAGAAATAGGTCG
AG
CGCAGGTGACTCTGTGAGGCCATACCTGATGCCACCTTACGCAGTACCTGAAAATTCGGGATCCAGAAAATCTCATCGA
CGTA
CAGGTCGCCGTTATGACTCTGCGCGGTGTTGGAGTTGGTGCCGAGAAAAATCAGTTTTGCGCCGTTATTGCCCAGGACA
ATCG
GGTCACCGGTCAGGTCAACGTCAACCAGACGGGCAAAGGCGATGATGTATTCGCGGAACACATACGCCTGTGTTTTACT
GGCC
GACAGAAAAATCTGGTTATGACCGGTTTTCAGGGCACGCAGCAGCGCCTCGCGGGAAAAATAAAACGTCGCGCCAATCT
GGC
GGGATTTCAGGATATCGCGGATGCGGTGCTCAAGCCCGGCACGATACCAGTGCAGCTGATAGTCGAAAGACTGCTCAAA
GAA
IV
AATCTGCTCCAGCTTTTCGATGGCCTCGTCACTGAAAAAATTCTTTTTCGGTTTGCGCCGTCCGCCTTTGTTACGGTTA
GCGACG n TTCGGATTAAGGTCTGCCTCGTTGCCGGTCTGGCTGTAGCGGTTGACCCGTGCCAGTCGTTCAATCTGGCGTCCCAGCA
GGTCA t=1 tl ATTTCCTTGAAGTCACCGCCGGTTTTCTGTGGTTTGATGATGAGCTGGGTCAGCCGCGCTTCCAGACTCATTTCGACAC

o ATGGGGGCAACGCTGTCCCAGCCGTCGCGCTGTTTCCAGCTCTGCACTGTCGGGCGTTTCATCTGCAACATGGCGGCAA
TCTGC 'a o GGCACGGAAAACCCCTGCCAGTACAGCAGCGCCGCCTGACGACGCGGGTCGTGTAAAAGAGTGGTGTCTGTGGTGATGG
TCA tt n.) un TGAATACCTCGCCGTGATGAATACACGGCAAGGCTACTGAGTCGCGCCCCGCGATTCGCTAAGGTGCTGTTGTGTCAGT
GATA

AGCCATCCGGGACTGATGGCGGAGGATGCGCATCGTCGGGAAACTGATGCCGACATGTGACTCCTCTAATCACTATTCA
GGAC n.) o n.) TCCTGACAATGGCAAAAAAAGTCTCAAAATTCTTTCGTATCGGCGTTGAGGGTGACACCTGTGACGGGCGTGTCATCAG
TGCG
i-J
CAGGATATTCAGGAAATGGCCGAAACCTTTGACCCGCGTGTCTATGGTTGCCGCATTAACCTGGAACATCTGCGCGGCA
TCCT .6.
.6.
n.) GCCTGACGGTATTTTTAAGCGTTATGGCGATGTGGCCGAACTGAAGGCCGAAAAGATTGACGATGATTCGGCGCTGAAA
GGC oe AAATGGGCGCTGTTTGCGAAAATCACCCCGACCGATGACCTTATCGCGATGAACAAGGCCGCGCAGAAGGTCTACACCT
CAAT
GGAAATTCAGCCGAACTTTGCCAACACCGGCAAATGTTATCTGGTGGGTCTGGCCGTCACCGATGACCCGGCAAGCCTC
GGCA
CGGAATACCTGGAATTCTGCCGCACGGCAAAACACAACCCCCTGAACCGCTTCAAATTAAGCCCTGAAAACCTGATTTC
AGTGG
CAACGCCTGTTGAGCTGGAATTTGAAGACCTGCCTGAAACCGTGTTCACCGCCCTGACCGAAAAGGTGAAGTCCATTTT
TGGCC
GCAAACAGGCCAGCGATGATGCCCGTCTGAATGACGTGCATGAAGCGGTGACCGCTGTTGCTGAACATGTGCAGGAAAA
ACT
GAGCGCCACTGAGCAGCGCCTCGCTGAGATGGAAACCGCCTTTTCTGCACTTAAGCAGGAGGTGACTGACAGGGCGGAT
GAA
P
ACCAGCCAGGCATTCACCCGCCTGAAAAACAGTCTCGACCACACCGAAAGTCTGACCCAGCAGCGCCGCAGCAAAGCCA
CCGG

CGGTGGCGGTGACGCCCTGATGACGAACTGCTGACCGGCGTCAGTCAGTCCGGGAAAACCTTCACGATTAACCCTTAAT
TTCA
un GGAAAAACTATGCGCCAGGAAACCCGCTTTAAATTTAATGCCTACCTGTCCCGTGTTGCCGAACTGAACGGCATCGACG

.6.
.3 TGATGTGTCGAAAAAATTCACCGTTGAACCGTCGGTCACCCAGACCCTGATGAACACCATGCAGGAGTCCTCTGACTTT

CCGCATCAATATTGTGCCGGTCAGCGAAATGAAAGGGGAAAAAATTGGTATCGGTGTCACCGGCTCCATCGCCAGCACT
ACCG
.31 .3 ACACTGCCGGTGGTACCGAGCGTCAGCCGAAGGACTTCTCGAAGCTGGCGTCAAACAAGTACGAATGCGACCAGATTAA
CTTC
GATTTTTATATCCGCTACAAAACGCTGGACCTGTGGGCGCGTTATCAGGATTTCCAGCTCCGTATCCGTAACGCCATTA
TCAAAC
GCCAGTCCCTTGATTTCATCATGGCCGGTTTTAACGGCGTGAAGCGTGCCGAAACCTCTGACCGCAGCAGCAATCCGAT
GTTGC
AGGATGTGGCGGTCGGCTGGCTGCAGAAATACCGCAATGAAGCACCGGCGCGCGTGATGAGCAAGGTCACTGACGAGGA
AG
GCCGCACCACCTCTGAGGTTATCCGCGTGGGTAAGGGCGGTGATTATGCCAGCCTTGATGCACTGGTGATGGATGCGAC
CAAC
AACCTGATTGAACCGTGGTATCAGGAAGACCCTGACCTTGTGGTGATTGTGGGGCGTCAGCTACTGGCGGACAAGTATT
TCCC
IV
CATCGTCAACAAGGAGCAGGACAACAGCGAAATGCTGGCCGCTGACGTCATCATCAGCCAGAAACGCATCGGTAACCTA
CCA n GCGGTACGCGTCCCGTACTTCCCGGCGGATGCGATGCTCATCACGAAGCTGGAAAACCTGTCCATCTACTACATGGATG
ACAG t=1 tl CCATCGCCGCGTGATTGAGGAAAACCCGAAACTCGACCGCGTGGAGAACTACGAGTCAATGAACATTGATTACGTGGTG

o GACTACGCCGCCGGTTGTCTGGTGGAAAAAATCAAGGTCGGTGACTTCTCCACACCGGCTAAGGCGACCGCAGAGCCGG
GAG 'a o CGTAACCGATGACGAGTCCCGCACAGCGCCACATGATGCGGGTCTCGGCAGCGATGACCGCGCAGCGGGAAGCCGCCCC
GCT tt n.) un GCGACATGCAACTGTCTATGAGCAGATGCTGGTTAAGCTCGCCGCAGACCAGCGCACACTGAAAGCGATTTACTCAAAA
GAGC

TGAAGGCCGCAAAAAAACGCGAACTGCTGCCGTTCTGGTTGCCGTGGGTGAACGGCGTGCTGGAGCTGGGCAAAGGTGC
AC n.) o n.) AGGATGACATTCTGATGACGGTCATGCTGTGGCGTCTGGATACCGGCGATATTGCCGGTGCGCTGGAGATTGCCCGTTA
TGCC
i-J
CTGAAGTACGGTCTGACCATGCCGGGTAAACACCGCCGTACCCCGCCGTACATGTTCACCGAGGAGGTAGCGCTTGCGG
CCAT .6.
.6.
n.) GCGCGCTCACGCTGCCGGTGAGTCTGTGGATACCCGCCTGCTGACGGAGACCCTTGAACTGACCGCCACGGCTGACATG
CCTG oe ATGAAGTGCGCGCAAAGCTGCACAAAATCACCGGTCTGTTTCTGCGTGACGGTGGTGATGCCGCCGGTGCGCTGGCGCA
CCT
GCAACGTGCGACACAGCTCGACTGTCAGGCAGGCGTCAAAAAAGAGATTGAACGACTGGAGCGGGAGCTGAAACCGAAG
CC
GGAGCCGCAGCCCAAAGCGGCCACCCGCGCCCCGCGTAAGACCCGGAGCGTGACACCGGCAAAACGTGGACGCCCGAAA
AA
GAAAGCCAGTTAACAACCGAATGCGCCCCGCGCCAGGGCGGCACGCCGGTCAGTGACGGTGAATCACCTGACACTGCAC
CGG
CGTCCACCGCCCGACTTTTCAGAGGTAGTCATGATGACGCTGATTATTCCGCGAAAGGAGGCTCCCGTGTCCGGTGAGG
GTAC
GGTGGTCATCCCGCAACCGGCAGGCGACGAGCCGGTGATTAAAAACACGTTCTTTTTTCCCGATATCGACCCGAAGCGC
GTCC
P
GGGAACGTATGCGCCTTGAGCAGACCGTCGCCCCCGCCCGTCTGCGTGAGGCCATCAAGTCAGGCATGGCTGAAACGAA
TGC

GGAGCTGTACGAGTACCGCGAACAGAAAATTGCCGCCGGTTTTACGCGTCTGGCTGACGTCCCGGCGGACGATATCGAC
GGT
un GAAAGCATCAAGGTTTTTTACTACGAGCGCGCCGTGTGTGCGATGGCGACCGCGTCGCTTTATGAGCGTTATCGCGGTG

un .3 TGCCAGTGCGAAAGGCGACAAGAAGGCTGACAGCATTGACAGCACCATTGATGAGCTGTGGCGGGATATGCGCTGGGCG

GGCGCGCATCCAGGGCAAGCCGCGCTGCATCGTGAGTCAAATCTGATGAAGACCTTTGCGCTACAGGGCGACACGCTCG
ACG
.31 .3 CCATTTGTGTCCGCTATTACGGGCGCACTGAGGGCGTGGTTGAGACCGTGCTCGCCGCAAATCCGGGACTGGCTGAACT
GGG
GGCGGTGCTGCCACACGGCACCGCCGTCGAACTGCCCGACGTTCAGACCGCGCCCGTGGCTGAAACTGTCAATCTGTGG
GAG
TAACGCATGACAGCAGAAGAAAAAAGCGTCCTGTCGCTTTTCATGATTGGGGTGCTGATTGTTGTCGGCAAGGTGCTTG
CCGG
TGGTGAACCTATCACCCCGCGTCTGTTTATCGGGCGCATGTTGCTCGGTGGTTTTGTCTCGATGGTTGCCGGTGTTGTT
CTGGT
GCAGTTTCCTGACCTGTCACTGCCAGCGGTGTGCGGCATCGGCTCCATGCTGGGTATCGCCGGTTATCAGGTGATTGAG
ATTG
CCATTCAGCGCCGCTTTAAGGGCAGGGGGAAACAGTAATGCCGGTAATTAACACGCATCAGAATATCGCCGCCTTTCTC
GACA
IV
TGCTGGCCGTGTCCGAAGGGACGGCGAATCATCCACTGACGAAAAACCGGGGCTATGACGTGATAGTCACCGGACTGGA
CGG n GAAGCCGGAAATTTTCACCGACTACAGTGACCACCCGTTCGCACATGGCCGACCGGCGAAGGTGTTTAACCGTCGCGGT
GAAA t=1 tl AATCCACGGCCTCCGGTCGCTATCAGCAGCTTTACCTGTTCTGGCCGCATTACCGCAAACAGCTTGCCCTGCCGGATTT

o GTTGTCACAGGACAGACTCGCCATTCAGTTGATCCGCGAACGCGGAGCACTGGATGACATCCGGGCGGGACGCATTGAG
CGC 'a o GCCATTTCACGCTGTCGCAATATCTGGGCGTCCCTGCCGGGTGCCGGTTACGGTCAGCGTGAGCATTCACTGGAAAAAC
TGGT tt n.) un CACCGTCTGGCGTACCGCTGGCGGCGTACCGGCTTAAACGGAGTAAATACCATGAAGAAATTATCCCTTTCACTGATGC
TGAA

CGTGTCGCTGGCGCTGATGCTGGCACTGTCCCTGATTTACCCGCAGAGCGTGGCCGTCAATTTTGTCGCTGCCTGGGCG
ATTCT n.) o n.) GGCGACGGTTATCTGTGTGGTTGCCGGTGGTGTGGGCGTGTATGCCACTGAGTATGTGCTGGAACGCTACGGGCGGGAG
CTG
i-J
CCGCCGGAATCGCTGGCCGTGAAGATTGTCACGTCGCTGTTTTTGCAGCCGGTGCCGTGGCGCAGACGGGCGGCGGCTC
TGG .6.
.6.
n.) TAGTGGTGGTGGCGACGTTTATCTCGCTGGTCGCTGCCGGGTGGATTTTTACCGCGCTGATTTATCTTGTGGTGTCGCT
GTTTTT oe CCGGCTGATACGTAAAGCCTGTCGTCAGCGTCTTGAGGGGCGGGAACCATGTCAAGGCTGATGATTGTGCTGGTCGTGT
TGTT
ATCGCTGGCGGTGGCCGGTCTGTTTCTGGTGAAACACAAAAATGCCAGCCTGCGCGCCTCGCTGGACAGGGCGAACAAC
GTC
GCCAGCGGTCAGCAGACGACCATCACCATGCTGAAAAATCAGCTTCATGTTGCGCTCACCAGGGCAGATAAAAACGAGC
TGGC
GCAGGTGGCACTGCGTCAGGAACTGGAGAACGCCGCGAAACGTGAAGCACAGCGCGAGAAAACCATCACGAGGTTACTT
AAT
GAGAACGAAGATTTTCGCCGCTGGTACGGTGCTGACCTGCCTGATGCTGTGCGCCGGTTGCACCAGCGCCCCGCCTGCA
CCGA
CGCCAGTGATTGTCCCCAACGCATGCCCGAAAGTGAGCCTTTGCCCGATGCCGGGCAGTGACCCGCAGACGAACGGCGA
TTTA
P
AGTGCCGATATCCGGCAGCTTGAGAACGCGCTGGCACGCTGTGCCAGCCAGGTAAAAATGATTAAACACTGTCAGGACG
AAA

ACGATGCTCAAACCCGACAGCCTGCGCAGGGCGCTGACTGATGCCGTCACGGTGCTGAAAACTAACCCCGATATGCTGC
GGAT
un ATTCGTGGATAACGGGAGTATTGCCTCCACACTGGCGGCGTCGCTGTCATTCGAAAAGCGTTACACGCTCAATGTGATT

o .3 CCGACTTTACCGGTGATTTTGACCTGCTCATTGTGCCGGTGCTGGCGTGGCTGCGGGAAAATCAGCCCGACATCATGAC

GACGAAGGCCAGAAAAAGGGCTTCACGTTTTATGCAGACATCAACAATGACAGCAGCTTTGATATCAGTATCAGCCTGA
TGCT
.31 .3 GACCGAGCGCACGCTGGTCAGTGAGGTGGACGGCGCACTGCATGTGAAGAATATCTCGGAACCCCCGCCGCCGGAGCCG
GTC
ACCCGCCCGATGGAGCTGTATATCAATGGCGAACTGGTGAGTAAGTGGGATGAATGAGTTTAAGCGTTTTGAAGACCGG
CTG
ACCGGACTGATTGAATCGCTGTCACCGTCAGGGCGTCGGCGACTGAGTGCCGAACTGGCGAAACGTCTGCGGCAGAGTC
AGC
AGCGTCGGGTGATGGCACAGAAAGCCCCGGACGGCACACCCTACGCGCCACGCCAGCAGCAGAGCGTCAGAAAAAAGAC
CG
GTCGCGTTAAGCGAAAAATGTTTGCGAAACTTATTACCAGTCGTTTTTTGCATATCCGTGCCAGCCCGGAGCAGGCATC
AATGG
AATTTTACGGCGGGAAGTCGCCGAAAATCGCCAGTGTGCATCAGTTTGGTCTGTCGGAAGAAAACCGGAAAGACGGTAA
GAA
IV
AATTGATTATCCGGCGCGTCCCCTGCTCGGCTTTACCGGTGAGGATGTGCAGATGATTGAAGAGATTATCCTGGCTCAC
CTTGA n GCGTTAGTTTTATCCAGGCAGAGGCTGATGCGCAATTAAACATTGAGCGGCCATGCTGGTCGCTCAATGTTTAGAGGTT
TATG t=1 tl AGTGATTTTTATTTGATGCTTTGTATTCTAAAACCTTCTTATTGGCGTAAAAGAATTTTGTATATGACAGGAATATAAC

o TGAAGTGAAATAGACGAGGGATAGTATTAATAATGCTTTTTTGTGACTGTTATTATCTTTAATCTCCTGGCTTAACCAT
TCGGAG 'a o TCCTCCTCGTTTAGCTGTAAGAGCTTATTGCAGGCGATCTCAGGAAGTGTGTCTTTTATAAATACGTTTTGCAGTCTCT
TGCAAT tt n.) un CGGCAAGGCTATAAGTTTTATTAAATTCAACTGCTTTATTTTTGAAGGATAAAAGAACTTTGTCACTATAAACATAGTA
CATCAT

GTTTTTATATGGTATGCCTGTGGCATCCCTTACTATAACGGATTGTTCGTTGTGTATGTAACATGCGAGGAGAATGTAA
AAAAT n.) o n.) ACTGGCCAGAATTACAATTATTGTTTTAATTATGTGTGGTGGTTTTGTTATGTCACCCCAGATACGAGTAAGGAAAAAA
TACGA
i-J
TGTTTTTAGTTTTCCATCAATCAGTCCCTGCTGTATCATTCTCACATTTTCAATGCCTGATACATTGATTCCGTTAATT
ATTTTAAA .6.
.6.
n.) TAGTTGAATGTCGCGCCACTCGCGGTCAAGTCTTTTTAATTTTTTGTCTGAATATCCAAAATTGAAATAATGTGCAATA
AGCCTC oe ATAAGGTTACTTTTACCAAAGCTAAAAAATGCTAATACTGCAAAGCTACAAAGGAAAAAAACGATTAGCCCCCACACAT
TAGTC
ACATTATAGCTGACCATTACGCTCTCCTTGAATGTTGTCTGGTAGTTCTACAAATGAATCCAGATAGCATAACTTTTAT
ATATTGT
GCAATCTCACATGCATGAACACTCTCGCAAATATTCAGGAACTCGCGCGCGCACTGCGCAACATGATTCGCACTGGCAT
TATCG
TCGAAACCGACCTTAACGCCGGTCGCTGCCGCGTGCAGACCGGCGGCATGTGCACCGACTGGCTTCAGTGGCTGACCCA
TCGC
GCAGGACGTTCGCGCACATGGTGGGCACCTTCCGTGGGGGAACAGGTGCTGATTCTGGCCGTGGGTGGTGAACTCGACA
CGG
CGTTCGTTCTGCCGGGGATTTATTCCGGCGATAACCCCTCGCCGTCTGTGTCGGCGGATGCCCTGCATATCCGTTTCCC
TGACG
P
GGGCGGTGATTGAATATGAACCCGAAACCAGTGCACTCACGGTAAGCGGAATTAAAACGGCCAGCGTGACGGCTTCCGG
TTC

TGTTACTGCCACGGTGCCGGTGGTCATGGTGAAAGCATCAACCCGCGTCACCCTGGACACCCCGGAGGTGGTCTGCACC
AACA
un GGCTGATTACCGGCACGCTGGAAGTGCAGAAAGGCGGGACGATGCGCGGCAACATTGAACACACCGGCGGTGAACTCTC

--.1 .3 AAACGGTAAGGTACTGCATACCCATAAACACCCCGGCGACAGCGGCGGCACAACCGGGAGTCCTTTATGACAGCGCGTT

CGGAATGAATCGCAGTGATGGCCTGACTGTCACTGACCTTGAGCATATCAGCCAGAGTATCGGCGATATCCTGCGCACA
CCGG
.31 .3 TCGGCTCACGGGTGATGCGTCGTGATTACGGCTCGTTGCTGGCGTCAATGATTGACCAGCCGCAGACCCCGGCGCTTGA
GTTG
CAGATTAAAGTCGCCTGTTACATGGCAGTGCTGAAATGGGAACCCCGCGTCACCCTGTCATCCGTCACCACGGCGCGCA
GTTTT
GACGGGCGAATGACGGTCACGTTAACCGGCCAGCACAACGACACCGGCCAGCCACTTTCATTAACCATCCCTGTGAGTT
GAAA
CCATGCCGATTATCGACCTGAACCAGCTACCCGCACCGGATGTGGTCGAGGAGCTGGACTTTGAAAGCATTCTCGCTGA
ACGC
AAGGCGACACTGATTTCCCTTTACCCGGAAGATCAGCAGGAGGCGGTCGCCCGTACCCTGACACTGGAATCTGAGCCTC
TCGT
CAAACTGCTGGAAGAAAATGCTTATCGTGAGCTTATCTGGCGTCAGCGTGTGAATGAGGCCGCACGGGCGGTGATGCTG
GCC
IV
TGTGCCGCCGGTAATGACCTTGATGTGATTGGTGCCAATTACAACACCACGCGCCTGACTATCACCCCGGCAGATGATT
CGACC n ATCCCGCCGACACCGGCAGTGATGGAATCTGACACCGATTATCGTCTGCGTATTCAGCAGGCTTTTGAGGGCTTAAGCG
TCGC t=1 tl CGGGTCAGTGGGAGCCTATCAGTATCATGGTCGCAGTGCTGACGGGCGTGTCGCGGATATTTCTGTCACCAGTCCGTCT

o CCTGTGTCACCATCTCTGTGCTGTCACGTGAAAATAACGGCGTCGCATCCGAAGACCTGCTGGCTGTGGTGCGTAACGC
CCTTA 'a o ATGGCGAGGACGTCAGGCCGGTGGCCGACCGCGTGACCGTGCAGTCTGCCGCCATCGTTGAATACCAGATAAACGCCAC
GCT tt n.) un TTACCTTTACCCTGGTCCCGAAAGCGAACCCATCCGCGCTGCCGCTGTGAAAAAGCTGGAAGCGTATATCACGGCACAG
CACC

GGCTGGGGCGCGACATCCGTCTGTCTGCCATTTATGCCGCTTTGCATGTGGAAGGTGTGCAGCGTGTCGAACTGGCTGC
ACCA n.) o n.) CTGGCCGACATCGTGCTCAACAGTACGCAGGCGTCTTTCTGTACCGAATACCGCGTCGTGACCGGAGGCTCGGATGAGT
GATT
i-J
CGCGACTGCTGCCGACCGGCTCATCACCGCTTGAGGTCGCCGCCGCAAAAGCCTGTGCGGAAATTGAAAAAACGCCGGT
CAG .6.
.6.
n.) TATTCGTGAACTGTGGAACCCGGACACCTGTCCGGCAAATCTGCTGCCGTGGCTGGCGTGGGCGTTTTCGGTCGACAGG
TGGG oe ATGAAAAGTGGCCGGAAGCGACAAAACGCGCCGTTATCCGCGATGCCTATTTCATCCACTGTCATAAGGGCACGATAGG
TGCA
ATCCGGCGTGTGGTGGAGCCGCTCGGCTATCTCATCAACGTGACGGAGTGGTGGGAAAACAGTGACCCGCCCGGCACCT
TCC
GGCTTGATATTGGTGTACTGGAAAGCGGTATCACAGAGGCAATGTATCAGGAAATGGAACGGCTGATTGCTGATGCCAA
ACC
TGCAAGCCGTCACCTTATTGGCCTGAACATTACCCGGGACATTCCCGGCTATCTGTTCGCCGGTGGTGTGGCTTACGAC
GGCGA
TGTAATTACGGTTTACCCCGGATAAGTGAGGAATAATGAGCATAAAATTCAGAACCGTTATCACCACTGCCGGTGCAGC
AAAG
CTGGCAGCGGCAACCGCGCCGGGAAGGCGGAAGGTCGGCATTACCACGATGGCCGTCGGGGATGGCGGTGGTAAATTGC
CT
P
GTCCCGGATGCCGGACAGACCGGGCTTATCCATGAAGTCTGGCGACATGCGCTGAACAAAATCAGCCAGGACAAACGAA
ACA

GTAATTATATTATCGCCGAGCTGGTTATTCCGCCGGAGGTGGGCGGTTTCTGGATGCGTGAGCTTGGCCTGTACGATGA
TGCG
un GGAACGTTAATTGCCGTGGCGAACATGGCCGAAAGCTATAAGCCAGCCCTTGCCGAAGGCTCAGGACGTTGGCAGACCT

oe GCATGGTCATCATCGTCAGCAGTGTGGCCTCAGTGGAGCTGACCATTGACACCACAACGGTGATGGCGACGCAGGATTA

GATGACAAAATTGCAGAGCACGAACAGTCACGACGTCACCCGGACGCCTCGCTGACAGCAAAAGGTTTTACTCAGTTAA
GCAG
.31 .3 TGCGACCAACAGCACGTCTGAAACACTGGCCGCAACGCCGAAAGCGGTAAAGGCCGCGTATGACCTGGCTAACGGGAAA
TAT
ACCGCACAGGACGCCACCACAGCGCGAAAAGGCCTTGTCCAGCTTAGTAGCGCCACCAACAGCACGTCTGAAACGCTCG
CCGC
AACACCAAAAGCCGTTAAGACGGTAATGGATGAAACGAACAAAAAAGCGCCATTAAACAGCCCTGCACTGACCGGAACG
CCA
ACGACGCCAACTGCGCGACAGGGAACGAATAATACTCAGATCGCAAACACGGCTTTCGTTATGGCCGCGATTGCCGCCC
TTGT
AGACTCGTCGCCTGACGCACTGAATACGCTGAACGAGCTGGCGGCGGCGCTGGGCAATGACCCGAATTTTGCTACCACC
ATGA
CTAATGCGCTTGCGGGTAAGCAACCGAAAGATGCTACCCTGACGGCGCTGGCGGGGCTTGCTACTGCGGCAGACAGGTT
TCC
IV
GTATTTTACGGGGAATGATGTTGCCAGCCTGGCGACCCTGACAAAAGTCGGGCGGGATATTCTGGCTAAATCGACCGTT
GCCG n CCGTTATCGAATATCTCGGTTTACAGGAAACGGTAAACCGAGCCGGGAACGCCGTGCAAAAAAATGGCGATACCTTGTC
CGGT t=1 tl GGACTTACTTTTGAAAACGACTCAATCCTTGCCTGGATTCGAAATACTGACTGGGCGAAGATTGGATTTAAAAATGATG

o GGTGACACTGATTCATACATGTGGTTTGAAACGGGGGATAACGGCAATGAATATTTCAAATGGAGAAGCCGCCAGAGTA
CCA 'a o CAACAAAAGACCTGATGACGTTGAAATGGGATGCACTAAATATTCTTGTTAATGCCGTCATTAATGGCTGTTTTGGAGT
TGGTA tt n.) un CGACGAATGCACTAGGTGGTAGCTCTATTGTTCTTGGTGATAATGATACCGGATTTAAACAGAATGGAGACGGTATTCT
TGAT

GTTTATGCTAACAGTCAGCGTGTATTCCGTTTTCAGAATGGAGTGGCTATTGCTTTTAAAAATATTCAGGCAGGTGATA
GTAAA n.) o n.) AAGTTCTCGCTATCCAGCTCTAATACATCCACGAAGAATATTACCTTTAATTTATGGGGTGCTTCCACCCGTCCAGTGG
TTGCAG
i-J
AGTTAGGCGATGAGGCCGGATGGCATTTCTATAGCCAGCGAAATACAGATAACTCGGTAATATTTGCTGTTAACGGTCA
GATG .6.
.6.
n.) CAACCCAGCAACTGGGGAAATTTTGATTCCCGCTATGTGAAAGATGTTCGCCTGGGTACGCGAGTTGTTCAATTGATGG
CGCG oe AGGTGGTCGTTATGAAAAAGCCGGACACACGATTACCGGATTAAGAATCATTGGTGAAGTAGATGGCGATGATGAAGCC
ATC
TTCAGGCCGATACAAAAATACATCAATGGCACATGGTATAACGTTGCGCAGGTGTAAGTTATGCAGCATTTAAAGAACA
TTAA
GTCAGGTAATCCAAAAACAAAAGAGCAATATCAGCTAACAAAGAATTTTGATGTTATCTGGTTATGGTCCGAAGACGGA
AAAA
ACTGGTATGAGGAAGTGAAGAACTTTCAGCCAGACACAATAAAGATTGTTTACGATGAAAATAATATTATTGTCGCTAT
CACCA
GAGATGCTTCAACGCTTAATCCTGAAGGTTTTAGCGTTGTTGAGGTTCCTGATATTACCTCCAACCGACGTGCTGACGA
CTCAG
GTAAATGGATGTTTAAGGATGGTGCTGTGGTTAAACGGATTTATACGGCAGATGAACAGCAACAACAGGCAGAATCACA
AAA
P
GGCCGCGTTACTTTCCGAAGCGGAAAACGTTATTCAGCCACTGGAACGCGCTGTCAGGCTGAATATGGCGACGGATGAG
GAA

CGTGCACGACTGGAGTCATGGGAACGTTACAGCGTTCTGGTCAGCCGTGTGGATCCTGCAAATCCTGAATGGCCGGAAA
TGCC
un GCAATAAGTTGTATGACCTCTGTTGTGAACTTACATATCTATGGCACAGAGTAAAGCCTAATCTGAAGTCCACTCTGTG

o .3 AGCGGACCTTATAAACAAGAACAATGGTCATATCAGTGTGTTTTGATTGCATAGCTAACGTGCGTCTTCCTGTACAGAA

AGATGATAGGGCATAGGAGATGATTATTATCGCGTGTTTTAAAAATAGCTTTCTGCATCAAGTGTCACTTGCGAAGAGG
TATTG
.31 .3 GCGCTATGATTGGCATCACTCAGTTCAGATAATATTTTAAATATTTGGTAAATGTTCAGATTACACAATTCGGTCTGCG
TCCCGT
TAAAATTATCTACTATTAGTTATTACTATGAGGTGAATGGCAAGTGTTTTCCACTCGCCATTTTTGTTTTTTATTTTTT
TAAAGCCT
TTCCGACGTTTAAGGATTTTGCCAAGCCATAAAATATTTGATATTTCATTCCTGTTTTTATTTTTGGTGAGATATTTGC
GTAACTT
CTTTCTCTAAAACACCAACGTATAAACCTATTATTATCTTCGGTTGTTTCTATATAGCATATTACATTGAGACCAAAAA
GAAACTG
AAGGAAGTCATTTGCAGTTGTCATGAATTTTGGTTTTGTTGTGGAAATGGAACTAATATGAGTTTCAAGTTGTTTATAT
GCTAA
GAGGTATTCAGCGTAATCAAAAGTATCTTTTCCAGAGAGGAATTCAAAGAACTTTAAAAAAATCTCATAATGTTCACTT
CCATA
IV
ATAAAATGATAAATGATCTTTTATTTCTCCAAGTAAATAGTTAGAGTAATCTCTTTGCATGCTAGGACTTTCAAAGTCA
TCTAGA n ,-i CTAAATTGATTTTTCCCATCATCACCGCTGGCGTGTGATTTGATAATTTCGAGCATCCGTAATATATCTCTTGGGCGAT
AATATG t=1 tl ACCAGCGAAGGAAATTAATAAATGATGTGGGGTGGTTATAATTATCATGGACGTTGGGTGAATCCCAAGGGAAATAATG

o CCATGCCTGTCCCTTTTTAAGGGCCATGTCTTGCTGAGTCCTAAGCAGATGATCTAATACCTCAAATAATTGAGAGCTA
CGGTG 'a o GTTAACATATTCTGTCCGCCAGTCCAAGAATACTGAATTATCTCTGATTTTTGCGTTTTGATTTTGTAATCCTATGGAT
TCAAATA tt n.) un TATCTGGCCTGATCAAGAGAACGGCCTTCATTCTCCCTTTTCCTCCCTTTATACTTGGGAAGAAATCGTTGTTAATCTC
CCAGACT

GCGTGAGCTAATCCTTTTATACATTCTAGATAATCATCGTATGGAATTGAGGAAGGGCGGATATCAATACCATCGATAA
ATAAA n.) o n.) ATATGATTTTGAGATAAACGTATTTGGCTAAAGGCCTCTTCAAATTTTTTTTGTATATAAAATAAATTTATCTGAAATC
TGCTCTC
i-J
AGAAAAAGATAAAGACTCTCTTTCTTCACCTTTTAAGGTTGCATGCTTATGAAGTAGTTCGGCTGCAACTTTTGACTCT
TGAACA .6.
.6.
n.) AAACTGAGGGCTTGAATTATTTCTGGTGAAAATGCCTTAAGATAATACTCATCTATTGCATCATTAAGTGATGAGAATT
TTGAAT oe AATTTAGTAACTTTGAAATTTTCCCTTCTTTATCTAATACTTGTTTTGATATTAATAAATATAGAATAACTTTCCATAT
GCCAGAGA
AGTCAGACAAATTTAAATGCTTTTCAGATTTAAGAGTGATAAATTTTTGATATTCTGTCTCTCTTATATATTTAGTTGT
AGCTAGC
GTATTGTTAATATTGTTATTTGAGAGATAGATAGAGTATGCGGTTTTACCAGTACCTTTCTCTCCTACAAGAAACGATA
TATTTG
GTTCACATAATCTAGATAGGTGATTATCACGTATGAACACTTTGTTAAGTAATTCTTTATTCTCCCTTCTCTTATAATT
TTCAGCAT
CAGCATAGCCAAGAGTTAGATCTTTGATTGGTATCATTATTACCTCAACTAAAGCAATTGAAAAACATATGGTTATAAA
AACAT
AGCGCTGCTCTGTCGCATATTGTGGATATCAACATATGAAATGTAAAGGAACTATTTGTGGATGAATGAACCGAATCGC
TGTA
P
GCTTCGGTAGTCATCTTTCAGACTTGTATAAATGAACAACTTCCGCTTCTCGCTCAAAGGAAACTGTCAGATTTGATAG
CTTTTG

GGCTATGTAAACTGTCAGTCGGAAAATGAGTGAGTACAAATCAGGGCAGGTGAGCGAATTGCCCGCCTTTTCTTTACCG
GTGG
o TTGTGCTGTCGATTAGCCAACCGGGACAAATAGCCTGACATCTCCGGCGCAACTGAAAATACCACTCACCCATTAACCA

o .3 TTAAACGGATGAGTGACTATCATCACGGCGTGCAGGTGCTGGAGATTAACGAGGGCACCCGCGTCATTTCCACCGTATC

GCCATTGTCGGCATGGTCTGCACGGCCAGCGATGCAGATGCGGAAACCTTCCCCCTCAATAAACCTGTGCTGATTACCA
ATGTG
.31 .3 CAGAGCGCAATTTCAAAGGCCGGTAAAAAAGGCACGCTGGCGGCATCGTTGCAGGCCATCGCTGACCAGTCAAAACCGG
TCA
CCGTTGTCATGCGCGTGGAAGACGGCACCGGTGATGACGAGGAAACGAAACTCGCGCAGACCGTTTCCAATATCATCGG
CAC
CACCGATGAAAACGGTCAGTACACCGGACTAAAAGCCATGCTGGCGGCGGAGTCGGTAACCGGTGTTAAACCGCGTATT
CTC
GGCGTGCCGGGACTGGATACCAAAGAGGTGGCTGTTGCACTGGCATCAGTCTGTCAGAAGCTGCGTGCTTTCGGGTATA
TCA
GCGCATGGGGCTGTAAAACCATTTCCGAGGTGAAAGCCTATCGTCAGAATTTCAGCCAGCGTGAGCTGATGGTCATCTG
GCCG
GATTTCCTCGCATGGGATACGGTCACCAGTACCACCGCCACCGCGTATGCCACCGCCCGTGCGCTGGGGCTGCGCGCTA
AAAT
IV
CGACCAGGAGCAGGGCTGGCATAAAACGCTGTCCAATGTCGGGGTGAACGGTGTTACCGGCATCAGCGCATCTGTATTC
TGG n GATTTGCAGGAGTCCGGCACCGATGCTGACCTGCTTAACGAGTCAGGCGTCACTACGCTGATTCGCCGCGACGGTTTCC
GCTTC t=1 tl TGGGGTAACCGTACCTGCTCTGATGACCCGCTGTTCCTCTTTGAAAACTACACCCGCACCGCGCAGGTCGTGGCCGACA

o GCTGAGGCGCACATGTGGGCGGTGGACAAGCCCATCACTGCAACGCTGATTCGCGACATCGTTGACGGCATCAATGCCA
AATT 'a o CCGTGAGCTGAAAACAAACGGCTATATCGTGGATGCGACCTGCTGGTTCAGCGAAGAATCCAACGATGCGGAAACCCTC
AAG tt n.) un GCCGGAAAACTGTATATCGACTACGACTATACACCGGTGCCTCCTCTCGAAAACCTGACCCTGCGCCAGCGTATTACCG
ATAAA

TACCTGGCAAATCTGGTCACCTCGGTTAACAGCAATTAAGGAGCCTGACCGATGGCAATGCCGCGCAAACTCAAGTTAA
TGAA n.) o n.) CGTCTTTCTGAACGGCTACAGCTATCAGGGCGTTGCAAAGTCCGTCACGCTGCCAAAACTGACCCGTAAGCTCGAAAAC
TATCG
i-J
CGGTGCGGGGATGAACGGCAGCGCACCGGTAGACCTCGGCCTTGATGACGATGCGCTGTCAATGGAGTGGTCGCTCGGT
GGC .6.
.6.
n.) TTCCCGGATTCGGTTATCTGGGAGCTTTACGCCGCAACCGGTGTGGATGCCGTGCCGATTCGTTTTGCAGGCTCTTACC
AGCGC oe GACGATACCGGCGAAACGGTGGCCGTCGAAGTGGTCATGCGTGGACGTCAGAAAGAAATCGACACCGGCGAGGGTAAAC
AG
GGAGAAGACACTGAGTCGAAAATCTCCGTGGTCTGCACCTATTTCCGGCTGACGATGGACGGTAAGGAGCTGGTCGAAA
TTG
ACACCATCAACATGATTGAGAAGGTGAACGGCGTCGATCGGCTGGAGCAACACCGCCGCAATATCGGCCTGTGATTTTC
ATCC
GGTCAGCCTGGCTGGCCGGTTAACCCTGATTCAGAAGTGAGAAAACCATGAACAAAGAAAATGTCATTACCCTGGACAA
TCCG
GTCAAACGTGGTGAGCAGGTTATCGAACAGGTCACGCTGATGAAACCCAGTGCCGGGACGCTACGCGGTGTCAGTCTGG
CTG
CGGTTGCAAACTCCGAAGTCGATGCACTGATTAAGGTGCTGCCGCGCATGACGGCACCGATGCTGACCGAGCAGGAAGT
CGC
P
CGCGCTGGAACTGCCTGACCTTGTGGCGCTGGCCGGTAAGGTGGTCGGTTTTTTGTCGCCGAACTCGGTGCAGTGACGT
TTCC

GAAAAATCTCTCGGTCGATGACCTGATGGCGGATGTGGCAGTGATATTTCACTGGCCGCCATCAGAACTGTATCCCATG
AGCC
o TGACCGAACTCATCACATGGCGCGAAAAGGCGCTCCGGCGAAGCGGAAACACGAATGAGTAACAATGTAAAATTACAGG

1-, .3 TGCTCAGGGCTGTTGACCAGGCATCCCGCCCGTTTAAATCCATCCGCACAGCGAGCAAGTCGCTGTCGGGGGATATCCG

ACACAAAAATCACTGCGCGAGCTGAACGGTCACGCATCCCGTATTGAGGGATTCCGCAAGACCAGTGCACAGCTCGCCG
TGAC
.31 .3 TGGTCATGCACTTGAAAAGGCACGGCAGGAGGCCGAAGCCCTTGCCACACAGTTTAAAAACACCGAACGTCCGACCCGT
GCTC
AGGCGAAAGTCCTGGAATCCGCAAAGCGTGCGGCGGAGGACTTACAGGCGAAATATAACCGCCTGACAGATTCCGTTAA
ACG
CCAGCAGCGGGAACTGGCCGCTGTGGGAATTAATACCCGCAATCTTGCACATGATGAGCAGGGACTGAAAAACCGTATC
AGT
GAAACCACCGCACAGCTTAACCGTCAGCGTGATGCGCTGGTGCGTGTCAGTGCGCAACAGGCAAAACTTAACGCAGTAA
AAC
AGCGTTATCAGGCCGGAAAGGAACTGGCCGGAAATATGGCCTCAGTGGGCGCTGCCGGTGTGGGGATTGCGGCGGCGGG
AA
CGATGGCCGGTGTTAAGCTACTGATGCCCGGTTATGAGTTTGCGCAGAAAAACTCAGAATTACAGGCTGTGATCGGAGT
GGCA
IV
AAAGACTCCGCCGAAATGGCCGCACTCCGCAAGCAGGCGCGCCAGCTCGGCGACAATACCGCCGCCTCGGCAGATGATG
CAG n CCGGTGCGCAGATTATTATTGCGAAAGCCGGTGGGGATGTTGATGCCATTCAGGCGGCAACGCCGGTCACGCTGAACAT
GGC t=1 tl GCTGGCGAACCGTCGCACAATGGAAGAAAACGCCGCCCTGCTGATGGGGATGAAATCCGCCTTTCAGCTTTCAAACGAT

o TCGCTCATATCGGGGATGTTCTCTCCATGACGATGAACAAAACCGCCGCCGATTTTGACGGCATGAGCGATGCGCTGAC
CTAT 'a o GCCGCACCTGTGGCAAAAAATGCCGGTGTCAGCATTGAAGAAACCGCCGCAATGGTCGGGGCGCTGCATGATGCAAAAA
TCA tt n.) un CAGGCTCAATGGCGGGGACGGGAAGCCGTGCCGTGTTAAGCCGCCTGCAGGCACCGACGGGAAAAGCATGGGATGCACT
CA

AAGAGCTTGGAGTGAAAACCTCAGACAGCAAAGGAAACACCCGGCCAATATTTACCATTCTGAAAGAAATGCAGGCCAG
TTTT n.) o n.) GAGAAAAACCGGCTCGGTACTGCCCAGCAGGCTGAATACATGAAAACTATTTTCGGGGAGGAGGCCAGCTCAGCCGCTG
CCG
i-J
TGCTGATGACTGCCGCCTCAACCGGAAAGCTGGACAAACTGACCGCTGCGTTTAAAGCCTCAGACGGGAAGACCGCCGA
GCT .6.
.6.
n.) GGTAAATATCATGCAGGACAACCTAGGCGGTGACTTTAAAGCGTTTCAGTCCGCTTATGAGGCGGTGGGGACTGACCTG
TTTG oe ACCAGCAGGAAGGCGCGCTGCGTAAGCTCACGCAGACGGCCACAAAGTATGTGTTAAAACTCGACGGCTGGATACAGAA
AAA
CAAATCACTGGCGTCAACCATCGGCATCATTGCCGGCGGTGCACTGGCGCTTACTGGCATCATCGGTGCCATTGGCCTC
GTAGC
CTGGCCGGTTATCACCGGCATCAATGCCATCATCGCGGCAGCAGGCGCAATGGGGGCAGTCTTCACGACGGTTGGCAGT
GCT
GTTATGACCGCCATCGGGGCTATTAGCTGGCCGGTTGTGGCCGTGGTGGCTGCCATTGTCGCCGGTGCGTTGCTTATCC
GTAA
ATACTGGGAGCCTGTCAGCGCATTCTTTGGTGGTGTGGTTGAAGGGCTGAAAGCGGCATTTGCGCCGGTGGGGGAACTG
TTC
ACGCCACTTAAACCGGTTTTTGACTGGCTGGGCGAAAAGTTACAGGCCGCGTGGCAGTGGTTTAAAAACCTGATTGCCC
CGGT
P
CAAAGCCACCCAGGACACCCTGAACCGTTGCCGTGACACGGGCGTCATGTTCGGGCAGGCACTGGCTGACGCGTTGATG
CTG

CCGCTTAATGCGTTCAACAAACTGCGCAGTGGTATTGACTGGGTACTGGAAAAACTCGGTGTTATCAACAAAGAGTCAG
ACAC
o ACTTGACCAGACCGCCGCCAGAACTCATACCGCCACGTATGGTACCGGTGACTATATTCCGGCGACCAGCTCTTATGCA

n.) .3 TCAGGCTTATCAGCCGGTCACGGCACCGGCTGGCCGCTCTTATGTAGACCAGAGTAAAAACGAATATCACATCAGCCTG

GGGGGACTGCGCCGGGGACACAGCTTGACCGCCAGTTACAGGATGCGCTCGAAAAATACGAGCGGGATAAACGTGCGCG
CG
.31 .3 CCCGTGCCAGCATGATGCATGACGGTTAAGGAGGTGACGAAAAATGATGCTCGCGTTAGGTATGTTTGTTTTTATGCGC
CAGA
CGCTGCCACACCAGACCATGCAGCGTGAATCAGATTATCGCTGGCCGTCAAATTCCCGTATCGGTAAACGGGATGCCTT
TCAGT
TTCTCGGTGTGGGTGAGGAAAACATCACGCTGGCCGGTGTGCTTTATCCCGAACTGACCGGCGGCAAGCTGACGATGAC
CAC
GCTCAGGCTGATGGCAGAGGAGGGGCGGGCGTGGCCGTTGCTGGATGGCACCGGCATGATTTACGGCATGTATGTCATC
AGC
AGGGTGAGTGAAACAGGGAGTATTTTCTTTGCAGACGGCACACCCCGGAAAATTGATTTTACGCTGTCACTCACCCGCG
TTGA
TGAATCACTGGCCGCGCTTTATGGCGATATCGGTAAACAGGCGGAATCGCTCATCGGTAAGGCCGGCAGTATGGCGACC
AGA
IV
TTCACAGGTATGACGGGGGCGGGATAATGCTGGATGCGCTGACATTTGATGCAGGCAGTACGCTGACGCCGGATTACAT
GCT n GATGCTCGACAGCAGGGATATTACCGGCAATATCAGCGACCGTCTGATGAGCATGACCCTGACGGATAACCGGGGCTTT
GAG t=1 tl GCTGACCAGCTTGATATTGAACTGAACGATGCCGACGGGCAGGTCGGGCTGCCGGTTCGTGGCGCTGTCCTGACGGTGT

o CGGCTGGAAAGGTTTTGCCCTGGTATGCAAAGGGAAATTTACCGTTGATGAGGTTGAACACCGGGGCGCACCGGATGTA
GTC 'a o ACCATCCGCGCCCGGAGTGCAGATTTTCGCGGGACGCTCAATTCCCGCCGGGAAGGCTCCTGGCATGACACCACGCTCG
GTGC tt n.) un GATTGTTAAGGCGATAGCCACCCGTAACAGGCTGGAAGCCAGTGTCGCTCCGTCACTGGCCGGAATAAAAATTCCACAC
ATCG

ACCAGTCGCAGGAGTCTGATGCGAAATTCCTGACCCGTCTTGCAGAACGCAACGGCGGTGAGGTGTCGGTAAAAATGGG
AAA n.) o n.) ACTGTTGTTTCTCAAAGCGGGGCAGGGAGTGACGGCCAGCGGTAAAAAAATCCCGCAGGTCACCATAACCCGCAGCGAC
GGC
i-J
GACCGCCATCATTTTGCGATTGCTGACCGTGGAGCCTACACCGGTGTAACGGCAAAATGGCTACACACTAAAGACCCGA
AGCC .6.
.6.
n.) GCAAAAGCAGAAGGTAAAACTGAAACGCAAAAAGAAAGAGAAACACCTGCGCGCACTGGAGCACCCGAAAGCGAAACCG
GT oe CAGGCAGAAGAAAGCGCCTAAAGTACCGGAAGCGCGTGAAGGTGAATACATGGCCGGTGAGGCTGACAACGTTTTTGCC
CTG
ACCACGGTATATGCCACGAAAGCGCAGGCCATGCGCGCCGCTCAGGCGAAGTGGGATAAACTGCAACGGGGCGTTGCGG
AG
TTCTCTATCAGCCTGGCTACCGGTCGGGCAGATATTTACACGGAAACACCGGTCAAAGTGTCTGGCTTTAAGCGCGTCA
TAGAC
GAGCAGGACTGGACAATCACTAAGGTGACACATTTTCTGAATAATAGCGGCTTCACGACGTCCTTAGAGCTTGAGGTCA
GGCT
TTCTGATGTGGAGTACGAAACAGAAGATGATGAGTGATGTTTTTGTTTTATCTGTTTGTTTTGTAAGGATAAATTAACT
AAAAT
GGCACCATCAACAAAACCGGAAGAGGTGCTCGCGATGTTTCATTGTCCTTTATGCCAGCATGCCGCACATGCGCGTACA
AGTC
P
GCTATATCACTGACACGACAAAAGAGCGTTATCATCAGTGCCAGAACGTGAATTGCAGCGCCACGTTCATCACTTATGA
GTCG

GTACAGCGATACATCGTGAAGCCGGGAGAAGTCCACGCCGTAAGGCCGCACCCGTTGCCATCAGGGCAGCAAATTATGT
GGA
o TGTAATTACAAACAGAAAGCCCCTCAGTCGAGGGGCTTTTTTGTCGATGTGGTCAATGTGTGGACGTGACCAGAAATAA

.3 TTTTATTTCAATTTATTGTACGTAAAAAATAAGCCCGTGTAAGGGAGATTTAGGGTGTCACCAGTAGGGGCTTTCAACG

ATGCGGGTTTGAGCGGCATAAATTACCACTGAAAGCCCTTAAACGTTACTCTACTGTGGACACTGTGTGGACACTCTCG
GCCTC
.31 .3 AGTACCACCTCTTAGCGGATTAAGAGAAATGGCGTCCTGAAGGTACTCTGGCGCAAAATGAGCGTAAACCATAGTTTGC
TCAA
TCCGCGTGTGACCTAGTATCCGTTGTAGCGTGATAATACTTCCTCCATTAATCATGAAATGAGTGGCAAAGCTGTGCCT
TAGTG
CATGTGTGGCTTGCCCCATTGGCAAATCCGGTTTTATTGCTTTCATTGTTCGTCTGAAGCGAGGGTAATCAGCATCAGG
GAATA
AAAAACCTCGTTTGTTATCCGCGATCATTTTGGCAACAGCCTCTGAGATCGGGACGGTGCGTGGTTTGTTTGTTTTCGT
TTTAAC
AAACGTGACGCGGTTATGGATGATATTTTCTGCTTTCAAACGAGCTGCTTCTCCCCAACGTGCTCCTGTACTCAGGCAA
AGAAT
CGCAATCTTTTTATTGTCGCCGTCAAGTGCTGCAAGCAGTAAGGCAATTTCTTCCTGTGTTAGATAGCCTGTTTCTGGT
TTTTCCT
IV
CCTTAAGCCTTTTTGTCCCTCTGATAGGGTGCTCACCAAAGAATAACTCCGCTTCAATCAGGGCTGTAAACATGCCGCT
AATACA n TGTTAAATCACGATTGATACTCGAAGGTTTAATACCCTGACTTCTTCGGGTGGCGCAGTACTGGCTGATAAGCGATTTC
GTAAT t=1 tl TTGAAATGCGCATGGGTCATTCGTTATTTTTGTGAAGATTTCAATTTTTCCAAGATTAGATTTCCCATGCTCTTCGTGT

o TTAAATCCCACCAGATCTGTGTCAGCTCCGACAGACGTCGCTTGTCTGTTGGTTTTGATAGCCATTCTTTATTGTGGTG
GTTGTA 'a o CAACGTGTATTTCTCGAAAGCGACAGCTTCGCTTTTCTTATCAAACTTCCTACGGATGCGTTTTCCATTACGTCCAGTA
GGGCGG tt n.) un ATGTCCACTTCATATCGACCATCATCGAGTTTTTTGATTGCCATCAGAAAACCCTCCGAGTGGTGTGTTTTTTTGCGAC
TACTAAT

CGCTTTTTTCGTGGTGGCTGAAATTTAGCCACCAATAGTAGGCACTTGTGATGAATATATTCACGATGAATTGTTAACC
AGTCTT n.) o n.) TTGACCGGAGTGGGGCGACGTTGTTTCGTTTTGCCCAAAGTGTGCGAGAGCGGGCGCAATTTGCCCGGACTCAGGAGCG
ATC
i-J
TGATTGGTCATGAACCATAAAGTGTATTTGGTGAATTGTGGGGTCTGCAGGATGTTCATCATGACATCTGTTGGAGGTG
TTGA .6.
.6.
n.) ACGACCACTTTCATAGTAACTCAGCGTGCCATACGGAACCCCTGTTAAATCAGCAAGTTGTTGTCTGCTCAAATACTCT
GATTTT oe CGCATTAAGACTATCTTCTCGCTTATCGTGTTTGACATGGTGTTTAGATCTCAATAGTATTTAGTTTAGATGTAGATTG
TTTAGTG
CTTGGATGTGGGCACTAAAAGGCATTATAAGACATTAAACGCAATTCATGAGGGCTAGAGGACGACATGAGCAAGCAAG
TAA
CACTCATGACTGATGCGATTCCTTATCAGGAGTTCGCAAAACTAATAGGAAAATCGACAGGAGCGGTTCGTCGGATGAT
CGAT
AAAGGAAAGCTGCCTGTAATTGATATGACCGATCCACAATCAGCTTCAGGTCGTGCAGGTGAATATTGGGTATACCTTC
CGGC
ATGGAATAACGGACTAAAACTGGCTTATGAAAGCCGCCCTAAAGAGATTCGTGACGGCTGGTTGATGTGGTTAGGTCTC
GGTG
AACCACGTTAAGGAGAACCGTATGAATGAGCCTCGTTGTATTGCTCAGTTACTGCGTAACGAAAGCCCCAGGGCGATTG
ACTT
P
CACCATCACCCACGGTAAGGGGCGTAAGGGAATCATTATCCGCACCAAAAAACAGAGTCCGTTAAAAAAGGCTCTGACC
TTTC

TGAAAAGCCGGAGGGTATGGAAATGACAGTGATGACGCTCAATCTCGTTGAAAAACAGCCAGCAGCTATGCGCCGGATA
ATT
o GGTAAGCATCTTGCCGTTCCTCGCTGGCAGGATACATGTGATTATTATAATCAGATGATGGAGCGCGAACGGTTAACGG

.6.
.3 CTTCCATGCTCAGTTAAAACAGCGTCACGCAACGATGTGTTTTGAAGAAATGAACGACGTCGAACGTGAACGACTGGTA

CAATTGATGAATTGCGTGGTGCATTCTCAAAACGCCGTCAGGTTGGCGCAAGTGAGTATGCATATATTAGTTTTTTAAC
AGTCA
.31 .3 GTCAGCGTCGTACTTTATTTATGCATGCCGGATTGACTGAAAAAGAATTCAACCAGCCATACTGGCGAATTAATGAAGA
GTCAT
GTTACTGGCGTGATGCTTTATTCCGTGCATTACGTGAATTATTCAGTCTGTTTGAGTATGCACCGACAATTCTGACGTC
GGTAAA
ACCGGAGCAATATCTGCATTAAGTAATTAACCAGAGTTTTTAACGCACTTAATTGTGCGGGGCTTCTTTTTGCCTGGAG
AAAGT
TATGCATACAGTTTCTGAAAATCAGTGCGGTATATACGCATTACTGCTGCAACAGGCCAGAACCGAAGCACAGGCCGAC
GCTG
CGACGCGCTTTTCTTCTCATCTTGATGCCATGATTCGCCACATCACAAAGGCGGAGTTATCCCGCGTGGAGATAGTCGA
GCTGC
TCAGTCAGGAGTCGGAAAAATTTCACAATATCGGATTGTCTCGCGGGGAGGTGCTTTGATGTTCTGTTCTCGTGCAGTT
GTATT
IV
ACTGAATAACGCCTTAAAAATCGCCGTTATGAAAAATGGCGATTTGTCTCTTATTCAACTTGGTCTTGATAAAGAAAAA
CGCGA n ,-i AATAACTGAATCTGTTATCGCGATTTATCAGAGTGAATTAAACCTCCTGTCTGATGTGGTCAATTTACTTGTTAAACGC
GCTGTA t=1 tl TTTCACAAGCAAATTTCCTCCGTGGATGAACTGACGAAATTAACGACAGAAATCGCCAGCTATTGCGCTGATGAATTTA

o CTGAACGACAAAAGGAACTGGTAATGCCGGACAACGTAGATTTTATTCAGGAACAACAGGCTGAATTACTGGAGCGCCA
GATT 'a o AACGCGGCAAGGGTAAAACATTGCGGTGCTTCTGCGCTGGTTTGCGAAGAGTGTGACGCGCCAATACCTGCTGCCCGTC
GTGC tt n.) un GGCTTACCCGTCAGCCACGCGTTGTGTTTCCTGTCAGTCAGTCTTTGAAGCAAAAAACAAACATTACCGGAGAACGGCA
TGAG

TATTCGTATTGAAATTGGCGAACGTTATGTCGTTACCAGTGACAGCTTTCAGTTTATTCTCCACGAGAAAAAGAGAGCG
GAAAG n.) o n.) CGGTAAAAACGCCGGTCAGGAATGGCTGGCGGTGGTTGGTTATTACCCGAAATTAAGCCAGCTCGTTTCAGGCCTGATG
CATC
i-J
ACGATATTCTGACCGGAAGCGCAAAGTCTTTTGCTGATTTAAACGTGCAGGTTGAGCAACTCAGCAAGCGTTGTTCAGA
GGCT .6.
.6.
n.) TTTGGCTCATATGGCCGTTAAAGCCTCCGGGCGTTTTGTCCCTCCGTCAGCATTTGCCGCAGGCACCGGTAAGATGTTT
ACCGG oe TGCTTATGCATGGAACGCGCCACGCGAGGCCGTCGGGCGCGAAAGACCCCTTACACGTGACGAGATGCGTCAGATGCAA
GGT
GTTTTATCCACGATTAACCGCCTGCCTTACTTTTTGCGCTCGCTGTTTACTTCACGCTATGACTACATCCGGCGCAATA
AAAGCCC
GGTGCACGGGTTTTATTTCCTCACATCCACTTTTCAGCGTCGTTTATGGCCGCGCATTGAGCGTGTGAATCAGCGCCAT
GAAAT
GAACACCGACGCGTCGTTGCTGTTTCTGGCAGAGCGTGACCACTATGCGCGCCTGCCGGGAATGAATGACAAGGAGCTG
AAA
AAGTTTGCCGCCCGTATCTCATCGCAGCTTTTCATGATGTATGAGGAACTCAGCGATGCCTGGGTGGATGCACATGGCG
AAAA
AGAATCGCTGTTTACGGATGAGGCGCAGGCTCACCTCTATGGTCATGTTGCTGGCGCTGCACGTGCTTTCAATATTTCC
CCGCT
P
TTACTGGAAAAAATACCGTAAAGGACAGATGACCACGAGGCAGGCATATTCTGCCATTGCCCGTCTGTTTAACGATGAG
TGGT

GGACTCATCAGCTCAAAGGCCAGCGTATGCGCTGGCATGAGACGTTACTGATTGCTGTCGGGGAGGTGAATAAAGACCG
TTCT
o CCTTATGCCAGTAAACATGCCATTCGTGATGTGCGTGCACGCCGCCAAGCAAATCTGGAATTTCTTAAATCGTGTGACC

un .3 AACAGGGAAACCGGCGAGCGCATCGACCTTATCAGTAAGGTGATGGGCAGTATTTCTAATCCTGAAATTCGCCGGATGG

GATGAACACCATTGCCGGTATTGAGCGTTACGCCGCCGCAGAGGGTGATGTGGGGATGTTTATCACGCTTACCGCGCCT
TCAA
.31 .3 AGTATCACCCGACACGTCAGGTCGGAAAAGGCGAAAGTAAAACCGTCCAGCTAAATCACGGCTGGAACGATGAGGCATT
TAA
TCCAAAGGATGCGCAGCGTTATCTCTGCCATATCTGGAGCCTGATGCGCACGGCATTCAAAGATAATGATTTACAGGTC
TACG
GTTTGCGTGTCGTCGAGCCACACCACGACGGAACGCCGCACTGGCATATGATGCTTTTTTGTAATCCACGCCAGCGTAA
CCAGA
TTATCGAAATCATGCGTCGCTATGCGCTCAAAGAGGATGGCGACGAAAGAGGAGCCGCGCGAAACCGTTTTCAGGCAAA
ACA
CCTTAACCAGGGCGGTGCTGCGGGGTATATCGCGAAATACATCTCAAAAAACATCGATGGCTATGCACTGGATGGTCAG
CTCG
ATAACGATACCGGCAGACCGCTGAAAGACACTGCTGCGGCTGTTACCGCATGGGCGTCAACGTGGCGCATCCCACAATT
TAAA
IV
ACGGTTGGTCTGCCGACAATGGGGGCTTACCGTGAACTACGCAAATTGCCTCGCGGCGTCAGCATTGCTGATGAGTTTG
ACGA n GCGCGTCGAGGCTGCACGCGCCGCCGCAGACAGTGGTGATTTTGCGTTGTATATCAGCGCGCAGGGTGGGGCAAATGTC
CCG t=1 tl CGCGATTGTCAGACTGTCAGGGTCGCCCGTAGTCCGTCGGATGAGGTTAACGAGTACGAGGAAGAAGTCGAGAGAGTGG

o GGCATTTACGCGCCGCATCTCGGCGCGCGTCATATTCATATCACCAGAACGACGGACTGGCGCATTGTGCCGAAAGTTC
CGGT 'a o CGTTGAGCCTCTGACTTTAAAAAGCGGCATCGCCGCGCCTCGGAGTCCTGTCAATAACTGTGGAAAGCTCACCGGTGGT
GATA tt n.) un CTTCGTTACCGGCTCCCACACCTTCTGAGCACGCCGCAGCAGTGCTTAATCTGGTTGATGACGGTGTTATTGAATGGAA
TGAAC

CGGAGGTCGTGAGGGCGCTCAGGGGCGCATTAAAATACGACATGAGAACGCCAAACCGTCAGCAAAGAAACGGAAGCCC
GT n.) o n.) TAAAACCGCATGAAATTGCACCATCTGCCAGACTGACCAGGTCTGAACGATTGCAGATCACCCGTATCCGCGTTGACCT
TGCTC
i-J
AGAACGGTATCAGGCCTCAGCGATGGGAACTTGAGGCGCTGGCGCGTGGAGCAACCGTAAATTATGACGGGAAAAAATT
CAC .6.
.6.
n.) GTATCCGGTCGCTGATGAGTGGCCGGGATTCTCAACAGTAATGGAGTGGACATGATGGCAAAAATTCACGAGGTAAAGC
TGC oe ACGCAAAATATTTCGACCTTGTACTGGAAGGAAAGAAACGCGCAGAGTTTCGGAAAAAAGATCGTAATTATGAGCGCGG
GGA
CACGTTGATTTTGCATGAATGGGTGCAGGGTGTGTTTACGGGGCGAAAGGTTGAAGCCCGGATAACAGATGTTACTGAC
CTGT
CAGACTGGCTGGAAGATTATGTCTTGCTGCACAGAATACTAGGTGAGATACTGCCGCTGACTGTCAAGCTGGCAGTATC
CTCC
ACATTTTTTATAAATCTAAGGCGAAGAACGGTGGCAATATATTTGCTTTAAATTGTAAATAAGCAAATTATTCTAAGCC
ACAAAT
GATATCAATTGAGTATCGATTTTCGTTCGAATATAACCATAACGCCAAGCGTCAAATGTAAGTCATCTATATGTGATTC
GTATTC
ATATTGGTCAGCATCAGGAGATAGTAAATGCTCTGCTGCTGTAAAAACATCAATTGTAACAGATATATCGTCTGGAATT
TCTTC
P
ATTTAATTCGGATATGTATTTTTTATATTTGTTTTTTAATTTCCTTATCATGTCTTCCTTATGGTGGATTGTAGTGCAG
TAATTTTTT

TTAGTTGAATCATTGTATGAATGGAATGAACAGTTTAATGCTAGGCCAAGACTTGAATTTCCTGTAGGGCCTGTGTGTT
TAAAG
o TATTCAAGATTTCTTCTTATGTCAGAAAATAAAAGACTTTTACAGGGGGCATTTCCTTTAACTTCTATTGCACATATTG

o .3 GAAAAATGGTCTGCTGTCCAATATGGCAATATCAATTCTGCCTGATCTTGTTGTGTTTGTGTTTTTTCTGACAGTTTTT

ATATTTTTTGGGGGTCAGACCGTTTCGATAAAGGAACTGTTGCATTAATGAATTTATTTGTATGGTACTCAAATATAAC
TTTGTA
.31 .3 ATCACCATGGCGAAAAGATTTTATTTCTAACAACGATAGACCTATAGATACGGTAGCTACATATTCAGTGTTTATTTTT
GCACCA
GACCTGAAATCCATTAAATAAGTTGCGTCGGCTGACTTTTTCATTCCATCTTTTACAGCTTTCACAATATCCATGTTAT
TCATATC
AACACCTTTTATATTCCAGAATTATAAATCTTAATGATTGATTTGTTTGTGTGTTTAGGTTACTAGATTGACGTACTTA
TAGCACA
TTCTGATTTTGAAATCACCATCCTTGGGTAAAGCACAGCCTATCACTATTGGTTTAGTACTGTGCTGTAATAGTATCAG
ATTAAG
TTAAATCCATTTTATGAAATCTTCCATTTCATCATATTTTTTTATTACTGCTCTAACATCTTTGCTTTCATCACATAAA
CAATCTAAA
AGACTATCCCATTCAGTTTGTTTTTTACCAAATCCATAAACAGCCTCTATATCACCGGATTTCCATATATAGATTCCGT
TATCTTTC
IV
AATTTTTGATGTATTTCATGTATATGCTTTTGTGTTTTTTCATGGTTACATATTTTGATGAAGTCTTTGCTTGAAATGC
TTTTGAAT n ,-i GACTCAAAAGTAGTAACTTTTGTATTTAAACTAAGCTCTCCTGAGTTGATTGATTCAATCAAAGCAGTCAATAGATTAT
CACATT t=1 tl CAGTACTAAGTAAGTCTTTATGCTCTGTTAATAGAATATTTGATAAGAAATCACAATCAGCTAGAATCCTTGTTTTTAT

o GGCATTGATGATTTGTGACATCTTAAATAAACTACCCTTACCGTCAACGGCAACAATGCAGATTTTACTCGGGTTGAGT
TCATGT 'a o CCGTTAATTTTTTTATAAAGTGCATATAGAACGTTTGTCTCTGTTTTCCCTTCAACAAGCAAAACTTCTTCAGAAAATA
AAAGGTA tt n.) un TGATGAATTAGAAAGCGTGAATGCTGAGTGCAATTGCGGTGATGAAGATTTATATAATTCTTCGATTTTTTCAGATATA
GTCTTC

CTTGCTATGGTTCCATTAGAATCCTTACAAACCTGAATCGCATTTGCTGCATGCTTTGCAGAAAGCATACTGGCTGAGT
GAGTT w o w GATATTATAACCTGATACCCTGATTCACTTAATGTGACAAGTGATTCTCTGACAGAATTAATG
GCTGAAGGGTGTAAATATAAC
i-J
TCAG GTTCATCAATG AAAATCAAAGTGTTTG ATTTTTTTG ATTC G CTGTTTTCTTTTTTTATTTC G GC
CAG GTATTG AATTAATG C
4,.
w CATTTG AATG G AAC GTTGTGTTC CGTG ACCAAATC G G CTG ATATCTCTCATTACCG GTTCATCTTCC
CG AG ACTCAAAAACTTTC oe AG AGTG C CG G ATTTAAATATCTCATCTAATGTCG GTGTG G G AAAGTGTAACTTTACACTTAC GTCAGG
AAAAAATTG GTTTACT
TTTTTATTTACACCTG AGTCTATTTTATTAAG G CTTTCTAATCTGTTCTCACC GTTGTG AG AAAG
ATATTTAC CTATTTCTGATATG
TTTTTTG AAAATTTTTCTTCGTGTTCTTGTTTTATTTCAG AAACAATTG C G G AAAGTATCTTTC
CTATTGTG GTCGTGTTTTTG CAT
TTTGTTG AGTCTTCG ACAG CGTCAG ACATTG CAG G G ATATG AATTG GTTC CG G AAATATATTG G
AG ATTG CAC CATCTATG C CG
CCAGGGTTTTTCTTCCACGTGGTACCGTCATATACATCCAGACTTTTTTTGGCTTTTCCTGTTTCCTTATTAAATTCCT
GTCTTCTT
G CAAAG GTAAG AGTCC CGTCAATTATAAAC G G AG CTATTTTTTGTTG ATTCTCTTCTGTTAACAAAG
ACAG AGTATCATCTGTTA
P
TACCTTG AATAACG C CTTCAACG G ATACG G G GTGTGTAG G ATCGTACACATCTG ATTCTG
AAATCAAAG AG C CATCTAACAG C C .
, ACTTAATTGCTAAGATAATATTTGATTTTCCTGCGTTATTATAACCAACTAAAGCAGTGAAGGGGCGCAAAATAGCCGA
TGTTG
, 1¨
.3 o ACTTACAAG AACG AAAGTTG CTAATTG
AAACTG AAG CAAG AC GTACAGTCATTATTTTTC CTTAAATGTG CTATTTGTATG CAAT .

G AGTTCATACG AAACG CTTTTTTACATTTTATAGTCGTTGCATTCAAG G GTG CATG AG ATTG CATTAAG
G G AAACTGTG ATATG "
, , , GCTTGGCTTTTGACTGGAAATACTGATGGCTCATTAGTTTTATTAAGGTGCATTAAAACCG CC CC GTG AAGCG

, N, N, GCGGGGAAAGCAC
20 Native P4 (acc nr:
TTTGCCGCTGCCGCCGGGACCGGTCACCTCCAGAAAGAGCTGCCAGTCGTAGCGGTTTGCCAGCACCATAAACAGTGCA
GCCA
X51522) GAATCACGTCGCGTTTTTCCGCACGGCCACCGGCGGCACGGTCAAGCCAGCGCCAGAAGGCGGGGGCGTGGGTTTCCAG
CGT
TTCACCGTCCACCGGCGGGGTGAAATCCACATCGCACAGGGTGCGCATCCAGTGTGACGGACTGTGCGGGTGGAACGTG
CCG
TTCTGCGTGTCGAGCACGCCGTTACGAAAGCCAATCAGGCGGCGGGAGGGGGCTTCCTGCTGCGGAATAATCAGCTTCA
GGG
1-d TATCCACCACGGAGGCCACCTTCCCGGAGGAGAACGGCGCACGCAGACGCTGAAACAGCCCGGCCACATCCCGGGCAAA
GTC n CTGTGGCGGCAGCACCTTCCAGACACCATTTTCATAGCGGGACAGAAGCTGGCCGTTGGCATCGACCGCGAGCGCCTCG
CCGT t=1 1-d AATGCTCATAGATACGCATGGCCTTTTCGCTGGTACTCATGGCGGAAAACTCCGCTTCGCTCATGGTGTCGAACGGGCT
TTCAG w o w CCGGTGGCCGGATGGCATCGTAAATGGCCTTACGGGTGGCCTCCCCGCCGTACTGCGTGAAGGCATCATTCCAGTCACC
GAAG =
'a ACCGGCGGCAGGGCAACAACACCTTCACACGCATCTGCGGCTGCGGCGGCTTTTTTCTGGCCGTCACCACTGAGGTCAC
GGTC o 4,.
w w vi AGCGGCAAGGACAATCTGACAGGCGGGGTGCTTCTGCCGGGCAAGGCTGGCCAGAGAAAGGAGGTTCACGGAAGAAAGC
G

CCACCATCACCGTTTCACCGGTCAGGTGATGTACGGTAAGTGCGGTCGCGTATCCCTCCGCTATCCACAGACGTTTTCC
GGCCT n.) o n.) GATTCTGTCCTTCAAGGGTGTGACAGGTGCCCCTGACCTGTCCGCCTTTCAGGGTGCGCTTACGGCCGTCAGCACTGAT
TAACT
i-J
GAAGGTTAACCAGTTCGCCGCTGTCGTCATACAGTGGCACCACAAGGTCACCGGCGCGCCAGCTCACGCCACCGGCTCT
GTGT .6.
.6.
n.) GTGCCGGTCAGCATCCGGCATTCCCGGCCGGGAAAGCCCTTGCGGGTCAGGTAGGCGTTACCGGTTCCGGTACGGGTTT
TCGC oe CATCAGGGTTTGTGCCAGTGCGGCGGCGTTCTTCCGGGCAGCGTCTGTTTCATCAACGGCGGCGGTCGTCACTGCCGGG
TCAG
CCGGTGGCAGGCTGCCGGTCACGGCAGCCACCTTTGCGGCCGCGTCGGACGGGGAAACACCAAAAACCTTTTCAACCAG
TTTC
AGGCCGTCACCGGCACCACACTGATTGCAGTACCAGGTGCCGCGCCCCTCCCTGTCATCAAAACGGAAGCGGTCACTCC
CGCC
ACAGACCGGACAGGGCTGATGACGGTTCTTCAGCACCTGAATCCCCAGCGCCGGGAGAATACGCGGCCAGTGGCCGAGC
GCA
TGGCTGACGGTGGCGGTTACGTTCATTTTCATGGTGTTGTTCTCCTTCAGTGCAGTACCGGCGCTTTTATGTGACGGGC
ACAGA
GTTCATCCATCACAACCAGCCCGAGAAAGGACAGCGACGGCGCGGCCTTCAGGGGGCCGGATTCCATTAAATCTTCCAG
CAGG
P
GCACAGGCTATCTGACGCCCTTTTTCCTCACCGTGCTGGCGCAGATAAAAGCCTTCCAGCTCAGCGGCGATGGCCGCCT
CCAGT

GACTCAAGGGTGAGATGCGGGTAGCGGTGCTGACGTTCGCACACGGTCAGCCAGGCACAGGCGACAGCGCGACGGTAAA
GG
o GCAGCGCGTAAGACGGGCGGTAAGGGTGTTTTCATTTGCTTTTCTCCCTGTGACAGATGACTGCATTCCGTGCCGGTTG

oe ACTGATAAGGCATATCTGCGTCTCCTGAAGACGTGCGTATCCCTGCGCGAATACGCACATTTAATTTTTCGGGGGTCGT

ATTACAGATAATTGCGGTAACTGTTATCCGGGGTGGTTTCCGGGTCAGGCTCCGTGCGGGGAATTTCCCGCCATTCCCG
CGCCA
.31 .3 CCGGTGCTGCCCGGCTGACCGGAACAGTGTCCTGCGGGTAAATATCCAGATATTTTTCCCGCCATTTCTGTAATTCCGG
GTCTC
CGGCCATTTCTTTCAGTACCGCATGCCGGTTTACGGGGCTGCGTTTAAACAGGTCAGGACGGTCACAGGTAAATTCCCG
CAGA
AAACGCCCCAGCGGGATGTCTGTGGTGCGTCCGTCAGCGAGGATACGCACAAGGATACTGAATTTACGGCGGTACGGGT
TCC
AGACAATGTCCGGGCAGCGGTACGGCATTTCCCACGGAATACCGTCTTCCAGAATGCCGACCACGGCCACATCGGGAAA
ACCG
GCAGAACGGTAAATCTCACCGGGCTGGGGAAAATCAAACATGCGTCCTGTCTCCCCGGTCTTTCTGCTGGGCGAGAAAA
TCGC
GGCACAGGCCTTTGGCTTTCAGCTCATTCAGCACAAAATCAATATCTTCATTCAGGTAGCTGAAAATATGTGGAATGTA
GAGCT
IV
GATGCAGGCCGGAGAGTTCACGGTGAATCAAATCACCCCCAACAAACCGGGATACGGCGCTGGCGCGGTTGAGCTTATG
GTA n AGCCTCAATGCTGAGGTGTTCACGGGCGTCATGACGCGCTGAGACGGTCTGAGGGGCTTTTTTATTACGCACGGGACAC
CTCC t=1 tl ACCACCGGCAGACGGGCAGCAAGGGAGAGCACATAGTCACGGACAAGGGAACGGCGGGCACTGCGTTCATCACCGGCGA

o GTGCGAAGCATACAGATACGGGGATGACGGTCTGCGCGACGGACAGCCGCAAACACAAAGACAAATTCAGGGTGTGAGG
GG 'a o GTAAGGGTTGTAGCCATGATGGCAGCCTCCTGTGAATAGCAAATAACGCTATCGCCGGAGTTCTCACGCTCGATGGCGA
TAGC tt n.) un CCAGACGGGGGTGAGAATACCGGCTTCACAGGATACCGGCCAGCCCGGAGGCTGCCCCGCCTGAGCTACCATTGACTCT
GCG

GCATAATGAGCGGACGCGGGCAGGATGCACGGAATGCCATCTGCACGACTGACCACACACCACACCATAATCTGGCGCT
CTGT n.) o n.) GGCATTGATTGCGACACAAAAAAAGACGCGTGGCGCGTCATATGTCGCCTGTGAATTGCTCGGGTTCTCACGCCCGGCT
GCCG
i-J
ATTTTGCGGCAGGCGAAAAACTATATCCGCAAATGCCGGAAAAAGGCAAGCCAGAAAAAGGGAGTTTTTGCAGAGCGGG
CAT .6.
.6.
n.) CATCATGCGTCGTACCCCCGTTTGCGTCCGGCAATGCGTCCGGCCATCCATGCGGTGACTTCAGAGTGCAGCCAGGCCA
CATTT oe TTACCGCCAAGACTCACCTGCGGCGGAAATTCCCCCTTACGGATGAGTTCGTAGATGGTCGAGCGTGACAGGCCGCACA
GGTG
CATCACTTCCGGCACACGTAAAAAACGCTCCTGCGTGATGTCCGGCAGCGGCATCAGTGGCGTCACTGGGGCGGGAGAC
GGG
GAAGAAAAAACAGCTTGCATCGGGCTACCTCGTTAATGTCCATACAGCACCGGATAAGTCCGTCCGGCTTCGGGTAGCG
CTTT
ATTTTGTGAATATTTTCAGCAGACGCAACAGGGGGGATTTGTTCAGGCTGTCTTACAATGGCTGTGTGTTTTTTGTTCA
TCTCCA
CTTAAAGTCATTTAAAGCCACTTAAAGCAATTTGTAATTTTTATAGTGAAATACAAATCGTTTCTTCTTATTCATTCCC
GGCGAAT
TAATAAAAACAAACAGTAGTAAACAGCACAAAAAGCCCATCAACGGGTGAACAGTGGTGAACAGACGGTGAACAGTCAT
TAC
P
TGCGATTGTTCACCCTTTAACTTACTGTATTACTTATCTTTTTTATTAAGGTGAACAGAGGTGAACAGTAAAATATAAA
AAAACA

AACAGTAAGCCGGTTTTTCCTGCGACCTTTTCCTGGCTTGCCGGTCTGAGGATGAGTCTCCTGTGTCAGGGCTGGCACA
TCTGC
o AATGCGTCGTGTTGTTGTCCGGTGTACGTCACAATTTTCTTAACCTGAAGTGACGAGGAGCCGGAAAATGTCTGACCAC

o .3 CCCTGAATATCTGCAACCCGCACTGGCACAACTGGAAAAGGCCAGAGCCGCCCATCTTGAGAACGCCCGCCTGATGGAT

CCGTCACGGCCATTGAACGGGCAGAGCAGGAAAAAAATGCGCTGGCGCAGGCCGACGGAAACGACGCTGACGACTGGCG
CA
.31 .3 CGGCCTTTCGTGCAGCCGGTGGTGTCCTGAGCGACGAGCTGAAACAGCGCCACATTGAGCGCGTGGCACGCCGGGAGCT
GGT
ACAGGAATATGACAATCTGGCCGTGGTGCTGAATTTCGAACGTGAACGCCTGAAAGGGGCGTGTGACAGCACGGCCACC
GCC
TACCGGAAGGCACATCATCACCTTCTGAGTCTGTATGCAGAGCATGAGCTGGAACACGCCCTGAATGAAACCTGTGAGG
CGCT
TGTCCGGGCAATGCATCTGAGCATTCTGGTACAGGAAAATCCGCTCGCCAACACCACCGGCCATCAGGGCTACGTCGCA
CCGG
AAAAGGCTGTCATGCAGCAGGTGAAATCATCGCTGGAACAGAAAATTAAACAGATGCAAATCAGCCTCACCGGCGAGCC
GGT
TCTCCGGCTGACCGGACTGTCAGCGGCAACACTCCCGCACATGGATTATGAGGTGGCAGGCACACCGGCACAGCGCAAG
GTG
IV
TGGCAGGACAAAATAGACCAGCAGGGAGCAGAGCTTAAGGCCAGAGGGCTGCTGTCATGATTTACTGTCCGTCGTGTGG
ACA n TGTTGCTCACACCCGTCGCGCACATTTCATGGACGATGGCACCAAGATAATGATTGCACAGTGCCGGAATATTTATTGC
TCTGC t=1 tl GACATTTGAAGCGAGTGAAAGCTTTTTCTCTGACAGTAAAGATTCAGGAATGGAATACATTTCAGGCAAACAGAGATAC

o ATTCACTGACGTCAGCCTCCTGCGGTATGAAACGCCCGAAAAGAATGCTTGTTACCGGATATTGTTGTCGGAGATGTAA
AGGC 'a o CTTGCACTGTCAAGAACATCGCGGCGTCTGTCTCAGGAAGTCACCGAGCGTTTTTATGTGTGCACGGATCCGGGCTGTG
GTCT tt n.) un GGTGTTTAAAACGCTTCAGACCATCAACCGCTTCATTGTCCGCCCGGTCACGCCGGACGAACTGGCAGAACGCCTGCAT
GAAA

AACAGGAACTGCCGCCAGTACGGTTAAAAACACAATCATATTCGCTGCGTCTGGAATGAGGGCTGCCGGTTAACACCGG
CCGT n.) o n.) CGCCGCACACCGTATTTTTATTCTTCAGCATGATGAGAAAGAGATAACGATGGAAAGCACAGCCTTACAGCAGGCCTTT
GACAC
i-J
CTGTCAGAATAACAAAGCAGCATGGCTGCAACGCAAAAATGAGCTGGCAGCGGCCGAACAGGAATATCTGCGGCTTCTG
TCA .6.
.6.
n.) GGAGAAGGCAGAAACGTCAGTCGCCTGGACGAATTACGCAATATTATCGAAGTCAGAAAATGGCAGGTGAATCAGGCCG
CCG oe GTCGTTATATTCGTTCGCATGAAGCCGTTCAGCACATCAGCATCCGCGACCGGCTGAATGATTTTATGCAGCAGCACGG
CACAG
CACTGGCGGCCGCACTGGCACCGGAGCTGATGGGCTACAGTGAGCTGACGGCCATTGCCCGAAACTGTGCCATACAGCG
TGC
CACAGATGCCCTGCGTGAAGCCCTTCTGTCCTGGCTTGCGAAGGGTGAAAAAATTAATTATTCCGCACAGGATAGCGAC
ATTTT
AACGACCATCGGATTCAGGCCTGACGTGGCTTCGGTGGATGACAGCCGTGAAAAATTCACCCCTGCGCAGAACATGATT
TTTT
CGCGTAAAAGTGCGCAACTGGCATCACGTCAGTCAGTGTAAAATTCCCCGAAAATCCGCCCGTTTTTACTGAAAAAAGC
CATGC
ATCGATAAGGTGCATGGCTTTGCATGCGTTTTCCTGCCTCATTTTCTGCAAACCGCGCCATTCCCGGCGCGGTCTGAGC
GTGTC
P
AGTGCAACTGCATTAAAACCGCCCCGCAAAGCGGGCGGGCGAGGCGGGGAAAGCACCGCGCGCAAACCGACAAGTTAGT
TA

ATTATTTGTGTAGTCAAAGTGCCTTCAGTACATACCTCGTTAATACATTGGAGCATAATGAAGAAAATCTATGGCCTAT
GGTCC
--.1 AAAACTGTCTTTTTTGATGGCACTATCCTGAAAAATATGCAAAAAATAGATTGATGTAAGGTGGTTCTTGTCAGTGTCG

o .3 TCCTTAAGAATTCGTGGCATGAGAGAGTTAAAGGATGCTGAATCATGTATATGGATTAATTGGCGTTGTTGGGACCATA

CTATTGCTACTAGCTTCATCCGCAACCAAGATATTTCACAATGGTTGCTCGTTTGTTCTGGATGGTTAGCTGCTTTACT
TATTGGA
.31 .3 TGGTTTACACACAGAACGATTAAAGCAATTAGCAACAACCACACGAATGTTATAAAAAGTAATATGGAGGTAATCAACA
GTCA
TAATGAATCCAATCAAAACTTGATGGCTGAAAACAAAGAATTGATTAAAGAACTTGCAAGAACTTCAGAACAGAAAGAG
AAGA
TGGAAAGTATAGCTGCTTATCTTGCTACTCAGAACCCCCAAATTAACGCTATGCCAAGAACAGCAAGCCGACCAGAAAA
TATCG
ATTCGGAGGCTAATTAAATATGAAAGTATATTTTGAAAATTACTCATACTATCCAGCCCTAAGAACACGATCAGCAGAA
ATGAC
CGGACTGAATAATTTATCATATGAGAATAAGAAAAAAATATTACCTTTAATTTCTTTGGGGAAATGGCCTCGCTCGGAA
GAAAT
ACAGGTATCGTTAGATAAAAGTTTAGAAGTGATGAGTAATCTCCCATTTATTTTGGATGTCACAAAGGATAATTCTCAT
CATTGC
IV
GCGTCAAGCTTCGAACTACTCTCTCCTGAGAATGGTTTTAAAAACTGGATAGAGTTTTGTTCCAGAAATGATAATATTA
TTCCAG n ,-i TAGTCCAGATGCCGGATTCAGCAAAACTTAGAGATATATCTATCCAAGCCAGGGTTTTAGAGGAACTTAAAGGTTCAAT
TGCAT t=1 tl TTAGAATTAGAAATCTAAATACAGACATTAATAAAACCCTGACGTCACTAGTATCGATGAACTCTCCAGAAAATGCCAT

o CATAGATTTGGGCTACATTAGAGGGAATGTATCTGCTATAACAGCTGCAGCAATAAATTCGATTAATCAGATTAGAACT
GAGAT 'a o TCCTGAAGCCATAATTAGTGTGCTAGCAACAAGCTTCCCTAGTTCAGTTACAAATTTTTGCCGTGAAAATGGACAGTCC
GGATA tt n.) un TATTGATGTTATTGAGAGAGAATTGCATCAAAATATTGGTGGAAGTGATGTCGCTATTTATGGCGATCATGGTTCAATT
CATTC

AGTAGTGTATGACAACATAATTGGGCGATATGTTCCTAGAATTGATATAGCGTTAAATGATTCATGGTATTTCGAGCGC
CGCCC n.) o n.) TGGGATGAATAAAGAAGGGTTCATTGAAGCTGCGAAATCAATTTTGGCAGAGTATCCACATTATCAAAGGGAGGACTCT
TGG
i-J
GGAGCTGCAATGATAAGAAATGCTGCTATAGGGGATATAGCAGGTATGGGAAGTCCAGCTAAATGGATTGCTGTTCGAG
TGA .6.
.6.
n.) ATCTACACTTAAATAAGCAAATTGAGCTATCTGAAGCTCTACAGTATGGCTTTGATCATGATGAGGAAGATCTTATCTA
ATAAA oe AAACTAGGGAAGGTTAGCCTTCCCTAGCCCACAACCCTTTTAAGTACTTTTTCCGGAATATAGGAAACATCATCGCCAT
ATTTCC
TAACTATAGAGTCAACATCAACCTTATGGTATTCATTTAAAGATTTCTCCGAACAAGCCTTAAACTCAAACTTAACCAA
AGAATT
ATTCCTTCTTTTAGCTTTGCTCAATAAGGCAAGATCGTTTATAGATATTTCTTGATTACCATGGAGTCTAGATAAAAAA
TCATTAG
ATGTCGCATGATACCTATTTTTAATAGATGTAATGACAAATTCTCTAATCTTGTTTAAAGATAACTTAGATGCCAAAAC
CTCAAG
ACAATCCCTATGTAGTTCATTTGAAAATGTAATTCCATACTGATTAAGCAGATCTTTAAGCTCAGACTTAAGCAAAAAA
CCCAAA
TAAACAAATTTATTTAAGCGAGAGCAAACCCTTCCTTTTTGAATTATTTTAAAGCGTACGCTACTATTATTAGATTGAA
ATTCTAC
P
AACGCCAACATCTGATGAAACCCGTTTAGATACTTCACTGGTAAATTTTGGCGCGCACACAATTGTAACTTTTTCGAAG
CTAGAT

TTAAAAATTTTTAACTGACCATCCAAGCGCTTAAGAGAGTCAAAATCGCTTTTTATTTCAAAAGCTTGTAGGCTTCCAT
TGGCAA
--.1 TAGCCAAATCAGCTCTTCTGCTCCAGTTAGCAACAACCATTTCATTAATGATCGTTGCATCACTAAGCATGCCTTTTTT

1-, .3 CATTCAATTAGGGCTATTTTAACTTCCTGCTCTCGCAATAACTCAGCCATGTTAATCACACTCACTTCACGTTGATTTT

GCAACTAACAATTAAAAACTCCAGATTATGTTACTTAACACTCATTTTAAAAATCAACCAATAACTATTTTTATTTAAG
TATTTCTT
.31 .3 TGTATTTTGAATAATCATAAGGCTAATATATTTATTACGGTTCATATCCAAATAATCAGCCCACCACTGCATCATTAAA
CGCCGC
TCATCCAAATGCTCGGAGGTATGGATATATGCCGCACGTACATTATTACGCTCTGAGTGGCTCAGTTGCCTCTCTATCG
CGTCAT
CACTCCATAACCCCGACTCCCCCAACGCACCACGCGCCATAGTCCTAAACCCATGCCCACACACCTCGGTTTTAGTATC
ATAGCC
CATCGCACGCAATGCGCTGTTTACCGTGTTTTCACTCATAACCTTAGTTGCGTCATGATCCCCCGGAAAAAGCAGCTCT
TTATCA
CCACTAATCTGCTTTAACTGGTTTAATAAAATCATCGCCTGTCGACTAAGCGGAACGATATGTTCCTCTTTCATCTTCA
TGCCTCG
GTACGAATAACGCACACCTTTAATTTCTTCTCGCTTTGCAGGTATACGCCAAAGAGATTTATCGAAGTCGAATTCATCC
CAACGT
IV
GCGAAACGTAATTCACTGGAACGCACAAAAGTTAGCAAGGAAAGCTTGACCGCAATCCGTGTCATTACACGGCCACGAT
ATGC n ,-i AGCAAGACGTGCAAGAAACTCAGGGAATCGGCTAGAAGGTAAAGCAGGGTAATGTCGCGCTTTGGTTGTCGATAGCGCA
CCA t=1 tl GCCATATCGCTGGCTGGATTTGAGTCGATGTAATCGTTCTGTACGGCATAACGCATAATGGCTGTGACGCGCTGTTGCA

o CTGAGCGACATCGTGTTTGCCACTGGCATCAACTTTTTTAATCGGGGCTAACAGGTGGCTGGTTTTAAGCTGCCGAATG
TCGGA 'a o CGAACCGATATGAGGGAATATATAAAGCTCAAGATAGCGAAGAACGCGCGATCGATGGTCTTCACTCCAGCGCTTGTTA
CTGG tt n.) un CATGCCATTCTCGGGCGATAGCTTCAAAAGTATATGCCCCCGAATTCTCGGCCCGAGCTTCTTTTTGTACGACTTTTGG
GTCTAT

GCCCTGTACTAAAAGCTTTTTAGCTTCATCGCGTTTTGCTCTTGCCTGAGCAAGCGTCACAGTAGGCCAAACACCAAAC
GCAAG w o w ACGATCCTCTTTTTTGTCAGAGGGACGTCTGTATTTCATGCGCCAGTATTTGGAACCTTTGGCCGAAACCTCAAGATAC
AAACCA
i-J
CCACCATCGGCCATTTTGTAGGTTTTGTCTTTTGGCTTTGCGGTCTCGACCTGTCTGGCGTTGAGCTTCATTTGGGGGC
ACATTT
4,.
w CTAATCGAAGTTAAGATGCCCCCAATTATGCCCCCAATGATATCCGGATTTCAACGGACAACCTCGGAAGACGCAGGAC
GAAA oe AAATCGCTGCAAGCATTGATTTTAAAGGGATATTTGGACTTTCTCGGATGGTCTTGGAAGTAAGAATGGTGCCGAAGGC
CGGA
CTCAAACATCAAAATAAGTTAATGATAAAAAACAAATAATAAAACACAACAATGAAATATGCCCCCTTTTGTGCCCCCA
CTGTTT
TTCTGACCAATCTATTTTCAGCCCATCAATAAATCGGAAAGTTAAATCATTTTTAATCAGTAAGTTTGGATCCGTAGCT
CGGATC
CAAACCAGTGCATCTTTTATCCACATAAAAAATTTTTTTTCGAAAGAACTGTTCACACTGTTCACCTTTCTGTTTTCTC
CTTTTATT
TCAGAGTGATAGGTGGTGAATAATGGGTGAAGGGTGAACATTCGATTCTTCACCTCCGGCATTCTGCCGATGTGACTCA
TACC
GGTGATTAATCCTCCGCACTGAAATCACTCAGGAAGAAAAAAGTTTTTTTTGATTTGATTGTTCACACTGTTCACCTTT
CGTTTTT
P
CTCTTTTAATTTCAGTGTGATAACGGGTGAATATACGGTGAAGGGTGAACAGTGGATTGTTCACCTTCGGGGGATATCG
GGAT .
, AAAAAAAGACCGGCAGATGCCGGTCAGGTGGGTCAGGCTGTTGTAGGGTCGTCACATTTTGG CAG
CCAGTCGCCGTAGCTTTC
, 1¨
.3 CTCTTTCAGCGTCAGGTTGGTCTGTATCCCCTGTTTGGTATGGCGTTTCTCGTAATTCAGTCCGTATTCCTTCAGCATC
ACCGGCA .
w GCCCCAGCCCGAACATTTTCAGACTGAGTACATTCCGGTAGCCGTTTGCCTCCATGTAGGCCAGATAGGCGTGATAGAG
GTATT "
, , , TACGGTAATTGCGCGGGATGATACTGGCGTTCCCCATATACATGCCGCTGGTCTGCGGCAGGGTTTCCAGATAGCCGAT

, N, N, TCAAACGTCGGGTCGGCATCCCGTTTGATGTTCAGTGCCTCGTCTGAGTTCTGCTGGGACTGAAGCAGTGACCGGGCGA
GCAT
CGGGTCGCTGAACTTCTGCATCAGGTGACGCACGATGACCGCCAGCTCGCGGGTGATTTTGTCCTTAAGCTGCGGGTCG
CGCT
CCTGCGGGGCTATCTGTTCCGGGAAGTGAATAATCACCCGTCGGCGTGACACGCCGCCGCTGCGGTCGGTGAAGCGCAT
CGG
GTTATTGTTCACGGCCAGAATCACCGCCGGGATGTGCGTGGAGTACGCATCCCGGTATTTCGGGTCAACGGACACCGCA
TCGC
CGCCGGTGATGGCCTTGAGTCCGGCACCGTCGCCGCTCCATTTTTCCTGGTCCGGCAGGCGTATCAGTGAGAAGCCAGT
TAAC
GCGGCACGTTCACGCGGGGATTCCAGCGTCTCGATGGTGGCCGACGTGGCGTTATCCTCCCCGGCCAGCAGGGTGGCTA
TTTC 1-d GGCCATGATACT
n ,-i m ,-o 21 Sequence removed TTTGAGCGGCATAAATTACCACTGAAAGCCCTTAAACGTTACTCTACTGTGGACACTGTGTGGACACTCTCGG
CCTCAGTACCA w o w from P2 (replaced CCTCTTAGCGGATTAAGAGAAATGGCGTCCTGAAGGTACTCTGGCGCAAAATGAGCGTAAACCATAGTTTGCTCAATCC
GCGT =
'a GTGACCTAGTATCCGTTGTAGCGTGATAATACTTCCTCCATTAATCATGAAATGAGTGGCAAAGCTGTGCCTTAGTGCA
TGTGT o 4,.
w w vi with kanamycin GGCTTGCCCCATTGGCAAATCCGGTTTTATTGCTTTCATTGTTCGTCTGAAGCGAGGGTAATCAGCATCAGGGAATAAA
AAACC

marker; Example 3) TCGTTTGTTATCCGCGATCATTTTGGCAACAGCCTCTGAGATCGGGACGGTGCGTGGTTTGTTTGTTTTCGTTTTAACA
AACGTG w o w ACGCGGTTATGGATGATATTTTCTGCTTTCAAACGAGCTGCTTCTCCCCAACGTGCTCCTGTACTCAGGCAAAGAATCG
CAATCT
i-J
TTTTATTGTCGCCGTCAAGTGCTGCAAGCAGTAAGGCAATTTCTTCCTGTGTTAGATAGCCTGTTTCTGGTTTTTCCTC
CTTAAGC
4,.
w CTTTTTGTCCCTCTGATAGGGTGCTCACCAAAGAATAACTCCGCTTCAATCAGGGCTGTAAACATGCCGCTAATACATG
TTAAAT oe CACGATTGATACTCGAAGGTTTAATACCCTGACTTCTTCGGGTGGCGCAGTACTGGCTGATAAGCGATTTCGTAATTTG
AAATG

CCAAGATTAGATTTCCCATGCTCTTCGTGTTTACCCTTTAAATCCC
ACCAGATCTGTGTCAGCTCCGACAGACGTCGCTTGTCTGTTGGTTTTGATAGCCATTCTTTATTGTGGTGGTTGTACAA
CGTGTA
TTTCTCGAAAGCGACAGCTTCGCTTTTCTTATCAAACTTCCTACGGATGCGTTTTCCATTACGTCCAGTAGGGCGGATG
TCCACT
TCATATCGACCATCATCGAGTTTTTTGATTGCCATCAGAAAACCCTCCGAGTGGTGTGTTTTTTTGCGACTACTAATCG
CTTTTTT
CGTGGTGGCTGAAATTTAGCCACCAATAGTAGGCACTTGTGATGAATATATTCACGATGAATTGTTAACCAGTCTTTTG
ACCGG
P
AGTGGGGCGACGTTGTTTCGTTTTGCCCAAAGTGTGCGAGAGCGGGCGCAATTTGCCCGGACTCAGGAGCGATCTGATT
GGTC .
, ATGAACCATAAAGTGTATTTG GTGAATTGTG G G GTCTG CAG GATGTTCATCATGACATCTGTTG G AG
GTGTTG AACGACCACTT
, 1¨
.3 TCATAGTAACTCAGCGTGCCATACGGAACCCCTGTTAAATCAGCAAGTTGTTGTCTGCTCAAATACTCTGATTTTCGCA
TTAAGA .
CTATCTTCTCGCTTATCGTGTTTGACATGGTGTTTAGATCTCAATAGTATTTAGTTTAGATGTAGATTGTTTAGTGCTT
GGATGTG "
, , , GGCACTAAAAGGCATTATAAGACATTAAACGCAATTCATGAGGGCTAGAGGACGACATGAGCAAGCAAGTAACACTCAT

, N, N, TGATGCGATTCCTTATCAGGAGTTCGCAAAACTAATAGGAAAATCGACAGGAGCGGTTCGTCGGATGATCGATAAAGGA
AAG
CTGCCTGTAATTGATATGACCGATCCACAATCAGCTTCAGGTCGTGCAGGTGAATATTGGGTATACCTTCCGGCATGGA
ATAAC
GGACTAAAACTGGCTTATGAAAGCCGCCCTAAAGAGATTCGTGACGGCTGGTTGATGTGGTTAGGTCTCGGTGAACCAC
GTTA
AGGAGAACCGTATGAATGAGCCTCGTTGTATTGCTCAGTTACTGCGTAACGAAAGCCCCAGGGCGATTGACTTCACCAT
CACC
CACGGTAAGGGGCGTAAGGGAATCATTATCCGCACCAAAAAACAGAGTCCGTTAAAAAAGGCTCTGACCTTTCTGAAAA
GCCG
GAGGGTATGGAAATGACAGTGATGACGCTCAATCTCGTTGAAAAACAGCCAGCAGCTATGCGCCGGATAATTGGTAAGC
ATCT 1-d TGCCGTTCCTCGCTGGCAGGATACATGTGATTATTATAATCAGATGATGGAGCGCGAACGGTTAACGGTTTGCTTCCAT
GCTCA n ,-i GTTAAAACAGCGTCACGCAACGATGTGTTTTGAAGAAATGAACGACGTCGAACGTGAACGACTGGTATGTGCAATTGAT
GAAT t=1 1-d w TGCGTGGTGCATTCTCAAAACGCCGTCAGGTTGGCGCAAGTGAGTATGCATATATTAGTTTTTTAACAGTCAGTCAGCG
TCGTA =
w o CTTTATTTATGCATGCCGGATTGACTGAAAAAGAATTCAACCAGCCATACTGGCGAATTAATGAAGAGTCATGTTACTG
GCGTG 'a o ATG CTTTATTCCGTG CATTACGTGAATTATTCAGTCTGTTTGAGTATG
CACCGACAATTCTGACGTCGGTAAAACCG GAG CAAT
w w vi ATCTGCATTAAGTAATTAACCAGAGTTTTTAACGCACTTAATTGTGCGGGGCTTCTTTTTGCCTGGAGAAAGTTATGCA
TACAGT

TTCTGAAAATCAGTGCGGTATATACGCATTACTGCTGCAACAGGCCAGAACCGAAGCACAGGCCGACGCTGCGACGCGC
TTTT n.) o n.) CTTCTCATCTTGATGCCATGATTCGCCACATCACAAAGGCGGAGTTATCCCGCGTGGAGATAGTCGAGCTGCTCAGTCA
GGAGT
i-J
CGGAAAAATTTCACAATATCGGATTGTCTCGCGGGGAGGTGCTTTGATGTTCTGTTCTCGTGCAGTTGTATTACTGAAT
AACGC .6.
.6.
n.) CTTAAAAATCGCCGTTATGAAAAATGGCGATTTGTCTCTTATTCAACTTGGTCTTGATAAAGAAAAACGCGAAATAACT
GAATC oe TGTTATCGCGATTTATCAGAGTGAATTAAACCTCCTGTCTGATGTGGTCAATTTACTTGTTAAACGCGCTGTATTTCAC
AAGCAA
ATTTCCTCCGTGGATGAACTGACGAAATTAACGACAGAAATCGCCAGCTATTGCGCTGATGAATTTAAAAAACTGAACG
ACAA
AAGGAACTGGTAATGCCGGACAACGTAGATTTTATTCAGGAACAACAGGCTGAATTACTGGAGCGCCAGATTAACGCGG
CAA
GGGTAAAACATTGCGGTGCTTCTGCGCTGGTTTGCGAAGAGTGTGACGCGCCAATACCTGCTGCCCGTCGTGCGGCTTA
CCCG
TCAGCCACGCGTTGTGTTTCCTGTCAGTCAGTCTTTGAAGCAAAAAACAAACATTACCGGAGAACGGCATGAGTATTCG
TATTG
AAATTGGCGAACGTTATGTCGTTACCAGTGACAGCTTTCAGTTTATTCTCCACGAGAAAAAGAGAGCGGAAAGCGGTAA
AAAC
P
GCCGGTCAGGAATGGCTGGCGGTGGTTGGTTATTACCCGAAATTAAGCCAGCTCGTTTCAGGCCTGATGCATCACGATA
TTCT

GACCGGAAGCGCAAAGTCTTTTGCTGATTTAAACGTGCAGGTTGAGCAACTCAGCAAGCGTTGTTCAGAGGCTTTTGGC
TCAT
--.1 ATGGCCGTTAAAGCCTCCGGGCGTTTTGTCCCTCCGTCAGCATTTGCCGCAGGCACCGGTAAGATGTTTACCGGTGCTT

.6.
.3 TGGAACGCGCCACGCGAGGCCGTCGGGCGCGAAAGACCCCTTACACGTGACGAGATGCGTCAGATGCAAGGTGTTTTAT

CGATTAACCGCCTGCCTTACTTTTTGCGCTCGCTGTTTACTTCACGCTATGACTACATCCGGCGCAATAAAAGCCCGGT
GCACGG
.31 .3 GTTTTATTTCCTCACATCCACTTTTCAGCGTCGTTTATGGCCGCGCATTGAGCGTGTGAATCAGCGCCATGAAATGAAC
ACCGAC
GCGTCGTTGCTGTTTCTGGCAGAGCGTGACCACTATGCGCGCCTGCCGGGAATGAATGACAAGGAGCTGAAAAAGTTTG
CCG
CCCGTATCTCATCGCAGCTTTTCATGATGTATGAGGAACTCAGCGATGCCTGGGTGGATGCACATGGCGAAAAAGAATC
GCTG
TTTACGGATGAGGCGCAGGCTCACCTCTATGGTCATGTTGCTGGCGCTGCACGTGCTTTCAATATTTCCCCGCTTTACT
GGAAA
AAATACCGTAAAGGACAGATGACCACGAGGCAGGCATATTCTGCCATTGCCCGTCTGTTTAACGATGAGTGGTGGACTC
ATCA
GCTCAAAGGCCAGCGTATGCGCTGGCATGAGACGTTACTGATTGCTGTCGGGGAGGTGAATAAAGACCGTTCTCCTTAT
GCCA
IV
GTAAACATGCCATTCGTGATGTGCGTGCACGCCGCCAAGCAAATCTGGAATTTCTTAAATCGTGTGACCTTGAAAACAG
GGAA n ACCGGCGAGCGCATCGACCTTATCAGTAAGGTGATGGGCAGTATTTCTAATCCTGAAATTCGCCGGATGGAGCTGATGA
ACAC t=1 tl CATTGCCGGTATTGAGCGTTACGCCGCCGCAGAGGGTGATGTGGGGATGTTTATCACGCTTACCGCGCCTTCAAAGTAT

o CGACACGTCAGGTCGGAAAAGGCGAAAGTAAAACCGTCCAGCTAAATCACGGCTGGAACGATGAGGCATTTAATCCAAA
GGA 'a o TGCGCAGCGTTATCTCTGCCATATCTGGAGCCTGATGCGCACGGCATTCAAAGATAATGATTTACAGGTCTACGGTTTG
CGTGT tt n.) un CGTCGAGCCACACCACGACGGAACGCCGCACTGGCATATGATGCTTTTTTGTAATCCACGCCAGCGTAACCAGATTATC
GAAAT

CATGCGTCGCTATGCGCTCAAAGAGGATGGCGACGAAAGAGGAGCCGCGCGAAACCGTTTTCAGGCAAAACACCTTAAC
CAG n.) o n.) GGCGGTGCTGCGGGGTATATCGCGAAATACATCTCAAAAAACATCGATGGCTATGCACTGGATGGTCAGCTCGATAACG
ATAC
i-J
CGGCAGACCGCTGAAAGACACTGCTGCGGCTGTTACCGCATGGGCGTCAACGTGGCGCATCCCACAATTTAAAACGGTT
GGTC .6.
.6.
n.) TGCCGACAATGGGGGCTTACCGTGAACTACGCAAATTGCCTCGCGGCGTCAGCATTGCTGATGAGTTTGACGAGCGCGT
CGAG oe GCTGCACGCGCCGCCGCAGACAGTGGTGATTTTGCGTTGTATATCAGCGCGCAGGGTGGGGCAAATGTCCCGCGCGATT
GTC
AGACTGTCAGGGTCGCCCGTAGTCCGTCGGATGAGGTTAACGAGTACGAGGAAGAAGTCGAGAGAGTGGTCGGCATTTA
CGC
GCCGCATCTCGGCGCGCGTCATATTCATATCACCAGAACGACGGACTGGCGCATTGTGCCGAAAGTTCCGGTCGTTGAG
CCTC
TGACTTTAAAAAGCGGCATCGCCGCGCCTCGGAGTCCTGTCAATAACTGTGGAAAGCTCACCGGTGGTGATACTTCGTT
ACCG
GCTCCCACACCTTCTGAGCACGCCGCAGCAGTGCTTAATCTGGTTGATGACGGTGTTATTGAATGGAATGAACCGGAGG
TCGT
GAGGGCGCTCAGGGGCGCATTAAAATACGACATGAGAACGCCAAACCGTCAGCAAAGAAACGGAAGCCCGTTAAAACCG
CAT
P
GAAATTGCACCATCTGCCAGACTGACCAGGTCTGAACGATTGCAGATCACCCGTATCCGCGTTGACCTTGCTCAGAACG
GTATC

AGGCCTCAGCGATGGGAACTTGAGGCGCTGGCGCGTGGAGCAACCGTAAATTATGACGGGAAAAAATTCACGTATCCGG
TCG
--.1 CTGATGAGTGGCCGGGATTCTCAACAGTAATGGAGTGGACATGATGGCAAAAATTCACGAGGTAAAGCTGCACGCAAAA

un .3 TTCGACCTTGTACTGGAAGGAAAGAAACGCGCAGAGTTTCGGAAAAAAGATCGTAATTATGAGCGCGGGGACACGTTGA

TGCATGAATGGGTGCAGGGTGTGTTTACGGGGCGAAAGGTTGAAGCCCGGATAACAGATGTTACTGACCTGTCAGACTG
GCT
.31 .3 GGAAGATTATGTCTTGCTGCACAGAATACTAGGTGAGATACTGCCGCTGACTGTCAAGCTGGCAGTATCCTCCACATTT
TTTAT
AAATCTAAGGCGAAGAACGGTGGCAATATATTTGCTTTAAATTGTAAATAAGCAAATTATTCTAAGCCACAAATGATAT
CAATT
GAGTATCGATTTTCGTTCGAATATAACCATAACGCCAAGCGTCAAATGTAAGTCATCTATATGTGATTCGTATTCATAT
TGGTCA
GCATCAGGAGATAGTAAATGCTCTGCTGCTGTAAAAACATCAATTGTAACAGATATATCGTCTGGAATTTCTTCATTTA
ATTCGG

ATTGTATGAATGGAATGAACAGTTTAATGCTAGGCCAAGACTTGAATTTCCTGTAGGGCCTGTGTGTTTAAAGTATTCA
AGATT
IV
TCTTCTTATGTCAGAAAATAAAAGACTTTTACAGGGGGCATTTCCTTTAACTTCTATTGCACATATTGGAATGTCGAAA
AATGGT n ,-i CTGCTGTCCAATATGGCAATATCAATTCTGCCTGATCTTGTTGTGTTTGTGTTTTTTCTGACAGTTTTTTTTGAGAATA
TTTTTTGG t=1 tl GGGTCAGACCGTTTCGATAAAGGAACTGTTGCATTAATGAATTTATTTGTATGGTACTCAAATATAACTTTGTAATCAC

o GAAAAGATTTTATTTCTAACAACGATAGACCTATAGATACGGTAGCTACATATTCAGTGTTTATTTTTGCACCAGACCT
GAAATC 'a o CATTAAATAAGTTGCGTCGGCTGACTTTTTCATTCCATCTTTTACAGCTTTCACAATATCCATGTTATTCATATCAACA
CCTTTTAT tt n.) un ATTCCAGAATTATAAATCTTAATGATTGATTTGTTTGTGTGTTTAGGTTACTAGATTGACGTACTTATAGCACATTCTG
ATTTTGA

AATC AC CATC CTT G G GTAAAG C AC AG CCTATCACTATTG G TTTA G TACTG T G CTG
TAATAG TAT CA G ATTAA G TTAAATC C ATTT n.) o n.) ATTACTGCTCTAACATCTTTGCTTTCATCACATAAACAATCTAAAAGACTATCCCA
i-J
TTCAGTTTGTTTTTTACCAAATCCATAAACAGCCTCTATATCACCGGATTTCCATATATAGATTCCGTTATCTTTCAAT
TTTTGATG .6.
.6.
n.) TATTTCATGTATATG CTTTT GT G TTTTTTCATG GTTACATATTTTGATGAAGTCTTTG CTTGAAATG
CTTTTG AATG A CTCAAAA G oe TA G TAACTTTTG TATTTAAACTAAG CTCTC CTG A GTTG ATTG ATT CAATC AAAG C AG T CAATA
G ATTATC AC ATT CA GTA CTAA G
TAAGTCTTTATG CTCTGTTAATAGAATATTTGATAAGAAATCACAATCAG CTAGAATCCTTGTTTTTATG C C G
AT G G CATTGATG
ATTTGTG AC AT CTTAAATAAA CTAC CCTTACC GTC AACG G CAA CAATG CAGATTTTACTCG G
GTTGAGTTCATGTCCGTTAATTT
TTTTATAAAGTG C ATATA G AAC G TTT GT CT CTG TTTTC C CTT CAAC AA G
CAAAACTTCTTCAGAAAATAAAAG GTATGATGAATT
AG AAAG CGTGAATG CTG AG TG CAATTG CG GTGATG AAGATTTATATAATTCTTC G ATTTTTTC AG
ATATAGTCTTCCTTG CTATG
GTTCCATTAGAATCCTTACAAACCTGAATCG CATTTG CTGCATG CTTTG CAGAAAG CATACTG G CTG
AGTG AG TTG ATATTATA
P
AC CTG ATAC CCTG ATTCA CTTAATGTG AC AA GTG ATTCT CTG ACAG AATTAATG G CTGAAG G
GTGTAAATATAA CT CAG GTTC A

TCAATGAAAATCAAAGTGTTTGATTTTTTTG ATTCG CTGTTTTCTTTTTTTATTTCG G C CA G G TATTG
AATTAATG CCATTTGAAT L."
--.1 GGAACGTTGTGTTCCGTGACCAAATCGGCTGATATCTCTCATTACCGGTTCATCTTCCCGAGACTCAAAAACTTTCAGA

o .3 G GATTTAAATATCTCATCTAATGTCG GT G TG G G AAAG T G TAACTTTA CA CTTAC GT CA G G

AC AC CT G AG T CTATTTTATTAA G G CTTT CTAATCTG TT CTC AC C G TT GT G A G AAAG
ATATTTA C CTATTTCTG ATAT G TTTTTTG A
.31 .3 AAATTTTTCTTC GTG TTCTTG TTTTATTTC AG AAA CAATTG C G G AAAG TAT CTTTC CTATTGTG
GTCGTGTTTTTG CATTTTG TTG A
GTCTTCGACAG CGTCAGACATTG CAG G GATATGAATTGGTTCCG GAAATATATTG G AG ATTG CAC
CATCTATG C CG CCAG G GT
TTTTCTTCCACGTGGTACCGTCATATACATCCAGACTTTTTTTGGCTTTTCCTGTTTCCTTATTAAATTCCTGTCTTCT
TGCAAAGG
TAAG AGTC CC GT CAATTATAAACG GAG CTATTTTTTGTTG ATTCTCTTCTG TTAA CAAAGACAG AG
TAT CATCTGTTATAC CTTG
AATAACGCCTTCAACGGATACGGGGTGTGTAGGATCGTACACATCTGATTCTGAAATCAAAGAGCCATCTAACAGCCAC
TTAAT
TG CTAAGATAATATTTGATTTTCCTGCGTTATTATAACCAACTAAAG CAGTGAAG G G G CG CAAAATAG C
CG ATG TTG ACTTA CA
IV
AG AAC G AAAG TTG CTAATTG AAA CT G AA G CAAG A C G TACA G TC ATTATTTTT C
CTTAAAT G TG CTATTTG TAT G CAATG AG TTC n ,-i ATACGAAACGCTTTTTTACATTTTATAGTCGTTGCATTCAAGGGTGCATGAGATTGCATTAAGGGAAACTGTGATATGG
CTTGG t=1 tl CTTTTGACTG GAAATACTGATG G CTCATTAGTTTTATTAAG GTG CATTAAAACCG C CC CGTG AAG

o 'a o .6.
n.) n.) un AAAGCACGGCGAGGCGGGGAAAGCACTGCGCGCTGACGGTGGTGCTGATTGTATTTTTTCAGCGTCTCAGCGCGTCGTG
ACG

GCACTTAGTCTGCCCGTTGAGGCGTTGTGTGTCTGCGGGGTGTTTTGTGCGGTGGT
w o w o 22 WilcktypecloDF13 GCGCTGCGGACACATACAAAGTTACCCACAGATTCCGTGGATAAGCAGGGGACTAACATGTGAGGCAAAACAGCAGGGC
CGC
w 4.
4.
on GCCGGTGGCGTTTTTCCATAGGCTCCGCCCTCCTGCCAGAGTTCACATAAACAGACGCTTTTCCGGTGCATCTGTGGGA
GCCGT w m GAGGCTCAACCATGAATCTGACAGTACGGGCGAAACCCGACAGGACTTAAAGATCCCCACCGTTTCCGGCGGGTCGCTC
CCTC
TTGCGCTCTCCTGTTCCGACCCTGCCGTTTACCGGATACCTGTTCCGCCTTTCTCCCTTACGGGAAGTGTGGCGCTTTC
TCATAGC
TCACACACTGGTATCTCGGCTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTAAGCAAGAACTCCCCGTTCAGCCCGA
CTGCT
GCGCCTTATCCGGTAACTGTTCACTTGAGTCCAACCCGGAAAAGCACGGTAAAACGCCACTGGCAGCAGCCATTGGTAA
CTGG
GAGTTCGCAGAGGATTTGTTTAGCTAAACACGCGGTTGCTCTTGAAGTGTGCGCCAAAGTCCGGCTACACTGGAAGGAC
AGAT
TTGGTTGCTGTGCTCTGCGAAAGCCAGTTACCACGGTTAAGCAGTTCCCCAACTGACTTAACCTTCGATCAAACCACCT
CCCCAG
GTGGTTTTTTCGTTTACAGGGCAAAAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC
P
.
, 1-, 23 MutantcloDF13ori GCGCTGCGGACACATACAAAGTTACCCACAGATTCCGTGGATAAGCAGGGGACTAACATGTGAGGCAAAACAGCAGGGC
CGC , --.1 (higher copy number GCCGGTGGCGTTTTTCCATAGGCTCCGCCCTCCTGCCAGAGTTCACATAAACAGACGCTTTTCCGGTGCATCTGTGGGA
GCCGT
on cloD13_cop3;
GAGGCTCAACCATGAATCTGACAGTACGGGCGAAACCCGACAGGACTTAAAGATCCCCACCGTTTCCGGCGGGTCGCTC
CCTC , , , , single mutation TTGCGCTCTCCTGTTCCGACCCTGCCGTTTACCGGATACCTGTTCCGCCTTTCTCCCTTACGGGAAGTGTGGCGCTTTC
TCATAGC
versusmMcktypeis TCACACACTGGTATCTCGGCTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTAAGCAAGAACTCCCCGTTCAGCCCGA
CTGCT
shown underlined GCGCCTTATCCGGTAACTGTTCACTTGAGTCCAACCCGGAAAAGCACGGTAAAACGCCACTGGCAGCAGCCATTGGTAA
CTGG
andinbold GAGTTCGCAGAGGATTTGTTTAGCTAAACACGCGGTTGCTCTTGAAGTGTGCGCCAAAGTCCGGCTACACTGGAAGGAC
AGAT
TTGGTTGCTGTGCTCTGCGAAATCCAGTTACCACGGTTAAGCAGTTCCCCAACTGACTTAACCTTCGATCAAACCACCT
CCCCAG
GTGGTTTTTTCGTTTACAGGGCAAAAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATC
Iv n ,-i m ,-o t..) =
t..) =
'a c., 4,.
w w u, Table 7: Example Plasmids and Copy Numbers w _______________________________________________________________________________ ________________________________________ <
w i Common Vectors Copy Number OF11 Incompatibliity Group Control t.) OC
pUC -500-700 0,481 (derivative) A Relaxed pBR322 -15-20 pME31 A Relaxed PET -15-20 pBR322 A Relaxed pG EX -15-20 pBR322 A Relaxed pColE1 -15-20 ColE1 A Relaxed 0 IpR6K -15-20 R6 K ' C Stringent .

.
oe ,pACYC -10 p15A
B Relaxed .
0 ..
i ..
i IpSC101 -5 pSC 101 C Stringent ipB1Liescript -300-500 0 1E1 (derivative) and Fl"
A Relaxed pGENI -300-500 pUC and Fl -A Relaxed .0 n ,-3 ril .0 w <
F, t.) t.) !A

Claims (24)

CLAIMS:

1. A host bacterial cell comprising a) a first DNA; and b) one or more second DNAs;
wherein (i) the DNAs together comprise all genes required to produce a transduction particle comprising a copy of the first DNA packaged by phage structural proteins;
(ii) the first DNA is devoid of at least one functional essential gene required to produce the particle; and wherein the one or more second DNAs comprises said functional essential gene(s);
(iii) the first DNA comprises a phage packaging signal for producing the particle; and (iv) the second DNA is devoid of a nucleotide sequence required for packaging the second DNA into transduction particles;
wherein the second DNA is required for packaging first DNA to produce particles, wherein the DNAs are operable in the cell for producing transduction particles comprising phage structural proteins that package copies of the first DNA.

2. The cell of claim 1, wherein the DNAs together encode all phage structural proteins required to produce a packaged transduction particle comprising a copy of the first DNA;
wherein the first DNA
encodes none or at least one, but not all, of the structural proteins; and wherein the one or more second DNAs encode the remainder of the structural proteins.

3. The cell of claim 1 or 2, wherein the first DNA is comprised by an episome that is devoid of said essential or structural protein gene(s) and/or the second DNA is comprised by an episome or a chromosome of the cell.

4. The cell of any preceding claim, wherein all of said essential genes or phage structural protein genes are comprised by the second DNA and the first DNA is devoid of said genes.

5. The cell of any preceding claim, wherein the first DNA encodes a guided nuclease or a component of a CRISPR/Cas system (optionally, a crRNA or a guide RNA).

6. The cell of any preceding claim, wherein the first DNA comprises a phage origin of replication and/or the first DNA comprises phage replication genes and phage lysis genes.

7. The cell of any preceding claim, wherein each transduction particle is a non-self replicative transduction particle.

8. The cell of any preceding claim, wherein the essential genes, structural protein genes and packaging signal are genes and a packaging signal of a tailed phage, optionally a P2, T4, T7, Phi92, lambda, K 1-or 933w phage.

9. The cell of any preceding claim, wherein the first DNA is comprised by a phage genome, wherein the phage genome is integrated in a plasmid; optionally wherein each particle is capable of infecting a target bacterium, the first DNA comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in the target bacterium, wherein the NSI replaces the essential gene(s) or structural protein gene(s) of the phage.

10. The cell of any preceding claim, wherein each particle is capable of infecting a target bacterial cell, the first DNA comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in the target bacterium, wherein the presence in the target cell of the NSI-encoded protein or RNA mediates target cell killing, or downregulation of target cell growth or propagation, optionally wherein the NSI encodes a guided nuclease or a component of a CRISPR/Cas system that is toxic to the target cell.

11. The cell of any preceding claim, wherein the packaging signal is a pac or cos sequence, or is a homologue thereof; or wherein the packaging signal is a direct terminal repeat (DTR).

12. The cell of any preceding claim, wherein the packaging signal comprises SEQ ID NO: 2 or a sequence that is at least 70, 80, 90, 95, 96, 97, 98 or 99% identical thereto, or is a homologue thereof from a different phage.

13. An isolated DNA, comprising a first DNA as defined in any preceding claim;
or comprising a second DNA as defined in any preceding claim, optionally wherein the isolated DNA is comprised by a plasmid.

14. A kit comprising (a) a cell wherein the cell comprises a second DNA as defined in any one of claims 1 to 12, but the cell does not comprise the first DNA as defined in any one of claims 1 to 12; and (b) a vector (optionally a plasmid) comprising the first DNA, wherein the vector is not comprised by the cell;
optionally, wherein the cell is a bacterial or archaeal cell.

15. A method of producing a transduction particle composition, the method comprising expressing phage structural proteins in a cell and replicating in the cell first DNA, wherein transduction particles are produced that comprise packaged first DNA; and optionally separating an amount of transduction particles from cellular material wherein an amount of purified transduction particles is obtained, wherein the cell is a cell of any one of claims 1 to 12 when dependent from claim 2.

16. A composition comprising a population of transduction particles obtainable by the method of claim 15.

17. The method of claim 15 or the composition of claim 16, wherein the second DNA is comprised by plasmid DNA and less than 5% of total DNA comprised by the composition is DNA
of said plasmid.

18. The cell, DNA or composition of any one of claims 1 to 13, 16 and 17 for administration to a human or animal subject for medical use.

19. The cell, DNA or composition of any one of claims 1 to 13, 16 and 17 for administration to a human or animal subject for treating an infection of target bacterial cells, wherein the particles are capable of infecting and killing the target cells, optionally wherein the infection is a gut, blood, lung or uterine tract microbiome infection.

20. The cell, DNA or composition of any one of claims 1 to 13, 16 and 17 for use in a contained method of treating a disease or condition of a human or animal subject, wherein the disease or condition is mediated by target bacteria and the target bacteria are comprised by the subject (optionally comprised by a gut, blood, lung or uterine tract microbiome), the method comprising administering the composition to the subject, whereby the target bacteria are exposed to antibacterial means encoded by the first DNA and killed, and propagation of the transduction particles is contained.

21. The composition of claim 16 or 17 for controlling in a human or animal subject the dosing of transduction particle treatment of a target bacterial cell infection in the subject, wherein the particles are capable of transducing first DNA into the target cells, the first DNA
encoding antibacterial means that is toxic to target cells whereby target cells are killed.

22. The composition of claim 18, 19, 20 or 21 for reducing the risk of acquisition of foreign gene sequence(s) by the particles in the subject.

23. A method of treating an environment ex vivo, the method comprising exposing the environment to a composition comprising a population of particles, wherein the particles are capable of transducing first DNA into target cells comprised by the environment, the first DNA
encoding antibacterial means that is toxic to target cells whereby target cells are killed, wherein the composition is a composition according to claim 16 or 17.

24. The method of claim 23, wherein the method is for (a) containing the treatment in the environment;
(b) reducing the risk of acquisition of foreign gene sequence(s) by the particles in the environment; and/or (c) controlling the dosing of the particle treatment in the environment.