Nucleic acid molecule and methods

Abstract

The invention relates to a nucleic acid molecule encoding a group II intron, wherein the group II intron encodes a retrotransposition-activated marker (RAM) and a counter-selection marker. The invention further relates to a nucleic acid molecule encoding: a first promoter operably-linked to a group II intron, wherein the group II intron does not encode a protein comprising reverse transcriptase activity, and a second promoter operably-linked to a nucleic acid sequence encoding an intron-encoded protein (IEP), wherein the group II intron encodes: a retrotransposition-activated marker (RAM), a counter-selection marker, and a targeting sequence; and related kits and methods.

Description

[0001]NUCLEIC ACID MOLECULE AND METHODS FIELD OF THE INVENTION The invention relates to a nucleic acid molecule. The invention also relates to a host cell encoding the nucleic acid molecule. The invention also extends to a kit comprising the nucleic acid molecule or comprising the host cell. The invention also encompasses a method of introducing a heterologous polynucleotide into DNA of a plurality of host cells, and a method of removing a polynucleotide sequence from DNA of a plurality of host cells. The invention further provides a cell produced by any method of the invention. BACKGROUND CRISPR Cas9 technologies have been deployed as counter-selectable markers in combination with homology arms to direct in-frame deletion events in prokaryotes which lack the ability to repair double stand DNA breaks through non-homologous end joining (NHEJ). This is advantageous over traditional methods which rely on integration of a vector at one of the homologous regions, followed by counter-selection against an element on the plasmid, because the wild type locus itself is selected against, rather than the integrated plasmid sequence. This in theory should mean that all colonies which survive induction will be cells in which the intended double crossover event has occurred, and the guide target has been lost from the genome. In practise however this technology has a number of limitations. Cas9 is extremely toxic when expressed in cells, likely due to its off-target effects on similar sequences to their intended targets, therefore tight repression is critical and cloning efforts in the E. coli host strain are usually hampered. Finding appropriate guide targets to the gene target in question is often laborious, and it is commonplace to design three or more alternative guide sequences when targeting a specific gene. This is to offset the likelihood of encountering either a non-specific guide which causes double stand breaks elsewhere in the genome, or one that is not able to cause double strand breaks at the target locus with high enough fidelity to be useful as a counter-selective pressure. Once colonies have arisen after counter-selection with Cas9, they are PCR screened at the targeted locus to identify intended knockouts from the escapees of the counter-selective pressure which remain wild type at the genomic locus. Exhaustive PCR screening is often required to isolate successful mutants from the wild type cells, and in most cases, there will be no screenable phenotype associated with the knockout mutants to enable a higher throughput screening method. The invention described below seeks to address all of the problems mentioned above. STATEMENTS OF THE INVENTION According to a first aspect of the invention, there is provided a (first) nucleic acid molecule encoding: (a) a first promoter operably-linked to a (single) group II intron, wherein the group II intron does not encode a protein comprising reverse transcriptase activity, and (b) a second promoter operably-linked to a nucleic acid sequence encoding an intron-encoded protein (IEP), wherein the group II intron encodes / comprises: (i) a retrotransposition-activated marker (RAM), (ii) a counter-selection marker, and (iii) a targeting sequence (e.g. for targeting a target site of the DNA of a host cell), optionally wherein, in use (e.g. in a host cell), (a transcript of) the group II intron is inserted into a target site of the DNA of the host cell by a protein of the IEP and the targeting sequence of the group II intron. The invention may be a host cell comprising a nucleic acid according to an aspect of the invention, e.g., the first aspect of the invention. Thus, according to a second aspect, there is provided a host cell comprising a (first) nucleic acid molecule encoding: (a) a first promoter operably-linked to a (single) group II intron, wherein the group II intron does not encode a protein comprising reverse transcriptase activity, and (b) a second promoter operably-linked to a nucleic acid sequence encoding an intron-encoded protein (IEP), wherein the group II intron encodes / comprises: (i) a retrotransposition-activated marker (RAM), (ii) a counter-selection marker, and (iii) a targeting sequence (e.g. for targeting a target site of the DNA of a host cell), optionally wherein, in use (e.g. in a host cell), (a transcript of) the group II intron is inserted into a target site of the DNA of the host cell by a protein of the IEP and the targeting sequence of the group II intron. According to another aspect of the invention, there is provided a single (first) nucleic acid molecule encoding a group II intron, wherein the group II intron encodes / comprises a retrotransposition-activated marker (RAM) and a counter-selection marker. The nucleic acid molecule according to this aspect of the invention may further encode an IEP. Preferably the group II intron does not encode an IEP, such as a protein comprising reverse transcriptase activity. The group II intron may encode / comprise a targeting sequence. A (first) nucleic acid molecule according to the invention, which may be referred to as NegaTron, may be for assisting with the insertion of a polynucleotide into DNA of a host cell and / or for assisting with the removal of a polynucleotide from DNA of a host cell. Preferably the group II intron of the invention does not encode an intron-encoded protein comprising reverse transcriptase activity. The RAM may be any RAM referred to herein. The counter-selection marker may be any counter-selection marker referred to herein. The group II intron may be operably-linked to a first promoter referred to herein. The group II intron may further encode / comprise a targeting sequence referred to herein. A nucleic acid molecule according to the invention may be used to assist with the genetic modification of the DNA of a host cell. The DNA of the host cell may be genetically modified by inserting a polynucleotide into the DNA of the host cell and / or by removing a polynucleotide from the DNA of the host cell. Insertion or removal of a polynucleotide may result in the phenotype of the host cell being altered. In embodiments in which a nucleic acid molecule according to the invention (e.g., the first aspect) is used to insert a polynucleotide into DNA of a host cell and remove a polynucleotide from DNA of a host cell, the nucleic acid molecule according to the invention may be for replacing a polynucleotide sequence of DNA in a host cell with a separate polynucleotide (e.g., a heterologous polynucleotide). Thus, the polynucleotide being inserted into the DNA of a host cell may be different from a polynucleotide being removed from the DNA of the host cell. Advantageously, the nucleic acid molecule of the invention, particularly the group II intron, enables subsequent precise, in-frame and scarless genome editing in a host cell. This, in turn, ensures that any genetic modifications performed using a nucleic acid molecule of the invention do not have any polar effects on adjacent genes. Group II introns are catalytic RNAs that excise themselves from RNA transcripts using a lariat intermediate and insert themselves into a new distal target site using an intron encoded protein (IEP). Despite being called “introns”, group II introns encode a protein that is known in the art as an IEP. In nature and in the present invention, the lariat intermediate of the group II intron and the IEP form a complex, referred to as a ribonucleoprotein (RNP). The RNP enables the group II intron to retrotranspose to a new location in the DNA of a recipient / host cell. Retrotransposition is assisted by the multi-functional nature of the IEP because IEPs possess reverse transcriptase activity, endonuclease activity and maturase activity, one or more of these activities enable group II introns to be inserted into DNA location. Retrotransposition is also assisted by the targeting sequence of the lariat intermediate of the group II intron. The targeting sequence ensures that the RNP binds to a specific site within the DNA of the host cell, i.e., adjacent to the target site. Thus, the RNP, specifically the activity of the targeting sequence and the protein of IEP, enable the group II intron to be inserted into a target site of the DNA of the host cell. Group II introns are found in bacteria, archaea and eukaryota. The group II intron referred to herein may be from bacteria, archaea or eukaryota. Preferably, the group II comprises a targeting sequence and an IEP, so that the group II intron is capable of retrotransposition to a specific target site. In one embodiment, the group II intron is LI.LtrB (from Lactococcus lactis). In another embodiment, the group II intron is Ecl5 (from Escherichia coli). In another embodiment, the group II intron is RmInt1 (from Sinorhizobium meliloti). In a further embodiment, the group II intron isTeI3c (from Thermosynechococcus elongatus).Preferably, the group II intron is LI.LtrB. Preferably the group II intron referred to herein does not encode an intron-encoded protein. Preferably the group II intron of the nucleic acid molecule according to the invention does not encode an intron-encoded protein comprising reverse transcriptase activity. Preferably the group II intron referred to herein does not encode a protein (an intron-encoded protein) comprising endonuclease activity and / or maturase activity. The nucleic acid molecule according to the invention may encode an IEP that is operably linked to a second promoter. This is so that once retrotransposition has occurred and plasmid loss achieved, transcription of the group II intron by the host cell results in a transcript that does not produce an IEP and is therefore incapable of undergoing retrotransposition to another site in the DNA of the host cell. A further advantage of the invention is that the group II intron also comprises a counter- selection marker. Consequently, following identification / isolation of host cells containing DNA that corresponds to the group II intron (in methods according to the invention this may or may not be achieved using a RAM), the isolated cells can be modified with a second nucleic acid molecule comprising a homologous recombination cassette. Only cells that have had their group II intron DNA, including the counter- selection marker (e.g., a meganuclease recognition site), removed by homologous recombination using the homology cassette of the second nucleic acid molecule are able to survive counter-selection pressure. In other words, any cells that have not had their group II introns removed by homologous recombination will be susceptible to counter- selection pressure due to the presence of the counter-selection marker. These cells will not survive the corresponding counter-selection pressure that may be applied. Thus, the invention allows quick and easy identification / isolation of cells that have successfully and stably undergone homologous recombination without having to screen the genomic target using PCR, as the loss of the RAM enables a second phenotypic screen which may be used to confirm the in-frame deletion and loss of the group II intron from the target locus. The counter-selection marker (or negative selection marker) is a nucleotide sequence (gene) that confers a selection disadvantage to cells that have been transformed with a nucleotide sequence compared to cells that have not been transformed with the nucleotide sequence or cells from which the nucleotide sequence has been removed. Thus, under appropriate growth conditions, a counter-selectable marker promotes the death of the cells harbouring it. For example, the counter-selection marker may be a nuclease recognition site, which is recognised by a nuclease (a counter-selection agent) that specifically cleaves the nuclease recognition site, thus resulting in the death of cells harbouring the counter-selection marker. In one embodiment, the counter-selection marker may be a nuclease recognition site. Preferably, the nuclease recognition site (e.g., a meganuclease recognition site or an endonuclease recognition site) is not native to the host cell. Preferably, there is a nuclease enzyme (counter-selection agent) that is specific for nuclease recognition site. The enzyme that is specific for the nuclease recognition site may therefore be unlikely to exhibit much, if any, off-target effects. The nuclease recognition site may be a meganuclease recognition site (a homing endonuclease) or an endonuclease recognition site. Preferably the nuclease recognition site is a meganuclease recognition site. Meganuclease recognition sites are preferred because they have longer recognition sequences, which are recognised by their corresponding enzymes, and therefore they typically do not occur natively in the genome of many species. Preferably the counter-selection marker is a I-SceI meganuclease recognition site or a PpoI meganuclease recognition site. A corresponding counter selection agent (enzyme) for the I-SceI meganuclease recognition site may be a I-SceI meganuclease. A corresponding counter selection agent for the I-PpoI meganuclease recognition site may be a I-PpoI meganuclease. The endonuclease recognition site, I-SceI, is preferred because it is estimated to occur only once in 7 × 1010 bp of a random sequence. Accordingly, this recognition site is rarely found natively in the genome of the cells, e.g., only 17 sites were found in all (total of 77880) completed genomes held in GenBank on 6th Sept 2022. Also, the I-SceI enzyme is highly specific to the fixed 18 bp recognition site and therefore does not exhibit off-target effects. In one embodiment, the nucleotide sequence of the I-SceI meganuclease recognition site is provided herein as SEQ ID NO. 1, as follows: TAGGGATAACAGGGTAAT [SEQ ID NO. 1] The counter-selection marker may be a I-PpoI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-PpoI meganuclease. In one embodiment, the nucleotide sequence of the I-PpoI meganuclease recognition site is provided herein as SEQ ID NO. 2, as follows: TAACTATGACTCTCTTAAGGTAGCCAAAT [SEQ ID NO. 2] The counter-selection marker may be a I-CeuI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-CeuI meganuclease. In one embodiment, the nucleotide sequence of the I-CeuI meganuclease recognition site is provided herein as SEQ ID NO. 3, as follows: TAACTATAACGGTCCTAAGGTAGCGA [SEQ ID NO. 3] The counter-selection marker may be a I-ChuI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-ChuI meganuclease. In one embodiment, the nucleotide sequence of the I-ChuI meganuclease recognition site is provided herein as SEQ ID NO. 4, as follows: GAAGGTTTGGCACCTCGATGTCGGCTCATC [SEQ ID NO. 4] The counter-selection marker may be a I-CpaI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-CpaI meganuclease. In one embodiment, the nucleotide sequence of the I-CpaI meganuclease recognition site is provided herein as SEQ ID NO. 5, as follows: CGATCCTAAGGTAGCGAAATTCA [SEQ ID NO. 5] The counter-selection marker may be a I-CpaII meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-CpaII meganuclease. In one embodiment, the nucleotide sequence of the I-CpaII meganuclease recognition site is provided herein as SEQ ID NO. 6, as follows: CCCGGCTAACTCTGTGCCAG [SEQ ID NO. 6] The counter-selection marker may be a I-CreI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-CreI meganuclease. In one embodiment, the nucleotide sequence of the I-CreI meganuclease recognition site is provided herein as SEQ ID NO. 7, as follows: CTGGGTTCAAAACGTCGTGAGACAGTTTGG [SEQ ID NO. 7] The counter-selection marker may be a I-DmoI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-DmoI meganuclease. In one embodiment, the nucleotide sequence of the I-DmoI meganuclease recognition site is provided herein as SEQ ID NO. 8, as follows: ATGCCTTGCCGGGTAAGTTCCGGCGCGCAT [SEQ ID NO. 8] The counter-selection marker may be a H-DreI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a H-DreI meganuclease. In one embodiment, the nucleotide sequence of the H-DreI meganuclease recognition site is provided herein as SEQ ID NO. 9, as follows: CAAAACGTCGTAAGTTCCGGCGCG [SEQ ID NO. 9] The counter-selection marker may be a I-HmuI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-HmuI meganuclease. In one embodiment, the nucleotide sequence of the I-HmuI meganuclease recognition site is provided herein as SEQ ID NO. 10, as follows: AGTAATGAGCCTAACGCTCAGCAA [SEQ ID NO. 10] The counter-selection marker may be a I-HmuII meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-HmuII meganuclease. In one embodiment, the nucleotide sequence of the I-HmuII meganuclease recognition site is provided herein as SEQ ID NO. 11, as follows: AGTAATGAGCCTAACGCTCAACAA [SEQ ID NO. 11] The counter-selection marker may be a I-LlaI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-LlaI meganuclease. In one embodiment, the nucleotide sequence of the I-LlaI meganuclease recognition site is provided herein as SEQ ID NO. 12, as follows: CACATCCATAACCATATCATTTTT [SEQ ID NO. 12] The counter-selection marker may be a I-MsoI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-MsoI meganuclease. In one embodiment, the nucleotide sequence of the I-MsoI meganuclease recognition site is provided herein as SEQ ID NO. 13, as follows: CTGGGTTCAAAACGTCGTGAGACAGTTTGG [SEQ ID NO. 13] The counter-selection marker may be a PI-PfuI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a PI-PfuI meganuclease. In one embodiment, the nucleotide sequence of the PI-PfuI meganuclease recognition site is provided herein as SEQ ID NO. 14, as follows: GAAGATGGGAGGAGGGACCGGACTCAACTT [SEQ ID NO. 14] The counter-selection marker may be a PI-PkoII meganuclease recognition site. Therefore, a corresponding counter selection agent may be a PI-PkoII meganuclease. In one embodiment, the nucleotide sequence of the PI-PkoII meganuclease recognition site is provided herein as SEQ ID NO. 15, as follows: CAGTACTACGGTTAC [SEQ ID NO. 15] The counter-selection marker may be a I-PorI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-PorI meganuclease. In one embodiment, the nucleotide sequence of the I-PorI meganuclease recognition site is provided herein as SEQ ID NO. 16, as follows: GCGAGCCCGTAAGGGTGTGTACGGG [SEQ ID NO. 16] The counter-selection marker may be a AniI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a AniI meganuclease. In one embodiment, the nucleotide sequence of the AniI meganuclease recognition site is provided herein as SEQ ID NO. 17, as follows: TTGAGGAGGTTTCTCTGTAAATAA [SEQ ID NO. 17] The counter-selection marker may be a PI-PspI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a PI-PspI meganuclease. In one embodiment, the nucleotide sequence of the PI-PspI meganuclease recognition site is provided herein as SEQ ID NO. 18, as follows: TGGCAAACAGCTATTATGGGTATTATGGGT [SEQ ID NO. 18] The counter-selection marker may be a I-ScaI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-ScaI meganuclease. In one embodiment, the nucleotide sequence of the I-ScaI meganuclease recognition site is provided herein as SEQ ID NO. 19, as follows: TGTCACATTGAGGTGCACTAGTTATTAC [SEQ ID NO. 19] The counter-selection marker may be a PI-SceI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a PI-SceI meganuclease. In one embodiment, the nucleotide sequence of the PI-SceI meganuclease recognition site is provided herein as SEQ ID NO. 20, as follows: ATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGCA [SEQ ID NO. 20] The counter-selection marker may be a I-SceII meganuclease recognition site. Therefore, a corresponding counter selection agent may be a SceII meganuclease. In one embodiment, the nucleotide sequence of the I-SceII meganuclease recognition site is provided herein as SEQ ID NO. 21, as follows: TTTTGATTCTTTGGTCACCCTGAAGTATA [SEQ ID NO. 21] The counter-selection marker may be a I-SceIII meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-SceIII meganuclease. In one embodiment, the nucleotide sequence of the I-SceIII meganuclease recognition site is provided herein as SEQ ID NO. 22, as follows: ATTGGAGGTTTTGGTAACTATTTATTACC [SEQ ID NO. 22] The counter-selection marker may be a I-SceIV meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-SceVI meganuclease. In one embodiment, the nucleotide sequence of the I-SceIV meganuclease recognition site is provided herein as SEQ ID NO. 23, as follows: TCTTTTCTCTTGATTAGCCCTAATCTACG [SEQ ID NO. 23] The counter-selection marker may be a I-SceV meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-SceV meganuclease. In one embodiment, the nucleotide sequence of the I-SceV meganuclease recognition site is provided herein as SEQ ID NO. 24, as follows: AATAATTTTCTTCTTAGTAATGCC [SEQ ID NO. 24] The counter-selection marker may be a I-SceVI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-SceVI meganuclease. In one embodiment, the nucleotide sequence of the I-SceVI meganuclease recognition site is provided herein as SEQ ID NO. 25, as follows: GTTATTTAATGTTTTAGTAGTTGG [SEQ ID NO. 25] The counter-selection marker may be a I-SceVII meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-SceVII meganuclease. In one embodiment, the nucleotide sequence of the I-SceVII meganuclease recognition site is provided herein as SEQ ID NO. 26, as follows: TGTCACATTGAGGTGCACTAGTTATTAC [SEQ ID NO. 26] The counter-selection marker may be a I-Ssp6803I meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-Ssp6803I meganuclease. In one embodiment, the nucleotide sequence of the I-Ssp6803I meganuclease recognition site is provided herein as SEQ ID NO. 27, as follows: GTCGGGCTCATAACCCGAA [SEQ ID NO. 27] The counter-selection marker may be a I-TevI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-TevI meganuclease. In one embodiment, the nucleotide sequence of the I-TevI meganuclease recognition site is provided herein as SEQ ID NO. 28, as follows: AGTGGTATCAACGCTCAGTAGATG [SEQ ID NO. 28] The counter-selection marker may be a I-TevII meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-TevII meganuclease. In one embodiment, the nucleotide sequence of the I-TevII meganuclease recognition site is provided herein as SEQ ID NO. 29, as follows: GCTTATGAGTATGAAGTGAACACGTTATTC [SEQ ID NO. 29] The counter-selection marker may be a I-TevIII meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-TevIII meganuclease. In one embodiment, the nucleotide sequence of the I-TevIII meganuclease recognition site is provided herein as SEQ ID NO. 30, as follows: TATGTATCTTTTGCGTGTACCTTTAACTTC [SEQ ID NO. 30] The counter-selection marker may be a PI-TliI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a PI-TliI meganuclease. In one embodiment, the nucleotide sequence of the PI-TliI meganuclease recognition site is provided herein as SEQ ID NO. 31, as follows: TAYGCNGAYACNGACGGYTTYT [SEQ ID NO. 31] The counter-selection marker may be a PI-TliII meganuclease recognition site. Therefore, a corresponding counter selection agent may be a PI-TliII meganuclease. In one embodiment, the nucleotide sequence of the PI-TliII meganuclease recognition site is provided herein as SEQ ID NO. 32, as follows: AAATTGCTTGCAAACAGCTATTACGGCTAT [SEQ ID NO. 32] The counter-selection marker may be a I-Tsp061I meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-Tsp061I meganuclease. In one embodiment, the nucleotide sequence of the I-Tsp061I meganuclease recognition site is provided herein as SEQ ID NO. 33, as follows: CTTCAGTATGCCCCGAAAC [SEQ ID NO. 33] The counter-selection marker may be a I-Vdi141I meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-Vdi141I meganuclease. In one embodiment, the nucleotide sequence of the I-Vdi141I meganuclease recognition site is provided herein as SEQ ID NO. 34, as follows: CCTGACTCTCTTAAGGTAGCCAAA [SEQ ID NO. 34] The counter-selection marker may be a I-NjaI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-NjaI meganuclease. In one embodiment, the nucleotide sequence of the I-NjaI meganuclease recognition site is provided herein as SEQ ID NO. 64, as follows: AAGTCTGGTGCCAGCACCCGC [SEQ ID NO. 64] The counter-selection marker may be a I-DirI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-DirI meganuclease. In one embodiment, the nucleotide sequence of the I-DirI meganuclease recognition site is provided herein as SEQ ID NO. 65, as follows: TGAGTCAAATTAAGCCGCAGGTTC [SEQ ID NO. 65] The counter-selection marker may be a I-BmoI meganuclease recognition site. Therefore, a corresponding counter selection agent may be a I-BmoI meganuclease. In one embodiment, the nucleotide sequence of the I-BmoI meganuclease recognition site is provided herein as SEQ ID NO. 66, as follows: GCCATGTCATTACTACGGGCTCTTACTC [SEQ ID NO. 66] The meganuclease recognition site may be one or more sites selected from the group consisting of: AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-DmoI, H-DreI, I-HmuI, I- HmuII, I-LlaI, I-MsoI, PI-PfuI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, PI-SceI, I- SceII, I-SecIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I- TevIII, PI-TliI, PI-TliII, I-Tsp061I, and I-Vdi141I. The meganuclease recognition site may comprise a nucleotide sequence substantially as set out in any one of SEQ ID NOs. 1-34. The meganuclease recognition site may be one or more sites selected from the group consisting of: AniI, I-CeuI, I-ChuI, I-CpaI, I-CpaII, I-CreI, I-DmoI, H-DreI, I-HmuI, I- HmuII, I-LlaI, I-MsoI, PI-PfuI, PI-PkoII, I-PorI, I-PpoI, PI-PspI, I-ScaI, PI-SceI, I- SceII, I-SecIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-Ssp6803I, I-TevI, I-TevII, I- TevIII, PI-TliI, PI-TliII, I-Tsp061I, I-Vdi141I, I-DirI, I-BmoI, and I-NjaI. The meganuclease recognition site may comprise a nucleotide sequence substantially as set out in any one of SEQ ID NOs. 1-34, or 64-66. In another embodiment, the nuclease recognition site may be an endonuclease recognition site. An endonuclease recognition site can refer to a site recognised by a traditional endonuclease (an enzyme that provides defence against foreign (bacterial) DNA). Preferably the endonuclease recognition site has a sequence of at least 8 bp. The counter-selection marker may be a AbsI endonuclease recognition site. Therefore, a corresponding counter selection agent may be a AbsI endonuclease. In one embodiment, the nucleotide sequence of the AbsI endonuclease recognition site is provided herein as SEQ ID NO. 35, as follows: CCTCGAGG [SEQ ID NO. 35] The counter-selection marker may be a SbfI / SdaI endonuclease recognition site. Therefore, a corresponding counter selection agent may be a SbfI / SdaI endonuclease. In one embodiment, the nucleotide sequence of the SbfI / SdaI endonuclease recognition site is provided herein as SEQ ID NO. 36, as follows: CCTGCAGG [SEQ ID NO. 36] The counter-selection marker may be a MreI endonuclease recognition site. Therefore, a corresponding counter selection agent may be a MreI endonuclease. In one embodiment, the nucleotide sequence of the MreI endonuclease recognition site is provided herein as SEQ ID NO. 37, as follows: CGCCGGCG [SEQ ID NO. 37] The counter-selection marker may be a MauBI endonuclease recognition site. Therefore, a corresponding counter selection agent may be a MauBI endonuclease. In one embodiment, the nucleotide sequence of the MauBI endonuclease recognition site is provided herein as SEQ ID NO. 38, as follows: CGCGCGCG [SEQ ID NO. 38] The counter-selection marker may be a SgrDI endonuclease recognition site. Therefore, a corresponding counter selection agent may be a SgrDI endonuclease. In one embodiment, the nucleotide sequence of the SgrDI endonuclease recognition site is provided herein as SEQ ID NO. 39, as follows: CGTCGACG [SEQ ID NO. 39] The counter-selection marker may be a SgrfI endonuclease recognition site. Therefore, a corresponding counter selection agent may be a SgrfI endonuclease. In one embodiment, the nucleotide sequence of the SgrfI endonuclease recognition site is provided herein as SEQ ID NO. 40, as follows: GCCCGGGC [SEQ ID NO. 40] The counter-selection marker may be a AsiSI / SfaAI endonuclease recognition site. Therefore, a corresponding counter selection agent may be a AsiSI / SfaAI endonuclease. In one embodiment, the nucleotide sequence of the AsiSI / SfaAI endonuclease recognition site is provided herein as SEQ ID NO. 41, as follows: GCGATCGC [SEQ ID NO. 41] The counter-selection marker may be a NotI endonuclease recognition site. Therefore, a corresponding counter selection agent may be a NotI endonuclease. In one embodiment, the nucleotide sequence of the NotI endonuclease recognition site is provided herein as SEQ ID NO. 42, as follows: GCGGCCGC [SEQ ID NO. 42] The counter-selection marker may be a FseI endonuclease recognition site. Therefore, a corresponding counter selection agent may be a FseI endonuclease. In one embodiment, the nucleotide sequence of the FseI endonuclease recognition site is provided herein as SEQ ID NO. 43, as follows: GGCCGGCC [SEQ ID NO. 43] The counter-selection marker may be a AcsI / SgsI endonuclease recognition site. Therefore, a corresponding counter selection agent may be a AcsI / SgsI endonuclease. In one embodiment, the nucleotide sequence of the AcsI / SgsI endonuclease recognition site is provided herein as SEQ ID NO. 44, as follows: GGCGCGCC [SEQ ID NO. 44] The endonuclease recognition site may be or comprise one or more sites selected from the group consisting of: AbsI, SbfI / SdaI, MreI, MauBI, SgrDI, SrfI, AsiSI / SfaAI, NotI, FseI and AcsI / SgsI. Thus, the endonuclease recognition site may comprise a nucleotide sequence substantially as set out in any one of SEQ ID NOs. 35-44. The counter-selection marker of the invention may be a nuclease recognition site (e.g., a meganuclease recognition site or an endonuclease recognition site). The nuclease recognition site may comprise a nucleotide sequence substantially as set out in any one of SEQ ID NOs. 1-44. The counter-selection marker may be a toxic marker gene (e.g., a gene encoding an enzyme that is capable of catalysing the synthesis of a substance that is toxic to a host cell in response to contact with an exogenous substrate). Numerous such genes are known in the art, and include: the Bacillus subtilis sacB gene (encoding levan sucrase), which results in sucrose sensitivity; galK gene (encoding galactokinase), which mediates galactose or galactose analogue toxicity; tetAR antibiotic resistance genes which causes sensitivity to fusaric or quinalic acids; rpsL genes (codes for a component of the 30S ribosome), which cause sensitivity to streptomycin in cells carrying a mutant rpsL allele; a mutant pheS gene (encodes the α subunit of the phenylalanine-tRNA sythetase), which causes sensitivity to phenylalanine analogues like p- chlorophenylalanine; and the codA gene (encodes cytosine deaminase) and upp gene (encodes uracil phosphoribosyltransferase), which bring about sensitivity to 5- fluorocytosine (5FC) and 5-fluorouracil (5FU), respectively. Of particular note are the enzymes involved in the de novo pyrimidine biosynthesis pathway which is highly conserved across microorganisms and higher forms of life, and in particular orotate phosphoribosyl transferase and orotidine-5'-monophosphate decarboxylase. In bacteria, the encoded genes are designated pyrE and pyrF, respectively. pyrE or pyrF may be a counter-selection marker in the invention. pyrE converts 5-FOA into 5-fluroorotidine monophosphate (5-FOMP), which, in turn, is converted to the toxic substance, 5-fluorouridine monophosphate (5-FUMP) by pyrF. Thus, either pyrE or pyrF can be used as a counter-selection marker by being introduced into host cells that lack pyrE (a gene that encodes an enzyme with orotate phosphoribosyl transferase activity) or pyrF (a gene that encodes an enzyme with orotidine-5'-monophosphate decarboxylase activity) followed by culturing the host cells that comprise selection marker in media that contains 5-fluoroorotic acid (5-FOA) in order to exert a selection pressure against the cells. Only host cells that have inactivated the RAM or removed the group II intron by homologous recombination will survive the selection pressure. Preferably the toxic marker gene is pyrE or pyrF. In embodiments in which the toxic marker gene is pyrE or pyrF, a kit according to the invention may further comprise 5-FOA. Thus, cells that have been transformed with a nucleic acid molecule according to the invention, wherein the counter-selection marker is defined by any one of SEQ ID NOs. 1 to 44, will not survive being exposed to a counter selection pressure in the form of a relevant nuclease (meganuclease or endonuclease) or exogenous substrate (e.g., FOA), unless the RAM is subsequently inactivated or the group II intron is removed by homologous recombination. Thus, the toxic marker gene may be one or more genes selected from the group consisting of: sacB, galK, tetAR, rpsL, pheS, coda, upp, pyrE and pyrF. The toxic marker gene may be pyrE or pyrF. The toxic marker gene may be pyrE. The toxic marker gene may comprise a nucleotide sequence substantially as set out in any one of SEQ ID NOs. 54, 56 or 58. The group II intron of the nucleic acid molecule according to the invention encodes a retrotransposition-activated marker (RAM). The skilled person will appreciate that a RAM is an inactive positive selection marker gene, which, when activated, codes for a protein that provides a selection advantage, e.g., an antibiotic resistance protein. The positive selection marker of the RAM only becomes activated once the group II intron has been retrotransposed (i.e., inserted into the DNA of a host cell and converted from RNA into double stranded DNA) without, for example, a group I intron being present due to self-splicing of the group I intron (in embodiments where the RAM comprises a group I intron). This allows a user of the invention to quickly and easily identify / isolate host cells that have had their DNA modified to incorporate a positive selection marker (as part of a RAM) and a counter selection marker by phenotypic detection of the retrotransposed and suitably modified group II intron. Consequently, the invention avoids the need to perform PCR to identify host cells that have been successfully and stably transformed. The marker gene of the RAM disclosed herein may encode or encompass a group I intron. The group I intron may disrupt expression of the marker gene of the RAM. Thus, the group I intron prevents a transcript of the marker gene from being translated into a functional protein in the host cell prior to retrotransposition. The marker gene of the RAM may encode or encompass a group I intron in an orientation that enables the group I intron to catalytically self-splice itself out from the group II intron RNA transcript. This may occur prior to or during retrotransposition. Thus, the group I intron may be orientated such that it self-catalytically splices out of the transcript of the group II intron leaving a region encoding a functional positive selection marker protein. The marker gene of the RAM may be orientated in the reverse direction relative to the orientation of the group II intron. The group I intron may be orientated in the opposite direction to the marker gene. The group II intron may be orientated in the opposite direction to the marker gene. The group I intron and the group II intron may be orientated in the same direction. Encoding the group I intron in an orientation that enables the group I intron to splice itself out from an RNA transcript of the group II intron (thus leaving a region encoding a functional positive selection marker in the antisense orientation), results in retrotransposition of the group II intron producing a functional marker gene in the DNA of the host cell. Only cells encoding, in their DNA, a functional protein of the marker gene will be capable of expressing a functional protein and therefore surviving the relevant positive selection pressure. Host cells encoding (in their DNA) a marker gene comprising a group I intron are capable of producing a corresponding transcript of the marker gene but incapable of expressing a functional protein of the marker gene. Thus, such cells are not capable of surviving the relevant positive selection pressure. Like group II introns, group I introns are self-splicing introns (i.e., introns that remove themselves from RNA). However, unlike group II introns, they do so without the assistance of an IEP. The group I intron may self-splice with or without the assistance of a cofactor, such as a guanosine cofactor. Preferably, the group I intron referred to herein is capable of self-splicing without a cofactor. The group I intron of the nucleotide sequence of the invention may be from a bacteriophage. Preferably the group I intron is from a T4 phage. More preferably the group I intron is td (the group I intron located in the thymidylate synthase gene (td) of T4 phage). In one embodiment, the nucleotide sequence of the group I intron is td, provided herein as SEQ ID NO. 45, as follows: TAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTCTCTAGT AGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAATAAATACTTCTATATTTAAAGAGGTATT TATGAAAAGCGGAATTTATCAGATTAAAAATACTTTCTCTAGAGAAAATTTCGTCTGGATTAGTT ACTTATCGTGTAAAATCTGATAAATGGAATTGGTTCTACATAAATGCCTAACGACTATCCCTTTG GGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATATAGTCTG CTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTGAACATA ATG [SEQ ID NO. 45] In embodiments in which the RAM comprises a group I intron, the group I intron may be flanked by exons. The exon sequences may remain within the transcript after self- splicing of the group I intron has occurred. The remaining exon sequence(s) enable the RAM to encode a functional marker gene protein in a host cell once retrotransposition has occurred (due to the marker gene encoding a functional positive selection marker in the antisense orientation). Thus, the exons ensure that in-frame, splicing of the group I intron occurs. This, in turn, leads to the expression of a functional protein of the marker gene in host cells. Thus, the group I intron may be flanked by exons that enable self- splicing of the group I intron (i.e., splicing the RNA sequence / transcript). The marker gene may code for an antibiotic resistance marker gene. The antibiotic resistance marker gene of the RAM may be a marker gene selected from the group consisting of: beta-lactamase resistance marker gene (e.g., ampicillin- or carbenicillin- resistance marker gene), an aminoglycoside resistance marker gene (e.g., kanamycin-, streptomycin-, or spectinomycin- resistance marker gene), a macrolide resistance marker gene (e.g., an erythromycin resistance marker gene), a tetracycline resistance marker gene, a trimethoprim resistance marker gene (e.g., a type II dihydrofolate reductase), a chloramphenicol and thiamphenicol resistance marker gene and a glycopeptide resistance marker gene (e.g., a bleomycin resistance marker gene). Preferably the antibiotic resistance marker gene is a macrolide resistance marker gene. Most preferably the antibiotic resistance marker is a chloramphenicol and thiamphenicol resistance marker gene or an erythromycin resistance marker gene. Preferably the RAM and the marker of the RAM do not occur natively in the genome of the host cell. Thus, the RAM may be a chloramphenicol / thiamphenicol-resistance-RAM (Cat-RAM), an erythromycin-resistance-RAM (Erm-RAM), a kanamycin-resistance-RAM (Kan- RAM) or a trimethoprim-resistance-RAM (TpR-RAM). Preferably the RAM is a Cat- RAM. The RAM may comprise any one of CatA (e.g. Cat-RAM), ErmB (e.g. ErmB-RAM), PyrE (e.g. PyrE-RAM) or Kan (e.g. Kan-RAM). The RAM may be Cat-RAM. Cat (chloramphenicol acetyltransferase) is an enzyme that is encoded by the antibiotic resistance marker gene, cat. cat and thus Cat confers resistance to the antibiotic(s) chloramphenicol and / or thiamphenicol. This is because cat genes modify chloramphenicol and thereby confer resistance to chloramphenicol. Under anaerobic conditions, chloramphenicol is reduced and so generally is not used in bacteria which are obligate anaerobes, such as Clostridia. Thiamphenicol is an analogue of chloramphenicol that is generally not reduced by anaerobiosis. Hence the cat antibiotic resistance marker gene confers resistance to the antibiotics thiamphenicol and chloramphenicol, depending on the nature of the host cell targeted. Bacterial cat genes are widespread in the environment, being carried by a wide range of Gram-positive and Gram-negative species and can broadly be divided into those encoding Type A and Type B chloramphenicol acetyltransferases (based on their DNA – see Schwarz et al., 2004, FEMS Microbiology Reviews 28(5):519-542). Thus, the Cat may be a type A CAT (CatA) or a type B CAT (CatB). Preferably the Cat is a type A Cat. The type A and type B chloramphenicol acetyltransferases may be subdivided into subtypes depending on their source and sequence. For example, type A chloramphenicol acetyltransferases (CatA) may be subdivided into CatA1 through to CatA16, and type B chloramphenicol acetyltransferases (CatB) may be subdivided into CatB1 through to CatB5. Preferably the Cat is a CatA, such as CatA9. The CatA protein may be from Paenibacillus sylvae. In one embodiment, the nucleotide sequence of CatA-RAM is provided herein as SEQ ID NO. 46, as follows: ATGAAGTTCAACAGGATTGACTTTGATGTTTGGAATAGGACTGAGGTGTTTAATCACTATATGAA TCAGAACACTTCATTTAGCCTgCATTATGTTCAGATAAGGTCGTTAATCTTACCCCGGAATTATA TCCAGCTGCATGTCACCATGCAGAGCAGACTATATCTCCAACTTGTTAAAGCAAGTTGTCTATCG TTTCGAGTCACTTGACCCTACTCCCCAAAGGGATAGTCGTTAGGCATTTATGTAGAACCAATTCC ATTTATCAGATTTTACACGATAAGTAACTAATCCAGACGAAATTTTCTCTAGAGAAAGTATTTTT AATCTGATAAATTCCGCTTTTCATAAATACCTCTTTAAATATAGAAGTATTTATTAAAGGGCAGT CCTACAATTTAGCACGGGATTGTCTACTAGAGAGGTTCCCCGTTTAGATAGATTACAAGTATAAG TCACCTTATACTCAGGCCTCAATTAacccaagagaTAGATGTTTCTGTGTTATACAGATTCATAA AACAACATAAGTATAGGTTCACTCCTGCACTTATATTCTTGATTACTACTGTAGTAAACAGCCAC CCTGCTTTTAGAACTAGTTATAATTGTGAGGGAGACTTAGGATACTGGGACAGATTAGAGCCTCT ATACACTATTTTTGACAGGAGATCAGAGAGTTTTAGCGCAGTTTGGACTAGTGTAACTAATGACT TCGAGACTTTCCATAGAGCATACACAGCAGATGTTGAAAAATACAACGGATCAGGATCTCTATTT CCTAAGACACCTGTTCCTGAGAATACTTTTTCATTCAGCGTTATACCATGGACTTCATTCACATC TTTTAACTTAAATATAAATAACAATTCAAGATATTTGCTTCCTATAATAACTGCTGGTAGATTGA TACACCAAGGAAATTTGATTTACCTTCCTATTAGCCTACAATTGCATCATGCAGTATGCGACGGT TACCACGCAGGTCTATTTATGAACTCTGTTCAAGAGTTAGCTCACCACCCAAACAGCTGTATTTT T [SEQ ID NO. 46] The nucleotides of the group I intron of the CatA-RAM defined by SEQ ID NO. 46 are underlined. The RAM is in the sense orientation in this sequence, and the group I intron is in the antisense orientation. The nucleotides of the exons that flank the group I intron (which ensure that in-frame, splicing of the group I intron occurs) are in lowercase and italics. In another embodiment, the amino sequence of CatA after in-frame, self-splicing of a group I intron has occurred is provided herein as SEQ ID NO. 47, as follows: MKFNRIDFDVWNRTEVFNHYMNQNTSFSLTQEIDVSVLYRFIKQHKYRFTPALIFLITTVVNSHP AFRTSYNCEGDLGYWDRLEPLYTIFDRRSESFSAVWTSVTNDFETFHRAYTADVEKYNGSGSLFP KTPVPENTFSFSVIPWTSFTSFNLNINNNSRYLLPIITAGRLIHQGNLIYLPISLQLHHAVCDGY HAGLFMNSVQELAHHPNSCIF* [SEQ ID NO. 47] The key amino acid residues that enable the marker gene protein to be disrupted by a group I intron are in italics and underlined in SEQ ID NO. 47. These amino acid residues allow the marker gene protein to include the exons needed to ensure that in-frame, splicing of the group I intron occurs. Erm-RAM is a RAM that confers, to host cells, resistance to the antibiotic, erythromycin. Thus, in one embodiment, the nucleotide sequence of Erm-RAM is provided herein as SEQ ID NO. 48, as follows: ATGgCATTATGTTCAGATAAGGTCGTTAATCTTACCCCGGAATTATATCCAGCTGCATGTCACCA TGCAGAGCAGACTATATCTCCAACTTGTTAAAGCAAGTTGTCTATCGTTTCGAGTCACTTGACCC TACTCCCCAAAGGGATAGTCGTTAGGCATTTATGTAGAACCAATTCCATTTATCAGATTTTACAC GATAAGTAACTAATCCAGACGAAATTTTCTCTAGAGAAAGTATTTTTAATCTGATAAATTCCGCT TTTCATAAATACCTCTTTAAATATAGAAGTATTTATTAAAGGGCAGTCCTACAATTTAGCACGGG ATTGTCTACTAGAGAGGTTCCCCGTTTAGATAGATTACAAGTATAAGTCACCTTATACTCAGGCC TCAATTAacccaagagaTGCTGGTGCTTCTGGTGCTGGTATGAACAAAAATATAAAATATTCTCA AAACTTTTTAACGAGTGAAAAAGTACTCAACCAAATAATAAAACAATTGAATTTAAAAGAAACCG ATACCGTTTACGAAATTGGAACAGGTAAAGGGCATTTAACGACGAAACTGGCTAAAATAAGTAAA CAGGTAACGTCTATTGAATTAGACAGTCATCTATTCAACTTATCGTCAGAAAAATTAAAACTGAA TACTCGTGTCACTTTAATTCACCAAGATATTCTACAGTTTCAATTCCCTAACAAACAGAGGTATA AAATTGTTGGGAGTATTCCTTACCATTTAAGCACACAAATTATTAAAAAAGTGGTTTTTGAAAGC CATGCGTCTGACATCTATCTGATTGTTGAAGAAGGATTCTACAAGCGTACCTTGGATATTCACCG AACACTAGGGTTGCTCTTGCACACTCAAGTCTCGATTCAGCAATTGCTTAAGCTGCCAGCGGAAT GCTTTCATCCTAAACCAAAAGTAAACAGTGTCTTAATAAAACTTACCCGCCATACCACAGATGTT CCAGATAAATATTGGAAGCTATATACGTACTTTGTTTCAAAATGGGTCAATCGAGAATATCGTCA ACTGTTTACTAAAAATCAGTTTCATCAAGCAATGAAACACGCCAAAGTAAACAATTTAAGTACCG TTACTTATGAGCAAGTATTGTCTATTTTTAATAGTTATCTATTATTTAACGGGAGGAAATAA [SEQ ID NO. 48] The nucleotides of the group I intron of the Erm-RAM defined by SEQ ID NO. 48 are underlined. The RAM is in the sense orientation in this sequence, and the group I intron is in the antisense orientation. The nucleotides of the exons that flank the group I intron (which ensure that in-frame, splicing of the group I intron occurs) are in lowercase and italics. In another embodiment, the amino sequence of Erm after in frame, self-splicing of a group I intron has occurred is provided herein as SEQ ID NO. 49, as follows: MDPRDAGASGAGMNKNIKYSQNFLTSEKVLNQIIKQLNLKETDTVYEIGTGKGHLTTKLAKISKQ VTSIELDSHLFNLSSEKLKLNTRVTLIHQDILQFQFPNKQRYKIVGSIPYHLSTQIIKKVVFESH ASDIYLIVEEGFYKRTLDIHRTLGLLLHTQVSIQQLLKLPAECFHPKPKVNSVLIKLTRHTTDVP DKYWKLYTYFVSKWVNREYRQLFTKNQFHQAMKHAKVNNLSTVTYEQVLSIFNSYLLFNGRK* [SEQ ID NO. 49] The key amino acid residues that enable the marker gene protein to be disrupted by a group I intron are in bold and italics in SEQ ID NO. 49. The key amino acid residues are part of an N-terminal addition (i.e., linker), which has been underlined. These amino acid residues allow the marker gene protein to encode the exons needed to ensure that in-frame, splicing of the group I intron occurs. The RAM may be Kan-RAM. Kan-RAM is a RAM that confers, to host cells, resistance to the antibiotic, kanamycin. In one embodiment, the nucleotide sequence of Kan-RAM is provided herein as SEQ ID NO. 50, as follows: ATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACCGCTGCGTAAAAGA TACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGTGGGAGAAAATGAAAACCTATATTTAA AAATGACGGACAGCCGGTATAAAGGGACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTA TGGCTGGAAGGAAAGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAA TCTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATGAACAAAGCCCTG AAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTTCACTCCATCGACATATCGGATTGT CCCTATACGAATAGCTTAGACAGCCGCTTAGCCGAATTGGATTACTTACTGAATAACGATCTGGC CGATGTGGATTGCGAAAACTGGGAAGAAGACACTCCATTTAAAgCATTATGTTCAGATAAGGTCG TTAATCTTACCCCGGAATTATATCCAGCTGCATGTCACCATGCAGAGCAGACTATATCTCCAACT TGTTAAAGCAAGTTGTCTATCGTTTCGAGTCACTTGACCCTACTCCCCAAAGGGATAGTCGTTAG GCATTTATGTAGAACCAATTCCATTTATCAGATTTTACACGATAAGTAACTAATCCAGACGAAAT TTTCTCTAGAGAAAGTATTTTTAATCTGATAAATTCCGCTTTTCATAAATACCTCTTTAAATATA GAAGTATTTATTAAAGGGCAGTCCTACAATTTAGCACGGGATTGTCTACTAGAGAGGTTCCCCGT TTAGATAGATTACAAGTATAAGTCACCTTATACTCAGGCCTCAATTAacccaagagaGCTGTATG ATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTTTTCCCACGGCGACCTGGGAGACAGC AACATCTTTGTGAAAGATGGCAAAGTAAGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGA CAAGTGGTATGACATTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATG TCGAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATATTATATTTTA CTGGATGAATTGTTTTAG [SEQ ID NO. 50] The nucleotides of the group I intron of the Kan-RAM defined by SEQ ID NO. 50 are underlined. The RAM is in the sense orientation in this sequence, and the group I intron is in the antisense orientation. The nucleotides of the exons that flank the group I intron (which ensure that in-frame, splicing of the group I intron occurs) are in lowercase and italics. In another embodiment, the amino sequence of Kan after in-frame, self-splicing of a group I intron has occurred is provided herein as SEQ ID NO. 51, as follows: MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKMTDSRYKGTTYDVEREKDMML WLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYEDEQSPEKIIELYAECIRLFHSIDISDC PYTNSLDSRLAELDYLLNNDLADVDCENWEEDTPFKDPRELYDFLKTEKPEEELVFSHGDLGDSN IFVKDGKVSGFIDLGRSGRADKWYDIAFCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILL DELF* [SEQ ID NO. 51] The key amino acid residues that enable the marker gene protein to be disrupted by a group I intron are in italics and underlined in SEQ ID NO. 51. These amino acid residues allow the marker gene protein to encode the exons needed to ensure that in-frame, splicing of the group I intron occurs. The RAM may be TpR-RAM. TpR-RAM is a RAM that confers, to host cells, resistance to the antibiotic, trimethoprim. In one embodiment, the nucleotide sequence of TpR- RAM is provided herein as SEQ ID NO. 52, as follows: ATGGAgCATTATGTTCAGATAAGGTCGTTAATCTTACCCCGGAATTATATCCAGCTGCATGTCAC CATGCAGAGCAGACTATATCTCCAACTTGTTAAAGCAAGTTGTCTATCGTTTCGAGTCACTTGAC CCTACTCCCCAAAGGGATAGTCGTTAGGCATTTATGTAGAACCAATTCCATTTATCAGATTTTAC ACGATAAGTAACTAATCCAGACGAAATTTTCTCTAGAGAAAGTATTTTTAATCTGATAAATTCCG CTTTTCATAAATACCTCTTTAAATATAGAAGTATTTATTAAAGGGCAGTCCTACAATTTAGCACG GGATTGTCTACTAGAGAGGTTCCCCGTTTAGATAGATTACAAGTATAAGTCACCTTATACTCAGG CCTCAATTAacccaagaaaACAATGGAGTCAGTACTCTAGTTGCTGGCCAGTTTGCGCTCCCAAT GGACCAACACAACAATGGAGTCAGTACTCTAGTTGCTGGCCAGTTTGCGCTTCCATCGCACGCCA CGTTTGGCCTGGGAGATCGCGTGCGCAAGAAATCTGGCGCCGCTTGGCAGGGTCAAGTTGTCGGG TGGTACTGCACAAAACTGACCCCTGAAGGCTATGCCGTCGAGTCCGAGTCTCACCCCGGTTCAGT ACAGATTTATCCTGTGGCTGCGCTTGAACGCGTGGCCTAA [SEQ ID NO. 52] The nucleotides of the group I intron of the TpR-RAM defined by SEQ ID NO. 52 are underlined. The RAM is in the sense orientation in this sequence, and the group I intron is in the antisense orientation. The nucleotides of the exons that flank the group I intron (which ensure that in-frame, splicing of the group I intron occurs) are in lowercase and italics. In another embodiment, the amino sequence of TpR after in-frame, self-splicing of a group I intron has occurred is provided herein as SEQ ID NO. 53, as follows: METQENNGVSTLVAGQFALPMDQHNNGVSTLVAGQFALPSHATFGLGDRVRKKSGAAWQGQVVGW YCTKLTPEGYAVESESHPGSVQIYPVAALERVA* [SEQ ID NO. 53] The key amino acid residues that enable the marker gene protein to be disrupted by a group I intron are in italics and underlined in SEQ ID NO. 53. These amino acid residues allow the marker gene protein to encode the exons needed to ensure that in-frame, splicing of the group I intron occurs. The RAM of the invention may comprise an antibiotic resistance marker gene. The antibiotic resistance marker gene may be a chloramphenicol acetyltransferase (Cat) gene, an erythromycin (Erm) marker gene, a kanamycin (Kan) marker gene or a trimethoprim (TpR) marker gene. Thus, the RAM of the invention may be or comprise a nucleotide sequence substantially as set out in any one of SEQ ID NOs. 46, 48, 50 or 52. The marker gene protein of the RAM (after retrotransposition has occurred) may comprise an amino acid sequence substantially as set out in any one of SEQ ID NOs. 47, 49, 51 or 53. The marker gene of the RAM may or may not be a gene that codes for antibiotic resistance. The marker of the RAM may be an auxotrophy marker gene. The auxotrophy marker gene may encode a polypeptide or protein that essential for growth, e.g., an enzyme that is needed to synthesize a particular organic compound (e.g., amino acids, lipids, cofactors) essential for growth. Thus, through the use of a suitably auxotrophic host, the auxotrophy marker gene is able to restore the cell to prototrophy. For example, delivery of a functional copy of a gene encoding beta-isopropylmalate dehydrogenase which is required for the biosynthesis of the amino acid leucine, into a host (host cell) in which the chromosomal gene encoding this enzyme has been deleted. For example, in bacteria, the encoding gene is designated leuB, whereas in yeast, the equivalent gene is LEU2. Alternatively, the RAM may comprise a gene encoding an enzyme involved in substrate utilisation, without which the host may be unable to grow. Thus, growth on xylan or xylose as the sole carbon source, for instance, may require a functional copy of a gene encoding a xylanase (xyn) or xylose isomerase (xylA) in a host lacking these enzymes. Thus, the marker gene of the RAM may be one or more genes selected from the group consisting of: leuB, LEU2, xylA, and xyn. The auxotrophy or substrate utilisation marker gene may be inactivated by the presence of a group I intron. The marker gene of the RAM may be a gene that encodes an enzyme involved in purine or pyrimidine biosynthesis. In the pyrimidine biosynthesis pathway, for instance, the enzymes, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase, play a pivotal role. In bacteria these two enzymes are respectively encoded by genes designated as pyrE and pyrF. The skilled person will appreciate that pyrE and pyrF are bi-directional selection marker genes and can be used as a positive selection marker (selecting for restoration of uracil prototrophy) as well as a counter- selection marker under the appropriate conditions (media with no uracil supplementation). Thus, the pyrE referred to here may be identical to the pyrE used in the RAM as a positive selection marker. Thus, the auxotrophy marker gene of the RAM may encode pyrF or pyrE. In yeast and fungi, the equivalent genes are designated ura3 (pyrF) and ura5 (pyrE). Thus, the auxotrophy marker gene of the RAM may encode ura3, pyrF, ura5 or pyrE. A corresponding RAM may be referred to as pyrE-RAM, ura5-RAM, pyrF-RAM or ura3-RAM. Any one of these RAMs may be used as a positive selection marker by introducing them into host cells that lack corresponding functional copies. Following this the cells are cultured in media that lacks uracil in order to exert a selection pressure against the cells. Only host cells that have been successfully transformed with pyrE (ura5) or pyrF (ura3) are capable of synthesising uracil, and thus surviving the selection pressure. Example 3 illustrates how insertion of a group I self- splicing intron into selected pyrE genes converts them into a RAM. The heterologous auxotrophy marker gene, pyrE, may encode the pyrE protein of Clostridium leptum. The pyrE marker gene of the RAM may be inactivated by the presence of a group I intron. Thus, in one embodiment, the nucleotide sequence of pyrE- RAM is provided herein as SEQ ID NO. 54, as follows: TTGTTGCTgCATTATGTTCAGATAAGGTCGTTAATCTTACCCCGGAATTATATCCAGCTGCATGT CACCATGCAGAGCAGACTATATCTCCAACTTGTTAAAGCAAGTTGTCTATCGTTTCGAGTCACTT GACCCTACTCCCCAAAGGGATAGTCGTTAGGCATTTATGTAGAACCAATTCCATTTATCAGATTT TACACGATAAGTAACTAATCCAGACGAAATTTTCTCTAGAGAAAGTATTTTTAATCTGATAAATT CCGCTTTTCATAAATACCTCTTTAAATATAGAAGTATTTATTAAAGGGCAGTCCTACAATTTAGC ACGGGATTGTCTACTAGAGAGGTTCCCCGTTTAGATAGATTACAAGTATAAGTCACCTTATACTC AGGCCTCAATTAacccaagagaGAATTCTTGAAATTTTAAAGGAGGCCGGCGTTCTTTTAGAAGG GCATTTCTTATTGACTTCTGGAAGGCACAGCAATAAATATTTACAGTGCGCTAAGGTTTTCCGGA ATACAAAATACAGCGAGGAGTTGTGCAGCGCTCTGGCTGAACAGTTCCGAAATGATGGGGTAGAG GTGGTCATCGGACCCGCTATGGGGGCGGTGCAGATGGCTTATGAGGTAAGCCGGGGTTTACATTG TGAAAACTTTTTTGCAGAACGAGACCAGGATGGAAAAATGTGCTTGCGTAGGGGATTCGAGGTTC AACCTGGACAAAAGGTGCTTCTGGTAGAGGATGTAGTCACTACCGGAGGGTCTGTTCGTGAGGTT CTGGAGCTTGTTAAAGCTGCCGGCGGCGATGTGGTAGGGATTGGTTCTATTGTTGACCGTACTGG AGGAAAAATTGATTTTGGCGTACCCTTTAAAGCGGTTATTTCTATGGAAGTGGAATCTTATGAGC CCAGCGAATGTCCATTGTGTAAGGAAGGTAAAATTCCGGTTATAAAACCGGGAAGCCGTAAAATT AAATGA [SEQ ID NO. 54] The nucleotides of the group I intron of the pyrE-RAM defined by SEQ ID NO. 54 are underlined. The RAM is in the sense orientation in this sequence, and the group I intron is in the antisense orientation. The nucleotides of the exons that flank the group I intron (which ensure that in-frame, splicing of the group I intron occurs) are in lowercase and italics. In one embodiment, the amino acid sequence of Clostridium leptum pyrE (after in frame, self-splicing of a group I intron has occurred) is provided herein as SEQ ID NO. 55, as follows: LLLTQERILEILKEAGVLLEGHFLLTSGRHSNKYLQCAKVFRNTKYSEELCSALAEQFRNDGVEV VIGPAMGAVQMAYEVSRGLHCENFFAERDQDGKMCLRRGFEVQPGQKVLLVEDVVTTGGSVREVL ELVKAAGGDVVGIGSIVDRTGGKIDFGVPFKAVISMEVESYEPSECPLCKEGKIPVIKPGSRKIK * [SEQ ID NO. 55] The key amino acid residues that enable the marker gene protein to be disrupted by a group I intron are in italics and underlined in SEQ ID NO. 55. These amino acid residues allow the marker gene protein to encode the exons needed to ensure that in-frame, splicing of the group I intron occurs. The heterologous pyrE gene may encode the PyrE protein of Clostridiales bacterium isolate CIM:MAG 1312. The pyrE marker gene of the RAM may be inactivated by the presence of a group I intron. Thus, in one embodiment, the nucleotide sequence of the RAM is pyrE-RAM provided herein as SEQ ID NO. 56, as follows: ATGAACAAAACAGATTTTATTGAATTCATGCTAGCATCCGATGTTTTACGTTTCGGAGATTTTGT CACTAAAAGCGGTAGACAAACACAGTATTTTGTAAATACCGGAAATTACCGCACCGGTGCACAAA TTTCAACACTTGCAAAGTATTATGCTGAACTTATAACGGAAACGGTAGGTTCAGATTTTGACGCG ATGTTCGGTCCGGCTTATAAAGGAATACCTCTTGTAACGGCTGTTGCGGCTTCACTTTACAGTAA TTTCGGCATAGACAAACCTTACTTTTTTAACCGCAAAGAAGCAAAGGATCACGGTGAAGGCGGAT CGTTGGTGGGATATAAGCCCAAAGACGGCGATAAAATAATTATAATTGAAGACGTTATTACCGCA GGTACTGCTGTTCGTGAAACTCTACCGATTCTTTTTGATGCGGCAAAAGTTGAAGTAAATAATAT TTTCATAAGCGTAAATCGCTGTGAAAAAGGCCTTTCAGAAAAAACAGCgCATTATGTTCAGATAA GGTCGTTAATCTTACCCCGGAATTATATCCAGCTGCATGTCACCATGCAGAGCAGACTATATCTC CAACTTGTTAAAGCAAGTTGTCTATCGTTTCGAGTCACTTGACCCTACTCCCCAAAGGGATAGTC GTTAGGCATTTATGTAGAACCAATTCCATTTATCAGATTTTACACGATAAGTAACTAATCCAGAC GAAATTTTCTCTAGAGAAAGTATTTTTAATCTGATAAATTCCGCTTTTCATAAATACCTCTTTAA ATATAGAAGTATTTATTAAAGGGCAGTCCTACAATTTAGCACGGGATTGTCTACTAGAGAGGTTC CCCGTTTAGATAGATTACAAGTATAAGTCACCTTATACTCAGGCCTCAATTAacccaagagaTTT TTGAACAATACGGTATAAAAGTCAATTCCGTTGTAAATGTTTGCGACGTACATAATTATCTTTTA CAAAAAGCCCAAACGGAGCTTGCAAAAAAAATGGAAGATTATATGGAAAAATATTGCATTGTATA A [SEQ ID NO. 56] The nucleotides of the group I intron of the pyrE-RAM defined by SEQ ID NO. 56 are underlined. The RAM is in the sense orientation in this sequence, and the group I intron is in the antisense orientation. The nucleotides of the exons that flank the group I intron (which ensure that in-frame, splicing of the group I intron occurs) are in lowercase and italics. Thus, in one embodiment, the amino acid sequence of Clostridiales bacterium pyrE (after in-frame, self-splicing of a group I intron has occurred) is provided herein as SEQ ID NO. 57, as follows: MNKTDFIEFMLASDVLRFGDFVTKSGRQTQYFVNTGNYRTGAQISTLAKYYAELITETVGSDFDA MFGPAYKGIPLVTAVAASLYSNFGIDKPYFFNRKEAKDHGEGGSLVGYKPKDGDKIIIIEDVITA GTAVRETLPILFDAAKVEVNNIFISVNRCEKGLSEKTATQEIFEQYGIKVNSVVNVCDVHNYLLQ KAQTELAKKMEDYMEKYCIV [SEQ ID NO. 57] The key amino acid residues that enable the marker gene protein to be disrupted by a group I intron are in italics and underlined in SEQ ID NO. 57. These amino acid residues allow the marker gene protein to encode the exons needed to ensure that in-frame, splicing of the group I intron occurs. The heterologous pyrE gene may encode the PyrE protein of Legionella anisa. The pyrE marker gene of the RAM may be inactivated by the presence of a group I intron. Thus, in one embodiment, the nucleotide sequence of the pyrE-RAM provided herein as SEQ ID NO. 58, as follows: ATGAATCAAATGAAGAGCGCATTCATAAAACTAGCTTTGGACTGTCAGGTATTAAAGTTCGGTGA ATTTACACTTAAAAGCGGAAGAATTAGCCCATATTTCTTCAACGCTGGACTATTTTATCAAGGAA ACGCTTTGAGGCAGCTAGGTCAATTTTACGCTAAGACACTACTAGAGCACCAAGCTGAGTTCGAT CATTTGTTCGGTCCAGCTTACAAAGGACTTCCTTTGGCTACAGCTACAGCAATAGCATTGGCAGA ATTGGGTCAGGAGACTACTGTAACATTTAACAGGAAAGAGGTGAAGAACCATGGAGAAGGAGGTC AATTGATAGGTGCTCCTCTTACTGGTAAAACTATTGTGATAGACGACGTGATTACAGCTGGAACT GCTTTCAGGGAGAGCCAAATACTAATAAAAGAAAATGGAGGACAACTAACAGGTGTTATTATTGC TCTAGATAGGTGCGAAAGGGGTCTgCATTATGTTCAGATAAGGTCGTTAATCTTACCCCGGAATT ATATCCAGCTGCATGTCACCATGCAGAGCAGACTATATCTCCAACTTGTTAAAGCAAGTTGTCTA TCGTTTCGAGTCACTTGACCCTACTCCCCAAAGGGATAGTCGTTAGGCATTTATGTAGAACCAAT TCCATTTATCAGATTTTACACGATAAGTAACTAATCCAGACGAAATTTTCTCTAGAGAAAGTATT TTTAATCTGATAAATTCCGCTTTTCATAAATACCTCTTTAAATATAGAAGTATTTATTAAAGGGC AGTCCTACAATTTAGCACGGGATTGTCTACTAGAGAGGTTCCCCGTTTAGATAGATTACAAGTAT AAGTCACCTTATACTCAGGCCTCAATTAacccaagagaGTGCACTTGCAGAGATAAAAGCACAGG GAATTCACGTATATAGCATTATTACACTTTTCGACCTAATAGATTACTTGAAGTCAACACATCAG AATGAGCAGGTAAAGAAGCTAGAAGCATATCAAGCAGTATATGGATGTTAG [SEQ ID NO. 58] The nucleotides of the group I intron of the pyrE-RAM defined by SEQ ID NO. 58 is underlined. The RAM is in the sense orientation in this sequence, and the group I intron is in the antisense orientation. The nucleotides of the exons that flank the group I intron (which ensure that in-frame, splicing of the group I intron occurs) are in lowercase and italics. Thus, in one embodiment, the amino acid sequence of the Legionella anisa pyrE (after in-frame, self-splicing of a group I intron has occurred) is provided herein as SEQ ID NO. 59, as follows: MNQMKSAFIKLALDCQVLKFGEFTLKSGRISPYFFNAGLFYQGNALRQLGQFYAKTLLEHQAEFD HLFGPAYKGLPLATATAIALAELGQETTVTFNRKEVKNHGEGGQLIGAPLTGKTIVIDDVITAGT AFRESQILIKENGGQLTGVIIALDRCERGLTQESALAEIKAQGIHVYSIITLFDLIDYLKSTHQN EQVKKLEAYQAVYGC* [SEQ ID NO. 59] The key amino acid residues that enable the marker gene protein to be disrupted by a group I intron are in italics and underlined in SEQ ID NO. 59. These amino acid residues allow the marker gene protein to encode the exons needed to ensure that in-frame, splicing of the group I intron occurs. The auxotrophy marker gene of the RAM may be pyrE or pyrF. The auxotrophy marker gene of the RAM may be pyrE. Thus, the RAM of the invention may comprise a nucleotide sequence substantially as set out in any one of SEQ ID NO. 54, 56, or 58. The auxotrophy marker gene of the RAM may be leuB, LEU2, xylA, xyn, pyrE, pyrF, ura3 or ura5. Thus, the RAM of the invention may comprise a nucleotide sequence substantially as set out in SEQ ID NO. 46, 48, 50, 52, 54, 56, or 58. The marker gene protein of the RAM (after retrotransposition has occurred) may comprise an amino acid sequence substantially as set out in SEQ ID NO. 47, 49, 51, 53, 55, 57, or 59. The skilled person will appreciate that any RAM disclosed herein may be combined with a counter-selection marker disclosed herein to produce a nucleic acid molecule according to the invention. For example, a Cat-RAM (e.g., SEQ ID NO. 46) may be combined with a counter selection marker according to any one of SEQ ID NOs. 1 to 44. An Erm-RAM (e.g., SEQ ID NO. 48) may be combined with a counter selection marker according to any one of SEQ ID NOs. 1 to 44. A Kan-RAM (e.g., SEQ ID NO. 50) may be combined with a counter selection marker according to any one of SEQ ID NOs. 1 to 44. A TpR-RAM (e.g., SEQ ID NO. 52) may be combined with a counter selection marker according to any one of SEQ ID NOs. 1 to 44. A pyrE-RAM (e.g., SEQ ID NO. 54, SEQ ID NO. 56, or SEQ ID NO. 58) may be combined with a counter selection marker according to any one of SEQ ID NOs. 1 to 44. Once a nucleic acid molecule according to the invention is incorporated into the DNA of a host cell, the group II intron (transcript) is produced as a result of transcription. Retrotransposition (of the group II intron transcript) involves the group II intron inserting itself into a first (top) strand of the DNA of the host cell. This is achieved by specific nucleotides of the lariat RNA of the RNP complex (the targeting sequence) base pairing with complementary nucleotides in the DNA of the host cell. Three specific groups of nucleotides in group II intron RNA are responsible for the base pairing, called EBS1 (Exon Binding Site 1), EBS2 (Exon Binding Site 2) and delta. EBS1 hybridizes with ISB1 (Intron Binding Site 1) in a first (top) strand of the DNA of the host cell. EBS2 hybridizes with ISB2 (Intron Binding Site 2) in a first (top) strand of the DNA of the host cell. Delta hybridizes with delta’ in a first (top) strand of the DNA of the host cell. Thus, the targeting sequence of the nucleic acid molecule according to the invention causes the transcript of the group II intron to hybridise / bind to specific complementary nucleotides in the DNA of the host cell. Preferably the targeting sequence comprises a sequence that is complementary, or sufficiently complementary, to a target sequence of DNA of a host cell such that it can specifically hybridise to the target sequence. Preferably, the targeting sequence of the nucleic acid molecule of the invention comprises an EBS1, EBS2 and / or delta nucleotide sequence. Preferably the targeting sequence comprises an EBS1 and EBS2. The skilled person will appreciate that the specific nucleotide sequence of EBS1, EBS2 and delta will depend on the location in which the group II intron RNA is intended to retrotranspose. The skilled person will appreciate that the specific sequence of EBS1, EBS2 and / or delta may be generated using any mutagenic technique known in the art. The skilled person will further recognise that suitable intron insertion sites in a target gene can be identified by screening, where they can be scored for optimal insertion based on how closely they match up to a database of successful intron targeting locations (e.g. of efficiently retargeted Ll.LtrB introns). The total length of sequence considered for an intron targeting region is typically 45 base pairs, and the EBS1 / IBS1 and EBS2 / IBS2 are at a fixed position within this 45 base pair sequence, and they are six nucleotides and five nucleotides in length respectively. Details of such sites are discussed in detail by Perutka et al. (Journal of Molecular Biology, Volume 336, Issue 2, 2004, Pages 421- 439), which is herein incorporated by reference. The target sequence may comprise any DNA sequence of a host cell. The target sequence may comprise a gene sequence, or part thereof. In one embodiment, the gene target maybe selected from Spo0A, codA, pyrF, pyrE, and mtlD. Insertion of the group II intron into a first (top) strand of the DNA of the host cell also involves the IEP of the RNP complex cleaving the first (top) strand of the DNA of the recipient / host cell. This is achieved by endonuclease activity of the IEP. Therefore, the IEP encoded by nucleic acid molecule according to the invention may comprise endonuclease activity. Once the group intron has been inserted into a first (top) strand of the DNA of the host cell, reverse transcriptase activity of the IEP protein causes synthesis of a first (bottom) strand of the DNA. Therefore, the IEP encoded by the nucleic acid molecule according to the invention may comprise reverse transcriptase activity. Host factors of the host cell complete the insertion process, such that the intron RNA is removed from the first strand of DNA and a second strand of DNA is synthesised. Preferably the IEP encoded by the nucleic acid molecule according to the invention comprises endonuclease activity and reverse transcriptase activity. The IEP encoded by the nucleic acid molecule according to the invention may or may not comprise reverse maturase activity. The IEP encoded by the nucleic acid molecule according to the invention may be the LtrA IEP, the EcI5 IEP or the RmInt1 IEP. Preferably the IEP encoded by the nucleic acid molecule is LtrA. In one embodiment, the nucleotide sequence of LtrA is provided herein as SEQ ID NO. 60, as follows: ATGAAACCAACAATGGCAATTTTAGAAAGAATCAGTAAAAATTCACAAGAAAATATAGACGAAGT TTTTACAAGACTTTATCGTTATCTTTTACGTCCAGATATTTATTACGTGGCGTATCAAAATTTAT ATTCCAATAAAGGAGCTTCCACAAAAGGAATATTAGATGATACAGCGGATGGCTTTAGTGAAGAA AAAATAAAAAAGATTATTCAATCTTTAAAAGACGGAACTTACTATCCTCAACCTGTACGAAGAAT GTATATTGCAAAAAAGAATTCTAAAAAGATGAGACCTTTAGGAATTCCAACTTTCACAGATAAAT TGATCCAAGAAGCTGTGAGAATAATTCTTGAATCTATCTATGAACCGGTATTCGAAGATGTGTCT CACGGTTTTAGACCTCAACGAAGCTGTCACACAGCTTTGAAAACAATCAAAAGAGAGTTTGGCGG CGCAAGATGGTTTGTGGAGGGAGATATAAAAGGCTGCTTCGATAATATAGACCACGTTACACTCA TTGGACTCATCAATCTTAAAATCAAAGATATGAAAATGAGCCAATTGATTTATAAATTTCTAAAA GCAGGTTATCTGGAAAACTGGCAGTATCACAAAACTTACAGCGGAACACCTCAAGGTGGAATTCT ATCTCCTCTTTTGGCCAACATCTATCTTCATGAATTGGATAAGTTTGTTTTACAACTCAAAATGA AGTTTGACCGAGAAAGTCCAGAAAGAATAACACCTGAATATCGGGAGCTCCACAATGAGATAAAA AGAATTTCTCACCGTCTCAAGAAGTTGGAGGGTGAAGAAAAAGCTAAAGTTCTTTTAGAATATCA AGAAAAACGTAAAAGATTACCCACACTCCCCTGTACCTCACAGACAAATAAAGTATTGAAATACG TCCGGTATGCGGACGACTTCATTATCTCTGTTAAAGGAAGCAAAGAGGACTGTCAATGGATAAAA GAACAATTAAAACTTTTTATTCATAACAAGCTAAAAATGGAATTGAGTGAAGAAAAAACACTCAT CACACATAGCAGTCAACCCGCTCGTTTTCTGGGATATGATATACGAGTAAGGAGATCTGGAACGA TAAAACGATCTGGTAAAGTCAAAAAGAGAACACTCAATGGGAGTGTAGAACTCCTTATTCCTCTT CAAGACAAAATTCGTCAATTTATTTTTGACAAGAAAATAGCTATCCAAAAGAAAGATAGCTCATG GTTTCCAGTTCACAGGAAATATCTTATTCGTTCAACAGACTTAGAAATCATCACAATTTATAATT CTGAACTCCGCGGGATTTGTAATTACTACGGTCTAGCAAGTAATTTTAACCAGCTCAATTATTTT GCTTATCTTATGGAATACAGCTGTCTAAAAACGATAGCCTCCAAACATAAGGGAACACTTTCAAA AACCATTTCCATGTTTAAAGATGGAAGTGGTTCGTGGGGGATCCCGTATGAGATAAAGCAAGGTA AGCAGCGCCGTTATTTTGCAAATTTTAGTGAATGTAAATCCCCTTATCAATTTACGGATGAGATA AGTCAAGCTCCTGTATTGTATGGCTATGCCCGGAATACTCTTGAAAACAGGTTAAAAGCTAAATG TTGTGAATTATGTGGGACGTCTGATGAAAATACTTCCTATGAAATTCACCATGTCAATAAGGTCA AAAATCTTAAAGGCAAAGAAAAATGGGAAATGGCAATGATAGCGAAACAACGTAAAACTCTTGTT GTATGCTTTCATTGTCATCGTCACGTGATTCATAAACACAAGTGA [SEQ ID NO. 60] Thus, LtrA may comprise a nucleotide sequence substantially as set out in SEQ ID NO. 60. The nucleotide sequence of one embodiment of a group II intron comprising a CatA- RAM is provided herein as SEQ ID NO. 61, as follows: GTGTAGTAGCCTGTGAAATAAGTAAGGAAAAAAAAGAAGTAAGTGTTATATATGATGATTATTTT GTAGATGTAGATAGGATAATAGAATCCATAGAAAATATAGGTTATACAGTTATATAAAAATTACT TTAAAAATTAATAAAAACATGGTAAAATATAAATCGTATAAAGTTGTGTAATTTTTAAGCTTATA ATTATCCTTACCTATCCCAAGGGTGCGCCCAGATAGGGTGTTAAGTCAAGTAGTTTAAGGTACTA CTCTGTAAGATAACACAGAAAACAGCCAACCTAACCGAAAAGCGAAAGCTGATACGGGAACAGAG CACGGTTGGAAAGCGATGAGTTACCTAAAGACAATCGGGTACGACTGAGTCGCAATGTTAATCAG ATATAAGGTATAAGTTGTGTTTACTGAACGCAAGTTTCTAATTTCGGTTATAGGTCGATAGAGGA AAGTGTCTGAAACCTCTAGTACAAAGAAAGGTAAGTTAACCCTTGGGACTTATCTGTTATCACCA CATTTGTACAATCTGTAGGAGAACCTATGGGAACGAAACGAAAGCGATGCCGAGAATCTGAATTT ACCAAGACTTAACACTAACTGGGGATACCCTAAACAAGAATGCCTAATAGAAAGGAGGAAAAAGG CTATAGCACTAGAGCTTGAAAATCTTGCAAGGGTACGGAGTACTCGTAGTAGTCTGAGAAGGGTA ACGCCCTTTACATGGCAAAGGGGTACAGTTATTGTGTACTAAAATTAAAAATTGATTAGGGAGGA AAACCTCAAAATGAAACCAACAATGGCAATTTTAGAAAGAATCAGTAAAAATTCACAAGAAAATA TAGACGAAGTTTTTACAAGACTTTATCGTTATCTTTTACGTCCAGATATTTATTACGTGGCGACG CGTTAGGGATAACAGGGTAATTATATACTAAAAAATACAGCTGTTTGGGTGGTGAGCTAACTCTT GAACAGAGTTCATAAATAGACCTGCGTGGTAACCGTCGCATACTGCATGATGCAATTGTAGGCTA ATAGGAAGGTAAATCAAATTTCCTTGGTGTATCAATCTACCAGCAGTTATTATAGGAAGCAAATA TCTTGAATTGTTATTTATATTTAAGTTAAAAGATGTGAATGAAGTCCATGGTATAACGCTGAATG AAAAAGTATTCTCAGGAACAGGTGTCTTAGGAAATAGAGATCCTGATCCGTTGTATTTTTCAACA TCTGCTGTGTATGCTCTATGGAAAGTCTCGAAGTCATTAGTTACACTAGTCCAAACTGCGCTAAA ACTCTCTGATCTCCTGTCAAAAATAGTGTATAGAGGCTCTAATCTGTCCCAGTATCCTAAGTCTC CCTCACAATTATAACTAGTTCTAAAAGCAGGGTGGCTGTTTACTACAGTAGTAATCAAGAATATA AGTGCAGGAGTGAACCTATACTTATGTTGTTTTATGAATCTGTATAACACAGAAACATCTATCTC TTGGGTTAATTGAGGCCTGAGTATAAGGTGACTTATACTTGTAATCTATCTAAACGGGGAACCTC TCTAGTAGACAATCCCGTGCTAAATTGTAGGACTGCCCTTTAATAAATACTTCTATATTTAAAGA GGTATTTATGAAAAGCGGAATTTATCAGATTAAAAATACTTTCTCTAGAGAAAATTTCGTCTGGA TTAGTTACTTATCGTGTAAAATCTGATAAATGGAATTGGTTCTACATAAATGCCTAACGACTATC CCTTTGGGGAGTAGGGTCAAGTGACTCGAAACGATAGACAACTTGCTTTAACAAGTTGGAGATAT AGTCTGCTCTGCATGGTGACATGCAGCTGGATATAATTCCGGGGTAAGATTAACGACCTTATCTG AACATAATGCAGGCTAAATGAAGTGTTCTGATTCATATAGTGATTAAACACCTCAGTCCTATTCC AAACATCAAAGTCAATCCTGTTGAACTTCATatgaatccctcctaatttatacgttttctctaac aacttaattatacccactattattatttttatcaatataACGCGTTGGGAAATGGCAATGATAGC GAAACAACGTAAAACTCTTGTTGTATGCTTTCATTGTCATCGTCACGTGATTCATAAACACAAGT GAATGTCGACAGTGAATTTTTACGAACGAACAATAACAGAGCCGTATACTCCGAGAGGGGTACGT ACGGTTCCCGAAGAGGGTGGTGCAAACCAGTCACAGTAATGTGAACAAGGCGGTACCTCCCTACT TCACCATATCATTTTCTGCAGCCCCCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACA TATATGGCTAGATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATAT GCGTTGATGCAATTTCTATGC [SEQ ID NO. 61] The RAM is in the sense orientation in this sequence. The nucleotides of the Clostridium sporogenes fdx promoter (the first promoter, which is responsible for driving group II intron expression) are in bold only. The nucleotides of the intron targeting region (which in this embodiment target Clostridium sporogenes spo0A) are doubly underlined. The nucleotides counter-selection marker, the I-SceI recognition sequence, are in bold and singly underlined. The nucleotides of the Cat marker gene are in italics and bold (and in the antisense orientation). The nucleotides of the exons flanking the group I intron are in italics and doubly underlined. The nucleotides of the group I intron are singly underlined only and in the antisense orientation. The nucleotides of the C. ac thl promoter (the third promoter), in the antisense orientation, are in lowercase only. The third promoter causes transcription of the selectable marker gene (Cat) once the group II intron has been retrotransposed into a target site in the DNA of a host cell. The first promoter is capable of causing the transcription of the group II intron. Therefore, the first promoter causes a host cell to produce a transcript of the first nucleic acid molecule including the group II intron. The first promoter may be a constitutive promoter or an inducible promoter. Preferably the first promoter is recognised (bound) by a sigma factor (housekeeping sigma factor or an alternative sigma factor) of the host cell. Thus, the promoter may be a native promoter or a promoter that has a nucleotide sequence substantially similar to a native promoter. The promoter may be a native promoter derived from a gene encoding an enzyme or protein involved in an essential metabolic process, for example, central metabolism. In anaerobic bacteria, the first promoter may therefore be derived from a gene encoding a ferredoxin. Therefore, in one embodiment, the first promoter may be that of the ferredoxin gene of Clostridium sporogenes, the Pfdx promoter. Ferredoxins are abundant iron-sulphur proteins responsible for electron transfer in a wide variety of metabolic reactions. The first promoter may be a native promoter derived from a gene encoding an enzyme involved in the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, or the citric acid cycle. Thus, the first promoter may be derived from a gene encoding thiolase, the enzyme responsible for catalysing the condensation of two acetyl-coenzyme A (CoA) molecules. Thus, in one embodiment, the first promoter may be that of the thiolase gene of Clostridium acetobutylicum (the Pthl promoter). In embodiments in which the host cell is a yeast such as Saccharomyces cerevisae, the first promoter may be Ptef1, the promoter of the translational elongation factor EF-1 alpha. The first promoter may be Ptef1, the promoter of glycolytic genes, such as 3- phosphoglycerate kinase. The first promoter may be Ptdh3, the promoter of the glyceraldehyde-3-phosphate dehydrogenase gene. The first promoter may be Padh1, the promoter of the alcohol dehydrogenase gene. In embodiments in which the host cell is a Gram-positive bacterium, the promoter may be Pthl or Pfdx. In embodiments in which the host cell is a Gram-negative bacterium, such as Escherichia coli, the first promoter may be Pspc, the promoter of the spc ribosomal protein operon. The first promoter may be the PL promoter of phage λ, the P1 and P2 promoters of the rrnB ribosomal RNA operon, the trp promoter of the tryptophan biosynthesis operon, the lac promoter of the lactose utilisation operon or the trp / lac hybrid promoters trc and tac. These promoters will be equally applicable to other Gram- negative hosts, such as those belonging to the genus Pseudomonas and Cupriavidus, where other more species-specific will also be suitable, such as the promoter regions of Cupriavidus necator genes involved in the synthesis of polyhydroxyalkanoates (PHAs) typified by PphaC the promoter of the phaC gene (encoding PHA synthase). The group II intron may comprise a (second) promoter operably linked to a nucleic acid sequence encoding an intron-encoded protein. Thus, the second promoter causes transcription of the IEP gene. Preferably the second promoter is the PltrA promoter. In one embodiment, the nucleotide sequence of PltrA is provided herein as SEQ ID NO. 62, as follows: ACTCGTAGTAGTCTGAGAAGGGTAACGCCCTTTACATGGCAAAGGGGTACAGTTATTGTGTACTA AAATTAAAAATTGATTAGGGAGGAAAACCTCAAA [SEQ ID NO. 62] The marker gene of the RAM may further comprise a (third) promoter operably-linked to a coding sequence encoding the positive selection marker gene. The positive selection marker gene may be functional or non-functional. Thus, the third promoter is capable of causing the transcription of the selectable marker gene once it has been retrotransposed into a target site in the DNA of a host cell (regardless of whether the group I intron has been spliced out of the RAM). The third promoter may be a constitutive promoter or an inducible promoter. Preferably the third promoter is recognised by a sigma factor of the host cell. Thus, the promoter may be a native promoter or a promoter that has a nucleotide sequence substantially similar to a native promoter. The promoter may be a native promoter derived from a gene encoding an enzyme or protein involved in an essential metabolic process, for example, central metabolism. The third promoter may therefore be derived from a gene encoding a ferredoxin. Therefore, in one embodiment, the third promoter may be that of the ferredoxin gene of Clostridium sporogenes, the Pfdx promoter. The third promoter may be a native promoter derived from a gene encoding a gene encoding an enzyme involved in the EMP pathway of glycolysis, the pentose phosphate pathway, or the citric acid cycle. Thus, the third promoter may therefore be derived from a gene encoding thiolase the enzyme responsible for catalysing the condensation of two acetyl-coenzyme A molecules. Therefore, in one embodiment, the third promoter may be that of the thiolase gene of Clostridium acetobutylicum, the Pthl promoter. In one embodiment, the nucleotide sequence of Pthl is provided herein as SEQ ID NO. 63, as follows: TATATTGATAAAAATAATAATAGTGGGTATAATTAAGTTGTTAGAGAAAACGTATAAATTAGGAG GGATTCAT [SEQ ID NO. 63] The (first) nucleic acid molecule may comprise a replicon. The replicon may be a Gram- negative replicon or Gram-positive replicon such as pCB102 (e.g., a conditional version of the pCB102 replicon). The replicon may be operably linked to a fourth promoter. Preferably the fourth promoter is an inducible promoter. Induction of the inducible promoter may cause loss of the first nucleic acid molecule (e.g., plasmid loss), which is typically faster than just subculturing the host cell containing the first nucleic acid molecule (e.g., plasmid) in non-selective conditions. Induction destabilises the replicon and thus removes the need for passage through non-selective culture conditions to lose the plasmid and stabilise the intron. This ultimately enables one to further increase the speed of the mutant generation process using the nucleic acid molecule of the invention. The invention may be a kit comprising a first nucleic acid molecule according to the invention and a second nucleic acid molecule encoding a homologous recombination cassette. Thus, according to a third aspect, there is provided a kit comprising a first nucleic acid molecule; and a second nucleic acid molecule encoding a homologous recombination cassette, wherein the first nucleic acid molecule encodes: (a) a first promoter operably-linked to a (single) group II intron, wherein the group II intron does not encode a protein comprising reverse transcriptase activity, and (b) a second promoter operably-linked to a nucleic acid sequence encoding an intron encoded protein (IEP), wherein the group II intron encodes / comprises: (i) a retrotransposition-activated genetic marker (RAM), (ii) a counter-selection marker, and (iii) a targeting sequence, wherein, in use, (a transcript of) the group II intron is inserted into a target site of the DNA of the host cell by a protein of the IEP and the targeting sequence of the group II intron. According to a fourth aspect, there is provided a kit comprising a host cell; and a second nucleic acid molecule encoding a homologous recombination cassette, wherein the host cell comprises a nucleic acid molecule encoding: (a) a first promoter operably-linked to a (single) group II intron, wherein the group II intron does not encode a protein comprising reverse transcriptase activity, and (b) a second promoter operably-linked to a nucleic acid sequence encoding an intron encoded protein (IEP), wherein the group II intron encodes / comprises: (i) a retrotransposition-activated marker (RAM), (ii) a counter-selection marker, and (iii) a targeting sequence, wherein, in use, (a transcript of) the group II intron is inserted into a target site of the DNA of the host cell by a protein of the IEP and the targeting sequence of the group II intron. The homology cassette of the kit according to the invention may comprise a first homology arm and a second homology arm, wherein the first homology arm is homologous with a first homology region in the DNA of a host cell and the second homology arm is homologous a second homology region in the DNA of the host cell. In one embodiment, the first and second homology arms are located directly adjacent to each other. Therefore, there may be no intervening sequence between the first and second homology arms. Use of the second nucleic acid molecule in conjunction with (e.g., after) the first nucleic acid molecule of the invention results in deletion / removal of a polynucleotide from the DNA of a host cell due to homologous recombination (e.g., heterologous or native polynucleotide). In an alternative embodiment, use of the second nucleic acid molecule in conjunction with (e.g., after) the first nucleic acid molecule of the invention results in in-frame insertion of the polynucleotide (e.g., a bookmark or a gene of interest) into the DNA of a host cell (from the second nucleic acid molecule) due to homologous recombination. Thus, the first and the second homology arms may flank a polynucleotide. The polynucleotide may encode a gene of interest. The polynucleotide may be a heterologous polynucleotide. The gene of interest may be a wild type gene of the host cell. The polynucleotide may be a bookmark. The bookmark may be a unique polynucleotide sequence to be left in place of a wild type allele in the DNA of a host cell. The bookmark may leave an in-frame non-functional protein in place of the wild type allele. The bookmark may be targeted by a group II intron, re-delivering the RAM and the counter- selection marker to a locus of the host cell. The bookmark may be targeted by a group II intron such that the locus can be restored to the wild type sequence (for example, during complementation studies), or such that a second polynucleotide can replace the bookmark. The bookmark may comprise a unique 45 base pair sequence that can be efficiently targeted by a specific group II intron. Alternatively, the bookmark may comprise a (second) meganuclease recognition site. Preferably the (second) meganuclease recognition site is different to that of the host cell or the first nucleic acid molecule. The second meganuclease site may allow the target site to be modified or corrected without redelivery of a group II intron (e.g., a first nucleic acid molecule or a group II intron according to the invention). Use of this second nucleic acid molecule in conjunction with the first nucleic acid molecule of the invention results in insertion of the polynucleotide (e.g., a bookmark or gene of interest) into the DNA of a host cell (from the second nucleic acid molecule) due to homologous recombination. The second nucleic acid molecule referred to herein may further comprise the necessary machinery to induce a selection pressure against host cells in which the group II intron (comprising the counter-selection marker) has been removed. Thus, the second nucleic acid molecule may further encode a (corresponding) counter selection agent. For example, in embodiments in which the counter-selection marker of the first nucleic acid molecule is a nuclease recognition site, the counter selection agent is the corresponding nuclease (e.g., meganuclease or endonuclease) of the nuclease recognition site. In embodiments in which the toxic marker gene is pyrE or pyrF, the kit according to the invention (i.e., the third or fourth aspect) may further comprise 5-FOA. In embodiments in which the counter selection marker is nuclease recognition site, the kit according to the invention may further comprise a (corresponding) counter selection agent. In embodiments in which the counter-selection marker of the first nucleic acid molecule is a gene encoding an enzyme that is capable of catalysing the synthesis of a substrate that is toxic to a host cell in response to contact with an exogenous substrate (i.e., a toxic marker gene), the counter selection agent is the exogenous substrate of the enzyme. The counter-selection agent may be encoded by the second nucleic acid molecule referred to herein. A polynucleotide sequence encoding the counter-selection agent (e.g., a nuclease or a substrate) may be operably-linked to a fifth promoter. Thus, the fifth promoter may be capable of causing expression of the counter selection agent in the host cell. Preferably the fifth promoter is an inducible promoter. Thus, the kit may further comprise an inducer that can specifically induce transcription at the fifth promoter. In embodiments in which the counter-selection marker is a nuclease recognition site, the second nucleic acid molecule may further encode an inducible (fifth) promoter operably-linked to a polynucleotide sequence encoding a nuclease that is capable of cleaving a specific nuclease recognition site. Thus, the second nucleic acid molecule may encode a homologous recombination cassette and an inducible (fifth) promoter operably-linked to a polynucleotide sequence encoding a nuclease that is capable of cleaving a specific corresponding nuclease recognition site. In embodiments in which the counter-selection marker is a toxic (enzyme) marker gene, the second nucleic acid molecule may further encode an inducible (fifth) promoter operably-linked to a polynucleotide sequence encoding an exogenous substrate of the toxic marker. Thus, the second nucleic acid molecule may encode a homologous recombination cassette and an inducible (fifth) promoter operably-linked to a polynucleotide sequence encoding a toxic (enzyme) marker gene. The inducible promoter may be the tetracycline promoter. The second nucleic acid referred to herein may comprise an inducible promoter system operably-linked to the polynucleotide sequence encoding the counter-selection agent. The inducible promoter system may be the synthetic anhydrotetracycline (Ptet) promoter system, the cumate- inducible promoter system, a riboswitch-controlled system (e.g., a system responsive to theophylline), an arabinose-controlled system (ParaBAD), a m-toluic acid-controlled system (e.g., a system comprising a PxylS promoter), a xylose-controlled system (comprising a Pxyl and / or ParaE promoter), a lactose controlled system (e.g., a system comprising a PbgaL Plac, Ptrc and / or Ptac promoter) or a rhamnose controlled system (e.g., a system comprising a PrhaBAD promoter). It will be apparent to the skilled person that the first, second, third, fourth and fifth promoters referred to herein should be different from each other. It will also be apparent that the first, second, third, fourth and fifth promoters must be capable of initiating transcription in the host cell. A kit according to the invention may be used to perform a method according to the invention. Thus, a nucleic acid molecule of the invention (e.g., the first, the second or the third aspects) may be used in a method according to the invention. A kit according to the invention may comprise a (first) nucleic acid molecule according to the invention. A kit according to the invention may further comprise a second nucleic acid molecule, wherein the second nucleic acid molecule comprises a homologous recombination cassette. The first nucleic acid molecule according to the invention may be used as part of a two- step process to genetically modify the DNA of a host cell (see Figure 1). The first step of the process comprises retrotransposition of a transcript of the group II intron of the nucleic acid molecule into DNA of a host cell. The location at which retrotransposition occurs is determined by the targeting sequence of the group II intron. In one embodiment, the second step of the two-step process exploits homologous recombination to insert a heterologous polynucleotide into the DNA of a host cell. Thus, the second step may comprise using a second nucleic acid molecule comprising a homology cassette. The homology cassette may comprise a pair of homology arms (a first homology arm and a second homology arm) that flank a polynucleotide to be inserted into the DNA of a host cell. The first homology arm of the homology cassette is homologous with a first homology region of the host cell DNA and second homology arm of the homology cassette is homologous with a second homology region of the host cell DNA, wherein the first homology region and the second homology region flank the location into which the group II intron is to be retrotransposed. Thus, once homologous recombination occurs, the first homology region through to the second homology region of the host cell DNA (thus including the flanked group II intron) is replaced with the homology cassette of the second nucleic acid molecule, including the flanked polynucleotide. This ultimately results in the polynucleotide being inserted into the DNA of the host cell. In another embodiment, the second step of the two-step process exploits homologous recombination to remove a polynucleotide from the DNA of a host cell. This is achieved by providing a homology cassette comprising a pair of homology arms (a 5’ homology arm and a 3’ homology arm) without an intervening or flanked polynucleotide sequence. Thus, once homologous recombination has taken place, the first homology region through to the second homology region of the host cell DNA (thus including the flanked group II intron) is replaced with the homology cassette of the second nucleic acid molecule. This ultimately results in the group II intron being removed from the DNA of the host cell. In another embodiment, the second step of the two-step process exploits homologous recombination to replace a polynucleotide from the DNA of a host cell. There is no theoretical upper limit to the size of the homology arms used and at the lower limit could be as small 14 base pairs which can still support homologous recombination, albeit at low efficiency. Thus, the first and second homology arms used herein are at least about 14 base pairs. Preferably, however, the first and second homology arms used herein are each about 500-1000 base pairs in length. Homology arms above 1000 base pairs do not exhibit significantly increased frequency or accuracy of homologous recombination relative to 1000 base pair regions. Homology arms below 500 base pairs exhibit reduced the efficiency and / or accuracy during homologous recombination. In one embodiment of the method according to the invention, the first nucleic acid molecule according to the invention may comprise the features of the second nucleic acid molecule referred to herein. Thus, the first and second nucleic acid molecules may be present in a single nucleic acid molecule. Preferably the second promoter is an inducible promoter. This ensures that once induction has stopped, the promoter is repressed to the extent that the intron is stabilised. Preferably the counter-selection marker is not functional within the nucleic acid molecule per se. According to a fifth aspect, there is provided a method of introducing a polynucleotide into the DNA of a plurality of host cells, the method comprising: (i) providing a plurality of host cells that all comprise a nucleotide sequence encoding a counter-selection marker; (ii) using homologous recombination to replace the nucleotide sequence encoding the counter-selection marker with a polynucleotide in one or more of the plurality of host cells; and (iii) applying a counter-selection pressure to the plurality host cells such that only host cells in which the counter-selection marker has been replaced, survive. Step (i), the step of providing a plurality of host cells that all comprise a counter- selection marker, may comprise using a RAM. Step (i) may further comprise applying a positive selection pressure to the plurality host cells such that only host cells in which retrotransposition of a functional positive selection marker gene has occurred will survive. The RAM may be a RAM referred to herein. The RAM may be retrotransposed into the plurality of host cells using a group I intron or a group II intron. Preferably, the RAM is retrotransposed using a group II intron. Thus, step (i) may comprise transforming a plurality of host cells with a (first) nucleic acid molecule according to the invention (e.g., a first aspect of the invention), and applying a positive selection pressure to the plurality host cells such that only host cells in which retrotransposition of a group II intron, which encompasses a RAM encoding a functional positive selection marker, survive. Preferably, prior to step (ii), the polynucleotide is flanked by a first homology arm and a second homology arm. Thus, step (ii) may comprise transforming one or more of the plurality of host cells with a (second) nucleic acid molecule that encode a homologous recombination cassette. The homology cassette may comprise a first and a second homology arm that flank the heterologous polynucleotide, wherein the first homology arm is homologous with the first homology region and the second homology arm is homologous with the second homology region, and wherein the first homology region and the second homology region flank the counter-selection marker. The polynucleotide may be a polynucleotide referred to herein. The first and second homology arms referred to herein may be at least about 14 base pairs. Preferably, however, the first and second homology arms used herein are each about 500-1000 base pairs in length. It will be appreciated that the method according to this aspect of the invention may be used to replace a polynucleotide sequence from DNA of a plurality of host cells with a heterologous polynucleotide. In the final step (step (iii)), host cells without the counter-selection marker may be host cells that comprise the heterologous polynucleotide in their DNA. Thus, in one embodiment, the method may comprise: 1. transforming a plurality of host cells with a first nucleic acid molecule according to the invention (e.g., first aspect of the invention), such that the group II intron is retrotransposed; 2. applying a positive selection pressure to the plurality host cells such that only host cells encoding a functional positive selection marker of the RAM survive; 3. using homologous recombination to replace the nucleotide sequence encoding the counter-selection marker with the heterologous polynucleotide in one or more of the plurality of host cells by transforming one or more of the plurality of host cells with a second nucleic acid molecule that encodes a homologous recombination cassette comprising a first and a second homology arm that flank the heterologous polynucleotide, wherein the first homology arm is homologous with the first homology region and the second homology arm is homologous with the second homology region, and wherein the first homology region and the second homology region flank a nucleotide sequence encoding the retrotransposed group II intron comprising the counter selection marker; and 4. applying a counter-selection pressure to the plurality host cells such that only host cells in which the counter-selection marker has been replaced survive. The heterologous polynucleotide may encode a bookmark or a gene of interest. The (second) nucleic acid molecule may be a (second) nucleic acid molecule referred to herein. The skilled person will appreciate that the size of the heterologous polynucleotide to be introduced into the host cells may be dependent on a combination of factors including the genetic engineering tools used and the identity of the host cell. According to a sixth aspect, there is provided a method of removing a polynucleotide sequence from DNA of a plurality of host cells, the method comprising: (i) providing a plurality of host cells that all comprise a nucleotide sequence encoding a counter-selection marker; (ii) using homologous recombination to remove the nucleotide sequence encoding the counter-selection marker from the one or more of the plurality of host cells; and (iii) applying a counter-selection pressure to the plurality host cells such that only host cells in which the counter-selection marker has been removed, survive. Step (i), the step of providing a plurality of host cells that all comprise a counter- selection marker, may comprise using a RAM. Step (i) may further comprise applying a positive selection pressure to the plurality host cells such that only host cells in which retrotransposition of a functional positive selection marker gene has occurred will survive. The RAM may be a RAM referred to herein. The RAM may be retrotransposed into the plurality of host cells using a group I intron or a group II intron. Preferably, the RAM is retrotransposed using a group II intron. Thus, step (i) may comprise transforming a plurality of host cells with a (first) nucleic acid molecule according to the first aspect of the invention, and applying a positive selection pressure to the plurality host cells such that only host cells in which retrotransposition of a group II intron, which encompasses a RAM encoding a functional positive selection marker, survive. Step (ii) may comprise transforming one or more of the plurality of host cells with a (second) nucleic acid molecule that encodes a homologous recombination cassette. Preferably, the homology cassette comprises a first second homology arm adjacent to a second homology arm (i.e., without an intervening or flanked nucleotide sequence). Preferably the first homology arm is homologous with the first homology region and the second homology arm is homologous with the second homology region, wherein the first homology region and the second homology region flank the counter-selection marker. The first and second homology arms referred to herein may be least about 14 base pairs. Preferably, however, the first and second homology arms used herein are each about 500-1000 base pairs in length. Thus, in the final step (step (iii)), host cells without the counter-selection marker may be host cells from which the polynucleotide sequence has been removed. Thus, in one embodiment, the method may comprise: 1. transforming a plurality of host cells with a first nucleic acid molecule according to the invention (e.g., the first aspect of the invention), such that the group II intron is retrotransposed; 2. applying a positive selection pressure to the plurality host cells such that only host cells encoding a functional positive selection marker of the RAM survive; 3. using homologous recombination to remove the nucleotide sequence encoding the counter-selection marker in one or more of the plurality of host cells by transforming one or more of the plurality of host cells with a second nucleic acid molecule that encodes a homologous recombination cassette comprising a first homology arm adjacent to a second homology arm (i.e., without an intervening or flanked nucleotide sequence), wherein the homology cassette comprises a first and a second homology arm that flank the heterologous polynucleotide, wherein the first homology arm is homologous with the first homology region and the second homology arm is homologous the second homology region, and wherein the first homology region and the second homology region flank a nucleotide sequence encoding the retrotransposed group II intron comprising the counter selection marker; and 4. applying a counter-selection pressure to the plurality host cells such that only host cells in which the counter-selection marker has been removed, survive. The skilled person will appreciate that the polynucleotide sequence to be removed and the counter selection marker will be positioned between the first and second homology regions. The counter selection marker or the retrotransposed group II intron may be within or adjacent to the polynucleotide sequence to be removed. The (second) nucleic acid molecule may be a (second) nucleic acid molecule referred to herein. The skilled person will appreciate that the positive selection pressure will correspond with the positive selection marker of the RAM. For example, the antibiotic resistance marker and the skilled person will appreciate that the counter-selection pressure will correspond with the counter-selection marker. The skilled person will appreciate that the steps of transforming one or more of the plurality of host cells can be achieved using any technique known in the art. For example, transformation may be achieved using electroporation, transfection, or conjugation. Preferably step (2) of a method according to the invention (e.g., the method according to the fifth or the sixth aspect) is completed before step (3) is initiated. Preferably step (3) is initiated once the first nucleic acid molecule has been lost or degraded. The first nucleic acid molecule may be lost / degraded either by using a pseudo-suicide replicon or by using a conditionally functional replicon. The pseudo-suicide replicon may be rapidly lost in the absence of a selective pressure (e.g., an antibiotic selective pressure). A conditional replicon may allow inducible loss of the first nucleic acid molecule through addition of an inducer controlling its expression for instance or alteration of environmental conditions, for instance temperature, to destabilise the replicon. This ensures that the first nucleic acid molecule is lost prior to inducing the counter selection pressure, such that the intron group II is stabilised through the loss of the IEP. Preferably step (2) and step (3) do not occur simultaneously. The first and the second nucleic acid molecules may be delivered simultaneously (or in parallel). Thus, step (1) and step (3) may occur simultaneously. However, delivering the first and second nucleic acid molecules simultaneously requires that the nucleic acid molecules possess compatible replicons and individual resistance markers. Also, the first nucleic acid molecule is preferably lost prior to induction of the counter selection pressure, such that the intron group II is stabilised through the loss of the IEP. In other words, in the absence of the IEP (once the nucleic acid molecule has been lost / degraded in the host cell), the group II intron is completely stable. Stability of the group II intron is required in order for the subsequent counter-selective pressure provided by, for example, the expression of I-SceI, to select for double crossover events permitted by the presence of the editing cassette. If the group II intron was still mobilizable in the presence of the counter-selective pressure, then cells in which the group II intron had been lost through activity of the IEP (e.g., LtrA) would also be seen after applying positive selection pressure, and these would have reverted to the wild type locus at the target site. By screening for loss of the first nucleotide molecule, which contains the intron and the IEP, prior to induction of the counter-selectable pressure, it is ensured that the only cells which survive induction are those in which the desired double crossover homologous recombination event has occurred via the editing cassette provided. In one embodiment of the method according to the invention, the first nucleic acid molecule according may comprise the features of the second nucleic acid molecule referred to herein. Thus, the first and second nucleic acid molecules may be present in a single nucleic acid molecule. Preferably the second promoter is an inducible promoter. This ensures that once induction has stopped, the promoter is repressed to the extent that the intron is stabilised. Preferably the counter-selection marker is not functional within the nucleic acid molecule per se. Thus, the counter-selection marker either be PyrE-RAM or a meganuclease that is also interrupted by the group I intron. The invention may further provide a cell produced by any method described herein. According to a seventh aspect, there is provided a nucleic acid molecule encoding a chloramphenicol acetyl transferase antibiotic resistance marker gene comprising a group I intron (Cat-RAM). According to an eighth aspect, there is provided a nucleic acid molecule encoding a group II intron comprising a Cat retrotransposition-activated marker (Cat- RAM) gene, wherein the Cat-RAM comprises a Cat antibiotic resistance marker gene comprising a group I intron. The Cat may be a CatA. The CatA may be a CatA1, CatA2, CatA3, CatA4, CatA5, CatA6, CatA7, CatA8, CatA9, CatA10, CatA11, CatA12, CatA13, CatA14, CatA15 or CatA16. The Cat may be a CatB. The CatB may be a CatB1, CatB2, CatB3, CatB4, or CatB5. Preferably the Cat is a CatA, such as CatA9. In one embodiment, the CatA antibiotic resistance marker gene comprises a nucleotide sequence substantially as set out in SEQ ID NO. 46. Thus, nucleic acid molecule encoding a group II intron may comprise a nucleic acid coding sequence substantially as set out in SEQ ID NO. 46. In one embodiment, the nucleotide sequence of the group II intron comprising the CatA- RAM is provided herein as SEQ ID NO. 61. Thus, the group II intron referred to herein may comprise a nucleotide sequence substantially as set out in SEQ ID NO. 61. The group I intron may disrupt expression of the Cat marker gene of the RAM. The group I intron may be in an orientation that enables the group I intron to splice itself out from an RNA transcript of the group II intron. This may occur prior to or during retrotransposition. Thus, the group I intron may be orientated such that it self- catalytically splices out of the transcript of the group II intron leaving a region encoding a functional positive selection marker. The marker gene of the RAM may be orientated in the reverse direction relative to the orientation of the group II intron. The group I intron may be orientated in the opposite direction to the marker gene. The group II intron may be orientated in the opposite direction to the marker gene. The group I intron and the group II intron may be orientated in the same direction. Encoding the group I intron in an orientation that enables the group I intron to splice itself out from an RNA transcript of the group II intron (thus leaving a region encoding a functional positive selection marker), results in retrotransposition of the group II intron producing a functional marker gene in the DNA of the host cell. Only cells encoding a functional marker gene in their DNA will be capable of expressing a protein of the marker gene and therefore surviving the relevant positive selection pressure. Preferably, the group I intron is capable of self-splicing without the assistance of a cofactor, such as guanosine. Preferably the group I intron referred to herein does not encompass or encode an IEP. The group I intron may be located in the (first) promoter of the group II intron. The group I intron may be located in a linker between linker between the ORF of the marker gene and its promoter. The group I intron may be located in a coding sequence of the marker gene. Thus, the coding sequence of the antibiotic resistance marker gene encompasses the group I intron. Ultimately, the group I intron may be located at any position within the marker gene coding sequence as long as expression of a functional protein (e.g., a functional antibiotic resistance protein) is disrupted and that following excision of the intron that the exon sequence that remains encodes the requisite, functional, protein of the marker gene. Consequently, the exon splice sites of the group I intron may incorporate the codons needed to form a functional antibiotic resistance protein. As the sequences specifying the requisite splice sites are unlikely to occur naturally in antibiotic resistance genes, they may be introduced into the coding region along with the group I intron sequence. Therefore, the group I intron may be flanked by exons that provide (exon) splice sites and codons needed to form a functional marker gene protein (once splicing / excision of the group I intron has occurred). By relying on codon redundancy, it is possible to ensure the correct amino acid sequence in the encoded protein is produced. In the case of the td group I intron, the exon sequence corresponds to G|ACCCAAGAGA, where the intron is inserted between the first (G) and the first (A) nucleotide (illustrated by the |). Consequently, translation of those 11 nucleotides in the inserted frame should result in amino acids that generate an active protein. The possible amino acid sequences that can accommodate the td group I exon sequence are shown in Table 1 below:- Table 1 Frame AA Sequence of three possible frames L / M / V / S / P / T / A / Q / K / E / W / R / G T Q E I / M / T / N / K / S / R 1 G ACC CAA GAG A R / G P K R 2 GA CCC AAG AGA D P R D / E 3 GAC CCA AGA GA In embodiments in which the group I intron is td, the exon sequences flanking the group I intron may be defined by frame 1, 2 or 3 of Table 1. The nucleotide sequence nor the intron insertion site vary with reading frame. It will be appreciated that most proteins do not naturally contain any combination of these amino acid sequences. In the case of the ermB, for instance, it was not possible to incorporate the necessary td exon splice sequences into the native gene without altering the amino acids sequence of the ErmB protein. Rather, an extension of 12 codons (i.e., a linker) was added to the 5’-end of the ermB gene that contained the necessary splice sites which resulted in the production of an ErmB protein that despite the extra 12 amino acids at the N-terminus of the protein retained functionality. It is conceivable that the amino acid sequence of an antibiotic resistant protein, despite codon redundancy, could exclude the inclusion of the necessary splice sites, or not tolerate the addition of a N-terminal amino acid extension. In this case conservative amino acid replacements (may be made to the protein (e.g., Ile for Leu that would result in a protein that retained the required activity and allowed incorporation of the requisite splice sites. Conservative amino acid replacements are when an amino acid is replaced with a different amino acid that has similar biochemical properties (e.g., charge, hydrophobicity and size). The skilled person will appreciate that examples of conservative amino acid replacements include, replacement of Asp (D) with Glu (E), replacement of Arg (R) with Lys (K), replacement of Ile (I) with Leu (L) or Val (V) and replacement of Ser (S) with Thr (T). The group II intron, the Cat-RAM, the Cat antibiotic resistance marker gene or the nucleic acid molecule according to the invention may not comprise a linker. The availability of an additional RAM conferring an alternative antibiotic resistance increases the breadth of organisms to which this technology may be applied, especially given that native erythromycin resistance is an issue for several clostridial strains, including clinically relevant Clostridioides difficile (formerly Clostridium difficile) strains. The absence of the linker also reduces the size of the group II intron, the Cat- RAM, the Cat antibiotic resistance marker gene or the nucleic acid molecule. The group I intron may be located between exons in the coding sequence of the Cat antibiotic resistance marker gene. The Cat-RAM according to the invention may comprise a group I intron (e.g., td or SEQ ID NO. 45) flanked by exon sequences that allow the group I intron to splice out of the RNA transcript. The resulting spliced transcript preferably encodes a functional positive selection protein. The group I intron may be a group I intron referred to herein, such as that of a T4 phage. The group I intron may be a td group I intron or a group I intron defined by SEQ ID NO. 45. The Cat antibiotic resistance marker gene may comprise a promoter operably linked to a coding sequence of the Cat antibiotic resistance marker gene. The promoter may be any promoter that is capable of causing the transcription of the Cat antibiotic resistance marker gene. The promoter may be a constitutive promoter or an inducible promoter. Preferably the promoter is recognised (bound) by a sigma factor (housekeeping sigma factor or an alternative sigma factor) of the host cell. Thus, the promoter may be a native promoter or a promoter that has a nucleotide sequence substantially similar to a native promoter. The promoter may be a native promoter derived from a gene encoding an enzyme or protein involved in an essential metabolic process, for example, central metabolism. The first promoter may therefore be derived from a gene encoding a ferredoxin. In one embodiment, the first promoter may be that of the ferredoxin gene of Clostridium sporogenes, the Pfdx promoter. Ferredoxins are abundant iron-sulphur proteins responsible for electron transfer in a wide variety of metabolic reactions. The promoter may be that of the thiolase gene of Clostridium acetobutylicum, the Pthl promoter. The Cat-RAM according to the invention has been shown to confer resistance to thiamphenicol in several clostridial species, and the presence of the group I intron within the coding sequence of the marker gene has successfully ablated its function (see Example 1). The invention (e.g., the nucleic acid molecule according to the first aspect or present in the host cell of the second aspect of the invention) may comprise a nucleic acid molecule according to the seventh or eighth aspect of the invention. The host cell referred to herein may be a prokaryote or eukaryote. The eukaryote may be a yeast. The prokaryote may be bacterium or archaea. Thus, the host cell may be a bacterium or yeast. The bacterium may be an aerobe or an anaerobe. The bacterium may be a Gram-positive bacterium or a Gram-negative bacterium. The bacterium may be any bacterial species, but is preferably a member of the bacterial phylum Firmicutes, which is composed of the class Clostridia (orders Clostridiales, Halanaerobiales, Natranaerobiales and Thermoanaerobacterales), the class Bacilli (orders Bacillales and Lactobacillales) and the class Mollicutes (orders Acholeplasmatales, Anaeroplasmatales, Entomoplasmatales, Haloplasmatales and Mycoplasmatales). Thus, the bacterium may be of the class Clostridia, the Bacilli or Mollicutes. Preferably the bacterium is of the class Clostridia. The bacterium may be within the order of Clostridiales, Halanaerobiales, Natranaerobiales, Thermoanaerobacterales, Bacillales, Lactobacillales, Acholeplasmatales, Anaeroplasmatales, Entomoplasmatales, Haloplasmatales or Mycoplasmatales. Preferably, the bacterium is of the order of Clostridiales. Within the order Clostridiales is the genus, Clostridium. Thus, the bacteria may be of the genus, Clostridium. Preferred species are C. aceticum, C. acetobutylicum, C. aerotolerans, C. autoethanogenum, C. baratii, C. beijerinckii, C. bifermentans, C. botulinum, C. butyricum, C. cadaveris, C. cellulolyticum, C. cellulovorans, C. chauvoei, C. clostridioforme, C. colicanis, C. difficile (now renamed Clostridioides difficile), C. drakei, C. estertheticum, C. fallax, C. feseri, C. formicaceticum, C. glycolicum, C. hiranonis, C. histolyticum, C. innocuum, C. kluyveri, C. ljungdahlii, C. lavalense, C. mayombei. C. methoxybenzovorans, C. novyi, C. oedematiens, C. paraputrificum, C. pasteurianum, C. perfringens, C. phytofermentans, C. piliforme, C. ragsdalei, C. ramosum, C. roseum, C. saccharoperbutylacetonicum, C. scatologenes, C. septicum, C. sordellii, C. sporogenes, C. sticklandii, C. tertium, C. tetani, C. thermocellum, C. thermosaccharolyticum, C. tyrobutyricum, C. paprosolvens, C. saccharobutylicum, C. carboxidovorans, C. scindens, and C. autoethanogenum. as well as other acetogenic anaerobes, such as, Acetobacterium woodii, Acetonema longum, Alkalibaculum bacchi, Blautia producta, Butyribacterium methylotrophicum, Eubacterium limosum, Pseudoramibacter alactolyticus, Oxobacter pfennigii, Moorella thermoacetica, Moorella thermoautotrophica, Thermoanaerobacter kiuvi. The bacterium may be Clostridium sporogenes or Clostridium autoethanogenum. The skilled person will appreciate that prior to contact with a nucleic acid molecule according to the invention, a host cell according to the invention preferably does not encode a counter selection-marker or the marker gene of a RAM of a nucleic acid molecule according to the invention. For example, in embodiments in which the toxic marker gene is pyrE or pyrF, the host cell preferably lacks an enzyme with orotate phosphoribosyl transferase activity (such as pyrE) or lacks an enzyme with orotidine- 5'-monophosphate decarboxylase activity (such as pyrF). In embodiments, which the RAM is Cat-RAM, the host cell preferably lacks an enzyme with chloramphenicol acetyltransferase activity. It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The term “substantially” can be a sequence that has at least 40% sequence identity with any one of the sequences referred to herein, for example 40% identity with the nucleotide identified as SEQ ID No.1 disclosed herein. The term “substantially” can be a sequence that has greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to is also envisaged. Preferably, the nucleotide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein. The nucleic acid molecules referred to herein, including the nucleic acid molecule according to the invention, may be a double stranded nucleic acid molecule, such as DNA. The DNA molecule may be a plasmid or a vector. The term “nucleic acid” referred to herein may be a (heterologous) fragment or part of a nucleic acid molecule, such as part of the DNA of a recipient / host cell. The term “comprising” may refer to “consisting of” or “consisting essentially of”. All of the embodiments and features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects or embodiments in any combination, unless stated otherwise with reference to specific combinations, for example, combinations where at least some of such features and / or steps are mutually exclusive. FIGURES For a better understanding of the invention, and to show embodiments of the invention may be put into effect, reference will now be made, by way of example, to the accompanying drawings, in which:- Figure 1 is schematic diagram of the two-step process for generation of In-frame deletions at a target chromosomal locus; Figure 2 is a representation of the intron containing vector and highlights the nature and positioning of the meganuclease recognition site relative to the RAM. This positioning is identical in both the CatRAM and the ErmRAM, and between use of the I-SceI recognition site versus the I-PpoI recognition site; Figure 3 is a representation of the intron containing vector used to mobilise the intron containing the ErmRAM to the codA locus in C. autoethanogenum, utilised below in Example 2; Figure 4 is a representation of the vector used to generate the in-frame deletion at the codA locus in C. autoethanogenum following isolation of the ErmRAM intron containing strain, utilised below in Example 2; Figure 5 shows a replica plate screen from Example 1 comparing growth of 16 single colonies from 200 ng.ml-1 aTc induction plates onto TYG vs TYG + Tm. A sensitivity to Tm indicates that the CatRAM has been lost from the population through I-PpoI induction and therefore selection of homology cassette mediated double crossover events; Figure 6 is a PCR screen from Example 1, whereby five colonies which had demonstrated sensitivity to thiamphenicol in the replica plate screen were subject to PCR screening at the spo0A locus, using oligonucleotides which bind outside of the homology cassette locus. Lane 1 contains NEB Quick-load 1kb plus ladder, Lanes 2-6 represent templates of individual colonies which were demonstrated to be sensitive to thiamphenicol during the replica plating stage. These are confirmed to generate the ~1.5 kb band expected from an in-frame deletion at the spo0A locus. Lane 7 uses wild type C. sporogenes genomic DNA as the template, and Lane 8 uses C. sporogenes- spo0A::catA-I-PpoI which still contains the intron at the spo0A locus as a control. Figure 7 is a replica plate screen from Example 2 comparing growth of 36 single colonies from 100 ng.ml-1 aTc induction plates on YTAF + Tm + aTc (1) versus YTAF + Tm + Cla. A sensitivity to Cla indicates that the ErmRAM has been lost from the population through I-SceI induction and therefore selection of homology cassette mediated double crossover events. Figure 8 is a PCR screen from Example 2 of eight clarithromycin sensitive colonies at the codA locus. Lane 1 contains NEB 2-log ladder, Lanes 2-9 represent individual colonies which were demonstrated to be sensitive to clarithromycin during the replica plating stage. Lane 10 is wild type and Lane 11 is a negative control with water as the template for amplification; Figure 9 is a representation of the intron containing vector used to mobilise the intron containing the PyrERAM to the codA locus in C. autoethanogenum, utilised below in Example 3; Figure 10 is a representation of the vector used to generate the in-frame deletion at the codA locus in C. autoethanogenum following isolation of the PyrERAM intron containing strain, utilised below in Example 3; Figure 11 is Colony PCR of the codA locus from colonies resistant to 3 mgµl-15-FOA. Colonies 1, 3, 4 and 5 are the intended in-frame deletions, generating a band size of 1.67 kb. Wild type is designated WT, generating the expected product size of 2.86 kb. Colony 2 represents an escapee still containing the intron, generating a band size of approximately 4.6 kb at the codA locus. C represents the negative control with dH2O as the template; and Figure 12 is a representation of the intron containing vector used to mobilise the intron containing the CatRAM to the codA locus in C. autoethanogenum, utilised below in Example 3. Figure 13 is a PCR screen from Example 4-8 demonstrating intron insertions of the ErmRAM and meganuclease cassette at the pyrF locus of C. sporogenes. Colony PCR of erythromycin resistant colonies using oligonucleotides which bind outside of the pyrF locus. Insertion of the intron harbouring the meganuclease cassette 1 generates a product size of 4.88 kb (A.), whilst the intron containing the meganuclease cassette 2 generates a band at 4.48 kb (B.). Wild type designated WT produces an amplicon size of 2.49 kb. An NEB Quick-load 1 kb plus ladder was used for these agarose gels. Figure 14 is a PCR screen from Example 4-8 demonstrating creation of inframe deletions. Colony PCR of the pyrF locus of C. sporogenes following anhydrotetracycline induction of each meganuclease independently, using oligonucleotides which bind outside of the homology cassette locus. Colonies with band sizes of 1.68 kb show the intended in-frame deletions, with wild type designated WT, generating the expected product size of 2.49 kb. With an absence of an antibiotic resistance screen, escapee colonies still containing the intron are present in some gels, generating a band size of approximately 4.5 or 4.8 kb dependent on the meganuclease cassette it was targeting. As a marker the Thermo Scientific GeneRuler 1 kb Plus DNA Ladder was used in the top two gels, whereas an NEB Quick-load 1kb plus ladder was used in the bottom three. Figure 15 is a PCR screen from Example 9-11 demonstrating intron insertions of the ErmRAM at the pyrF locus of C. botulinum. Colony PCR erythromycin resistant colonies using oligonucleotides which bind outside of the pyrF locus. Insertion of the intron generates a product size of 4.82 kb, with wild type designated WT and producing an amplicon size of 2.49 kb. An NEB Quick-load 1kb plus ladder was used as a marker.. Figure 16 is a PCR screen from Example 9-11. Colony PCR of the pyrF locus of C. botulinum following anhydrotetracycline induction of each meganuclease independently, using oligonucleotides which bind outside of the homology cassette locus. Colonies with band sizes of 1.66 kb show the intended in-frame deletions, with wild type designated WT, generating the expected product size of 2.49 kb. With an absence of an antibiotic resistance screen, escapee colonies still containing the intron are present in some agarose gels, generating a band size of approximately 4.82 kb. An NEB Quick-load 1kb plus ladder was used for these agarose gels. Figure 17 is a PCR screen from Example 12 demonstrating intron insertions of the ErmRAM at the mtlD locus of C. difficile. Colony PCR erythromycin resistant colonies using an oligonucleotide which binds outside of the mtlD locus (Cdif mtlD SCRR1) and one which binds within the ErmRAM (EBS Universal). Insertion of the intron generates a product size of 1.12 kb, with wild type designated WT and not producing an amplicon, due to the absence of the EBS Universal primer binding site. An NEB Quick- load 1kb plus ladder was used for these agarose gels. Figure 18 is a PCR screen from Example 12. Colony PCR of the mtlD locus of C. difficile following anhydrotetracycline induction of the I-DmoI meganuclease, using oligonucleotides which bind outside of the homology cassette locus. Colonies from which 1.28 kb amplicons were generated show intended in-frame deletions, with wild type designated WT, generating the expected product size of 2.42 kb. No escapee colonies still containing the intron were observed in this assay. An NEB Quick-load 1kb plus ladder was used for these agarose gels. Figure 19 is a PCR screen from Examples 13-14 demonstrating intron insertions of the KanRAM at the pyrE locus of E. coli. Colony PCR of kanamycin resistant colonies using oligonucleotides which bind outside of the pyrE locus. Insertion of the intron generates a product size of 4.37 kb, with wild type designated WT and producing an amplicon size of 2.32 kb. An NEB Quick-load 1kb plus ladder was used as a marker.. Figure 20 is a PCR screen from Examples 13-14. Colony PCR of the pyrE locus of E. coli following L-arabinose induction of each meganuclease independently, using oligonucleotides which bind outside of the homology cassette locus. Colonies with band sizes of 1.69 kb show the intended in-frame deletions, with wild type designated WT, generating the expected product size of 2.32 kb. With an absence of an antibiotic resistance screen, escapee colonies still containing the intron are present in the agarose gels following induction, generating a band size of approximately 4.37 kb. An intron containing control designated by ‘Int.’ generating the same 4.37 kb size band was loaded on the gel. An NEB Quick-load 1kb plus ladder was used as a reference in the gels. EXAMPLES Materials and Methods Strains, media and growth conditions All E.coli and Clostridium strains used in this study are listed in Table 1. E. coli 10- beta (New England Biolabs) was used as a general host for plasmid construction and propagation. The E. coli strain sExpress was used as the donor strain for conjugation (Woods et al. 2019). All E.coli strains were transformed through a heat shock protocol described previously, based on the instructions provided by NEB for the 10-beta cloning host. E. coli strains were grown at 37 °C, in Luria-Bertani (LB) medium supplemented with chloramphenicol (25 µg / mL), erythromycin (500 µg / mL) or kanamycin (50 µg / mL) where necessary. Growth media for the different Clostridium species are specified on Table 2. Media for clostridial strains were supplemented with the following antibiotic / inducer / supplement when appropriate: thiamphenicol (15 µg / mL), clarithromycin (6 µg / mL), erythromycin (10 µg / mL), anhydrotetracycline (100 ng / mL in the case of C. autoethanogenum, 200 ng / mL in the case of C. sporogenes), D- cycloserine (250 µg / mL), cefoxitin (8 µg / mL). Clostridium strains were grown at 37 °C in an anaerobic cabinet (MG1000 anaerobic workstation; Don Whitley Scientific Ltd). Reagents All PCR reactions were performed using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) in the case of amplification for cloning purposes, or DreamTaq Green PCR Master Mix (Thermo Fisher Scientific) for screening purposes. NEBuilder HiFi DNA Assembly was used for assembly of all constructs. Fragments for HiFi assembly were either generated using PCR amplification or in some cases generated using restriction enzymes. All restriction enzymes were purchased from New England Biolabs. Plasmid design, construction and transformation Oligonucleotide primers used for screening and plasmid construction were synthesized by IDT. The gBlock gene fragment service provided by IDT was used for synthesis of larger fragments (>80bp). Constructs were verified by Sanger sequencing using the TubeSeq service (Eurofins). All of the plasmids used in this study are listed in Table 3. Details of plasmid design and sequences are provided in supplementary information section below. Tables Table 1 - List of strains used in this study Reference Strain Relevant characteristics source DH10B derivative - Δ(ara-leu) 7697 araD139 fhuA E. coli 10-beta ΔlacX74 galK16 galE15 e14- ϕ80dlacZΔM15 recA1 NEB relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr- hsdRMS-mcrBC) BL21 derivative - fhuA2 [lon] ompT gal sulA11 Wo (mcr-73::miniTn10--Sods et E. coli sExpress R Tet )2 [dcm] R(zgb- al. 20 210::Tn10-- tS19. Te ) endA1 Δ(mcrC-mrr)114::IS10 Clostridium sporogenes Wild type strain ATCC NCIMB 10696 Clostridium Wild type strain DSMZ autoethanogenum JA1-1 Clostridium Annan et Mutant created using allele coupled exchange (ACE) autoethanogenum ΔpyrE al. 2019 Clostridioides difficile Ingle et al. Mutant created using CRISPR / Cas9 genome editing 630Δerm* 2019 Clostridium botulinum Sebaihia et Wild type strain ATCC 3502 al. 2007 Clostridium sporogenes NCIMB 10696 containing group C. sporogenes - This study, II intron inserted at the spo0A locus, encoding a spo0A::catA-I-PpoI example 1 functional CatA and an I-PpoI recognition site Clostridium autoethanogenum JA1-1 containing group II C. autoethanogenum - This study, intron inserted at the codA locus, encoding a functional codA::ermB-I-SceI example 2 ErmB and an I-SceI recognition site Clostridium autoethanogenum JA1-1 containing group II C. autoethanogenum - This study, intron inserted at the codA locus, encoding a functional codA::C.lep-pyrE example 3 PyrE derived from C. leptum. Clostridium sporogenes NCIMB 10696 containing group C. sporogenes - This study, II intron inserted at the pyrF locus, encoding a functional pyrF::ermB-PpoI-SceI- example 4- ErmB followed by I-PpoI, I-SceI, I-CreI, I-DmoI, I-NjaI CreI-DmoI-NjaI 8 recognition sites. C. sporogenes - Clostridium sporogenes NCIMB 10696 containing group This study, pyrF::ermB-PpoI-DirI- II intron inserted at the pyrF locus, encoding a functional example 4- BmoI-LtrWI-TevI-HmuI- ErmB followed by I-PpoI, I-DirI, I-BmoI, I-LtrWI, I- 8 DdiI TevI, I-HmuI, I-DdiI recognition sites. Clostridium botulinum ATCC 3502 containing group II C. botulinum - This study, intron inserted at the pyrF locus, encoding a functional pyrF::ermB-PpoI-SceI- example 9- ErmB followed by I-PpoI, I-SceI, I-CreI, I-DmoI, I-NjaI CreI-DmoI-NjaI 11 recognition sites. Clostridioides difficile 630Δerm* containing group II C. difficile 630Δerm*- intron inserted at the mtlD locus, encoding a functional This study, mtlD::ermB-PpoI-SceI- ErmB and I-PpoI, I-SceI, I-CreI, I-DmoI and I-NjaI example 12 CreI-DmoI-NjaI recognition sites E. coli - -pyrE::Kan-PpoI- E. coli 10-beta containing group II intron inserted at the This study, SceI-CreI-DmoI-NjaI mtlD locus, encoding a functional kan and I-PpoI, I-SceI, example I-CreI, I-DmoI and I-NjaI recognition sites 13-14 Table 2 – Media composition for Clostridium growth Name Composition (g / L) Organism TYG Tryptone 30 C. sporogenes grown in Yeast extract 20solid or liquid mediaSodium-thioglycolate 1 Agar (for solid media) 10 YTAF Yeast extract 16 C. autoethanogenum Tryptone 9 grown in solid or liquid Arginine 1.2 medium Fructose 10 MES buffer 10 pH to 5.8 Agar (for solid media) PETCMES NH4Cl 1.00 C. autoethanogenum KCl 0.10 grown in solid or liquid MgSO4.7H2O 0.20 medium, minimal KH2PO4 0.20 media for auxotrophy CaCl20.02 selection - See Annan et al. 2019 for details Nitrilotriacetic Acid 0.05 of stock additions Fe(SO4)2(NH4)2.6H2O 0.05 CH3COONa 0.25 MES buffer 20.00 L-Cysteine 0.20 Stock additions (ml / L): Trace metals (100x) 10 ml Wolfes vitamins (100x) 10 ml Resazurin (2 gL-1)(2000x) 0.5 ml pH to 5.8 BHIS Brain Heart Infusion 37 C. difficile grown in Yeast Extract 5 solid or liquid medium L-cysteine 1 Table 3 - List of plasmids used in this study Reference Plasmid Relevant characteristics source ClosTron vector with CatP resistance marker on the pMTL007C- backbone, targeting spo0A 249s insertion site in ATUM E2::CspoSpo0A249s C. sporogenes, contains the ErmRAM. NegaTron vector containing ErmB resistance marker on pMTL007E-C1-I- the backbone, targeting spo0A 249s insertion site in This study, PpoI::CspoSpo0A249s C. sporogenes, with the CatRAM and I-PpoI example 1 recognition site. pMTL8825-Ptet-I- pMTL88251 modular vector containing codon This study, PpoIOpt-CspoSpo0A optimised I-PpoI under control of the inducible tet example 1 promoter, and a homologous recombination cassette to create the spo0A in-frame deletion in C. sporogenes. ClosTron vector with CatP resistance marker on the pMTL007C- backbone, targeting codA 532s insertion site in ATUM E2::CautoCodA532s C.autoethanogenum, contains the ErmRAM. NegaTron vector with CatP resistance marker on the This study, pMTL007C-E2-I- backbone, targeting codA 532s insertion site in example 2, SceI::CautoCodA532s C. autoethanogenum, contains the ErmRAM and I-SceI Figure 3 recognition site. pMTL83151 modular vector containing codon optimised I-SceI under control of the inducible tet This study, pMTL8315-Ptet-I- promoter, and a homologous recombination cassette to example 2, SceIOpt-CautoCodA create the codA in-frame deletion in Figure 4 C. autoethanogenum. NegaTron vector with CatP resistance marker on the This study, pMTL007C- backbone, targeting codA 532s insertion site in example 3, P2::CautocodA532s C. autoethanogenum, contains the PyrERAM Figure 9 pMTL88251 modular vector containing a homologous This study, pMTL8825- recombination cassette to create the codA in-frame example 3, CautoCodAHA deletion in C. autoethanogenum. Figure 10 NegaTron vector containing CatP resistance marker on This study, pMTL007C-E2-MN- the backbone, targeting pyrF 595s insertion site in example 4-8 Cassette1::CspopyrF595s C. sporogenes, with the ErmRAM and I-PpoI I-SceI, I- and 9-11. CreI, I-DmoI, I-NjaI recognition sites. NegaTron vector containing CatP resistance marker on the backbone, targeting pyrF 595s insertion site in This study, pMTL007C-E2-MN- C. sporogenes, with the ErmRAM and I-PpoI, I-DirI, I- example 4- Cassette2::CspopyrF595s BmoI, I-LtrWI, I-TevI, I-HmuI, I-DdiI recognition 8. sites. pMTL88151 modular vector containing codon optimised I-PpoI under control of the inducible tet This study, pMTL8815-Ptet-I-PpoI - promoter, and a homologous recombination cassette to example 4 CspopyrF create the pyrF in-frame deletion in C. sporogenes and and 9. C. botulinum. pMTL88151 modular vector containing codon optimised I-DmoI under control of the inducible tet This study, pMTL8815-Ptet I-DmoI- promoter, and a homologous recombination cassette to example 5 CspopyrF create the pyrF in-frame deletion in C. sporogenes and and 10. C. botulinum. pMTL88151 modular vector containing codon optimised I-NjaI under control of the inducible tet This study, pMTL8815-Ptet-I-NjaI- promoter, and a homologous recombination cassette to example 6 CspopyrF create the pyrF in-frame deletion in C. sporogenes and and 11. C. botulinum. pMTL88151 modular vector containing codon pMTL8815-Ptet-I-DirI- optimised I-DirI under control of the inducible tet This study, CspopyrF promoter, and a homologous recombination cassette to example 7. create the pyrF in-frame deletion in C. sporogenes. pMTL88151 modular vector containing codon pMTL8815-Ptet-I-BmoI- optimised I-BmoI under control of the inducible tet This study, CspopyrF promoter, and a homologous recombination cassette to example 8. create the pyrF in-frame deletion in C. sporogenes. NegaTron vector containing CatP resistance marker on This study, pMTL007C-E2-MN- the backbone, targeting mtlD 204s insertion site in example 12, Cassette1::CdimtlD204s C. difficile 630Δerm*, with the ErmRAM and I-PpoI I- Figure 17. SceI, I-CreI, I-DmoI, I-NjaI recognition sites. pMTL88151 modular vector containing codon optimised I-DmoI under control of the inducible tet This study, pMTL8815-Ptet-I-DmoI- promoter, and a homologous recombination cassette to example 12, CdimtlD create the mtlD in-frame deletion in C. difficile Figure 18. 630Δerm*. NegaTron vector containing CatP resistance marker on This study, pMTL007C-K1-MN- the backbone, targeting frE 522a insertion site in E. example Cassette1::EcpyrE522a coli, with the KanRAM and I-PpoI I-SceI, I-CreI, I- 13-14. DmoI, I-NjaI recognition sites. pMTL88151 modular vector containing a E. coli codon optimised I-SceI under control of the arabinose pMTL8815-PBAD-I- This study, inducible promoter, and a homologous recombination SceI- EcpyrE example 13. cassette to create the pyrE in-frame deletion in E. coli 10-beta. pMTL88151 modular vector containing a E. coli codon optimised I-DmoI under control of the arabinose pMTL8815-PBAD-I- This study, inducible promoter, and a homologous recombination DmoI- EcpyrE example 14 cassette to create the pyrE in-frame deletion in E. coli 10-beta. Table 4 - Key Oligonucleotides used in this study: Oligonucleotide FunctionNUCLEOTIDE SEQUENCE 5’=›3’Screening primers: Enables screening of the C. sporogenes GTTTTTCAAGCCAATCTTTACTATC (SEQ ID Cspo spo0A SCRF1 spo0A genomic locus, forward NO. 68) primer Enables screening of the C. sporogenes CTTTAAGAAATGAAATAAAAGAAGG (SEQ ID Cspo spo0A SCRR1 spo0A genomic NO. 69) locus, reverse primer Enables screening of the C. Cauto codA SCRF1 autoethanogenum GTAAAATTATAGCTTGGAGAC (SEQ ID NO.70) codA genomic locus, forward primer Enables screening of the C. Cauto codA autoethanogenum GTTAACTTCCAAAGACTTAG (SEQ ID NO. 71) SCRR1 codA genomic locus, reverse primer Enables screening of the C. sporogenesCAAGTACATACAAGACATGCTC(SEQ ID NO. Cspo pyrF SCRF1 pyrF genomic locus, 72) forward primer Enables screening of the C. sporogenes GGTGATGATATAAGTCATATAGATTC (SEQ ID Cspo pyrF SCRR1 pyrF genomic locus, NO. 73) reverse primer Enables screening of the C. botulinum CAAGTACATACAAGACATGCTC (SEQ ID NO. Cbot pyrF SCRF1 pyrF genomic locus, 74) forward primer Enables screening of the C. botulinum GGTGATGATATAAGTCATATAGATTC Cbot pyrF SCRR1 pyrF genomic locus, (SEQ ID NO. 75) reverse primer Enables screening of the C. difficile mtlD TCTGATTTTTTTGCAAATACTACAGG (SEQ ID Cdif mtlD SCRF1 locus, forward NO. 76) primer Enables screening of ACAAATAATGAGTGAAATAAGTGG (SEQ ID Cdif mtlD SCRR1 the C. difficile mtlD locus, reverse primer NO. 77) Enables screening of the E. coli pyrE GTAGTACAGGCTGAATTTAATCTG (SEQ Ec pyrE SCRF1 locus, forward ID NO. 85) primer Enables screening of CTTGTCAGGATAGGAATAACC (SEQ ID Ec pyrE SCRR1 the E. coli pyrE locus, reverse primer NO. 86) Enables screening CGAAATTAGAAACTTGCGTTCAGTAAAC EBS Universal for insertion of the group II intron (SEQ ID NO. 78) Example 1: spo0A in-frame deletion in C. sporogenes using NegaTron containing a CatRAM and pMTL8825-Ptet-I-PpoIOpt-CspoSpo0A In the following example the group II intron targeting region was specific to the spo0A gene, a master regulator of sporulation previously demonstrated to be non-essential, within Clostridium sporogenes. The Perutka algorithm (Perutka et al., 2004) was used to select the target site within spo0A, which was positioned between base pairs 249|250s, the ‘s’ denominating that the intron will insert in the sense orientation relative to the gene target. The Perutka algorithm assigned this target insertion site a score of 8.382. Vector construction: The pMTL007C-E2::CspoSpo0A249s supplied by ATUM was modified through exchange of the CatP antibiotic resistance marker on the backbone of the vector for ErmB, using NEB HiFi assembly. The ErmRAM and its flanking FRT sites were also exchanged for a novel CatRAM, which was codon optimised and synthesised by IDT, with the group I intron positioned within the marker during synthesis., The promoter selected to drive expression of the RAM was the native thiolase promoter from C. acetobutylicum. The 29 base pair I-PpoI recognition sequence outlined above was incorporated downstream of the CatRAM sequence through oligonucleotide overhangs to adjacent amplified regions. The sequence of the novel RAM can be found above. The CatRAM variant was termed pMTL007E-C1-I-PpoI::CspoSpo0A249s. pMTL8825-Ptet-I-PpoIOpt-CspoSpo0A was constructed via NEBuilder HiFi assembly through oligonucleotide-based amplification of modules in the following way. The backbone sequence was based on the modular vector pMTL88251 (plasmid maps and vectors available at plasmid-vectors.com). The homology arms which constitute the editing cassette were designed to be approximately 750 bp in length, and represent the sequence flanking spo0A natively, including a small amount of coding sequence of spo0A (in this case two codons at the 5’ end and three codons at the 3’ end, including the stop codon) to ensure that a small non-functional protein product is still produced following deletion of spo0A, thus avoiding potential polar effects on downstream sequences. Furthermore a ‘bookmark’ sequence representing a 45 bp intron targetable sequence was inserted between the homology arms within the editing cassette, such that the deleted region could be retargeted with an alternative group II intron for future complementation or cargo delivery. The 45 bp nature of the bookmark sequence ensures that the non-functional protein product left in place of spo0A remains in-frame. The I- PpoI amino acid sequence was taken from Physarum polycephalum and codon optimised for clostridial use, synthesised by IDT. The tetracycline inducible module driving expression of I-PpoI was taken from pMTLCW21, and its sequence provided in the supplementary section. A terminator from pMTL007C-E2 was included downstream of I-PpoI to prevent read-through from the tetracycline inducible promoter into the Gram- positive replicon. Following assembly resultant E. coli cloning host colonies were screened using PCR and their sequences confirmed using Sanger sequencing. The resultant vector was named pMTL8825-Ptet-I-PpoIOpt-CspoSpo0A. Strain generation: The shuttle vector pMTL007E-C1-I-PpoI::CspoSpo0A249s was conjugated into C. sporogenes using E. coli sExpress as the conjugal donor strain (Woods et al, 2019) using the established methods described therein. Recipient cells were isolated on TYG agar plates using 6 µg.ml-1 clarithromycin to select for acquisition of the vector, and 250 µg.ml-1 D-cycloserine to counter-select the Gram-negative donor sExpress cells. Colonies were then re-streaked to selective TYG agar plates containing 7.5 µg.ml-1 thiamphenicol to isolate cells in which mobilisation and integration of the group II intron had occurred, with self-spliced excision of the group I intron leading to acquisition of resistance to thiamphenicol through restoration of function of the CatA resistance marker. Resistant colonies were replica plated to selective media containing clarithromycin to screen for loss of the vector by virtue of the pseudo-suicide Gram- positive replicon, and sensitive colonies subsequently amplified by PCR at the spo0A locus to confirm the insertion of the group II intron at the target site. The three PCR products were also subjected to Sanger sequencing to confirm the presence of the CatA marker and I-PpoI recognition site, and the insertional strains termed C. sporogenes- spo0A::catA-I-PpoI. All three Sanger sequencing reactions returned the expected sequence for the inserted CatRAM. The vector pMTL8825-Ptet-I-PpoIOpt-CspoSpo0A was conjugated into C. sporogenes- spo0A::catA-I-PpoI.using E. coli sExpress as the conjugal donor strain, and recipients selected for on the basis of acquired clarithromycin resistance as previously described. Resistant colonies were then re-streaked to TYG agar plates containing 6 µg.ml-1 clarithromycin and 200 ng.ml-1 anhydrotetracycline. Single colonies which arose were re-streaked to a second plate containing 6 µg.ml-1 clarithromycin and 200 ng.ml-1 anhydrotetracycline to ensure suitable induction of the counter-selectable marker. Resultant colonies were then screened using replica plates to TYG agar with clarithromycin, and to TYG plates containing thiamphenicol and clarithromycin (Figure 5). Colonies which were completely unable to proliferate on the thiamphenicol containing plates were candidates for successful in-frame deletions and were screened by PCR at the spo0A locus for confirmation. PCR amplification of the spo0A locus using primers which bind to genomic regions outside of the design of the homologous editing cassette, revealed that 100% of the thiamphenicol sensitive colonies screened were in-frame deletions (Figure 6). Furthermore, it was observed that none were mixed colonies. It was also observed from the replica plating stage of the experiment that some level of counter-selective pressure was observed for the vast majority of colonies picked from the induction plates, with those that were still resistant only displaying very weak growth from discrete colonies in the patch as compared to the thiamphenicol containing plate. This indicates that escapees from the counter-selective pressure are not seen to the same level as those observed from CRISPR-Cas9 mutagenesis, where colonies are often present in high numbers which have completely evaded the counter-selective pressure. Three of the PCR products amplified were submitted for Sanger sequencing, which confirmed the anticipated nature of the in-frame deletions. Example 2: codA in-frame deletion in C. autoethanogenum using NegaTron containing an ErmRAM and pMTL8315-Ptet I-SceI In the following examples 2 and 3, the group II intron targeting region was specific to the codA (CLAU_3964) gene in Clostridium autoethanogenum, an industrially relevant acetogenic chassis organism. CodA was selected as the deletion target as it has previously been shown to be non-essential and has a simple phenotypic screen in the form of acquisition of resistance to 5-fluorocytosine (5-FC), a trait that is not required for isolation of mutants due to the screenable nature of this technology, but one that is useful for additional confirmation of genotype at large scale. The Perutka algorithm (Perutka et al, 2004) was used to select the target site within codA, which was between base pairs 531|532s, the ‘s’ denominating that the intron will insert in the sense orientation relative to the gene target. The Perutka algorithm assigned this target insertion site a score of 8.203. Vector design: The pMTL007C-E2::CautoCodA532s ClosTron plasmid supplied by ATUM was modified in the form of removal of the two FRT sites flanking the ErmRAM, and introduction of an I-SceI recognition site immediately downstream of ErmB. The modifications were incorporated on oligonucleotides used to amplify the ErmRAM, and the existing backbone of the vector amplified using overlapping oligonucleotides for subsequent construction using NEBuilder HiFi DNA Assembly. The new vector was termed pMTL007C-E2-I-SceI::CautoCodA532s, and its sequence confirmed through Sanger sequencing. pMTL8315-Ptet-I-SceIOpt-CautoCodA was constructed via NEBuilder HiFi assembly through oligonucleotide-based amplification of modules in the following way. The backbone sequence was based on the modular vector pMTL83151. The homology arms which constitute the editing cassette were designed to be approximately 750 bp in length, and represent the sequence flanking codA natively, including a small amount of coding sequence of codA (in this case three codons at the 5’ end and six codons at the 3’ end, including the stop codon) to ensure that a small non-functional protein product is still produced following deletion of codA. A ‘bookmark’ sequence representing a 45 bp intron targetable sequence was also inserted between the homology arms within the editing cassette, such that the deleted region could be retargeted with an alternative group II intron for future complementation or cargo delivery. The 45 bp nature of the bookmark sequence ensures that the non-functional protein product left in place of codA remains in-frame. The I-SceI amino acid sequence was taken from Saccharomyces cerevisiae and codon optimised for clostridial use, synthesised by Entelechon (now acquired by Eurofins). The tetracycline inducible module driving expression of I-SceI was taken from pMTLCW21 (Woods et al, 2022). A terminator from pMTL007C-E2 was included downstream of I-SceI to prevent read-through from the tetracycline inducible promoter into the Gram-positive replicon. Following assembly resultant colonies were screened using PCR and their sequences confirmed using Sanger sequencing. This vector was termed pMTL8315-Ptet-I-SceIOpt-CautoCodA. Strain generation: The shuttle vector pMTL007C-E2-I-SceI::CautocodA532s was conjugated into C. autoethanogenum using E. coli sExpress as the conjugal donor strain (Woods et al, 2019) using the established methods described therein. Recipient cells were isolated on YTF agar plates using 7.5 µg.ml-1 thiamphenicol to select for acquisition of the vector, and 250 µg.ml-1 D-cycloserine to counter-select the Gram-negative donor sExpress cells. Colonies were then re-streaked to selective YTF agar plates containing 6 µg.ml-1 clarithromycin to isolate cells in which mobilisation and integration of the group II intron had occurred, with self-spliced excision of the group I intron leading to acquisition of resistance to clarithromycin through restoration of function of the ErmB resistance marker. Resistant colonies were replica plated to selective media containing thiamphenicol to screen for loss of the vector by virtue of the pseudo-suicide Gram- positive replicon, and sensitive colonies subsequently amplified by PCR at the codA locus to confirm the insertion of the group II intron at the target site. The PCR product was also submitted for Sanger sequencing to confirm the presence of the ErmB marker and I-SceI site, and the insertional strain termed C. autoethanogenum-codA::ermB-I- SceI. The vector pMTL8315-Ptet-I-SceIOpt-CautoCodA was conjugated into C. autoethanogenum-codA::ermB-I-SceI using E. coli sExpress as the conjugal donor strain, and recipients selected for on the basis of acquired thiamphenicol resistance as previously described. Resistant colonies were then re-streaked to YTF agar plates containing 7.5 µg.ml-1 thiamphenicol and 100 ng.ml-1 anhydrotetracycline. Single colonies which arose were then screened using replica plates to YTF agar with thiamphenicol and anhydrotetracycline, and to YTF plates containing thiamphenicol, anhydrotetracycline and clarithromycin. Colonies which were completely unable to proliferate on the clarithromycin containing plates were candidates for successful in- frame deletions (Figure 7), and were screened by PCR at the codA locus for confirmation (Figure 8). Following PCR amplification of the codA locus using primers which bind to genomic regions outside of the design of the homologous editing cassette, it was observed that 100% of the clarithromycin sensitive colonies were in-frame deletions. Furthermore, it was observed that none were mixed colonies. It was also observed from the replica plating stage of the experiment that some level of counter-selective pressure was observed for all 36 colonies picked from the induction plates, with those that were still resistant only displaying very weak growth from discrete colonies in the patch as compared to the thiamphenicol containing plate. Three of the PCR products amplified here were submitted for Sanger sequencing, which confirmed the anticipated nature of the in-frame deletions. Example 3: codA in-frame deletion in C. autoethanogenum using NegaTron containing C. leptum PyrERAM and pMTL8825-codAHA Vector design: The pMTL007C-E2::CautoCodA532s ClosTron plasmid supplied by ATUM was modified such that the ErmRAM and flanking FRT sites were completely removed and replaced with an entirely novel PyrERAM. The PyrERAM is derived from the native amino acid sequence coding for PyrE in Clostridium leptum, and following a similar logic to the novel CatRAM described earlier and demonstrated in Example 3, it was selected as the required exon sequences for the self-splicing group I intron could be introduced silently into the native coding sequence. The required sequence of exons “G|ACCCAAGAGA” could be introduced from base pair 503 in C. leptum pyrE without alteration of the coding sequence, and the remainder of the sequence was codon optimised for use in Clostridium acetobutylicum (IDT codon optimisation service). The group I intron sequence was then inserted between the exons in the antisense orientation as compared to the pyrE sequence, and the completed RAM synthesised by IDT as a gBlock fragment. The PyrERAM was cloned into the backbone of pMTL007C- E2::CautoCodA532s using HiFi assembly, and the resultant vectors sequence confirmed by Sanger sequencing, termed pMTL007C-P2::CautocodA532s. pMTL88251-C.autoCodAHA was constructed using an identical editing cassette as described in Example 2, whereby the homology arms were approximately 750 bp in length, and represent the sequence flanking codA natively, including a small amount of coding sequence of codA (in this case three codons at the 5’ end and 6 codons at the 3’ end, including the stop codon) to ensure that a small non-functional protein product is still produced following deletion of codA. A ‘bookmark’ sequence representing a 45 bp intron targetable sequence was also inserted between the homology arms within the editing cassette, such that the deleted region could be retargeted with an alternative group II intron for future complementation or cargo delivery. The 45 bp nature of the bookmark sequence ensures that the non-functional protein product left in place of codA remains in-frame. The editing cassette was introduced into the multiple cloning site of the modular vector pMTL88251, between the NotI and XhoI restriction sites, and cloned using NEB HiFi assembly. Strain generation: The shuttle vector pMTL007C-P2::CautocodA532s was conjugated into C. autoethanogenum ΔpyrE (generated previously using Allele-Coupled Exchange) (Annan et al. 2019), using E. coli sExpress as the conjugal donor strain (Woods et al, 2019) as described previously. Recipient cells were isolated on YTAF agar plates using 7.5 µg.ml-1 thiamphenicol to select for acquisition of the vector, and 250 µg.ml-1 D- cycloserine to counter-select the Gram-negative donor sExpress cells. Colonies were then re-streaked to minimal defined media PETCMES lacking uracil to isolate cells in which mobilisation and integration of the group II intron had occurred, with self-spliced excision of the group I intron leading to restoration of uracil prototrophy through restoration of function of the intron-delivered pyrE gene. Resultant colonies were replica plated to non-selective and selective media containing thiamphenicol to screen for loss of the vector by virtue of the pseudo-suicide Gram-positive replicon, and sensitive colonies subsequently amplified by PCR at the codA locus to confirm the insertion of the group II intron at the target site. The PCR product was also subjected to Sanger sequencing to confirm the presence of the undisrupted C. leptum pyrE, and the insertional strain termed C. autoethanogenum -codA::C.lep-pyrE. The vector pMTL88251-CautoCodAHA was conjugated into C. autoethanogenum- codA::C.lep-pyrE using E. coli sExpress as the conjugal donor strain, and recipients selected for on the basis of acquired clarithromycin resistance as previously described. Resistant colonies were then re-streaked to YTAF agar plates containing 3 µgml-1 5- fluoroorotic acid. Single colonies which arose were then screened by PCR at the codA locus to confirm that cells still containing the pyrE gene had been counter-selected against leaving those which had successfully undergone homologous recombination via the supplied editing template. A drawback of the PyrERAM method is that, unlike in Example 1 and 2, there is no additional phenotypic screen to confirm the successful homologous recombination mediated in-frame deletion of the counter-selectable element and intron. It is also more likely for escapees to arise which are resistant to 5- FOA counter-selective pressure, as a single point mutation in the pyrE or natively encoded pyrF gene can prevent the conversion of 5-FOA to the toxic compound 5- FUMP. Nevertheless, this method resulted in successful editing with an 80% success rate in the colonies screened, as can be seen from the results of the PCR screen of the codA locus using primers annealing to genomic regions outside of the recombination cassette (Figure 11). Supplementary information Tetracycline inducible promoter module The nucleotide sequence of the Tetracycline inducible promoter module is provided herein as SEQ ID NO. 67, as follows: ttaagacccactttcacatttaagttgtttttctaatccgcatatgatcaattcaaggccgaataagaaggctggctctg caccttggtgatcaaataattcgatagcttgtcgtaataatggcggcatactatcagtagtaggtgtttccctttcttcttt agcgacttgatgctcttgatcttccaatacgcaacctaaagtaaaatgccccacagcgctgagtgcatataatgcattc tctagtgaaaaaccttgttggcataaaaaggctaattgattttcgagagtttcatactgtttttctgtaggccgtgtacct aaatgtacttttgctccatcgcgatgacttagtaaagcacatctaaaacttttagcgttattacgtaaaaaatcttgcca gctttccccttctaaagggcaaaagtgagtatggtgcctatctaacatctcaatggctaaggcgtcgagcaaagcccg cttattttttacatgccaatacaatgtaggctgctctacacctagcttctgggcgagtttacgggttgttaaaccttcgat tccgacctcattaagcagctctaatgcgctgttaatcactttacttttatctaatctagacatcattaattcctcctttttgttg acattatatcattgatagagttatttgtcaaactagttttttatttcgatgccctggacttcatgaaaaactaaaaaaaatattgaca ctctatcattgatagagtataattaaaataagcttgatcgtagcggacgtccgcgagagaccttaaatatattgaagaggagga aatacat [SEQ ID NO. 67] The nucleotides in bold represents tetR in the antisense orientation, the lowercase nucleotide section represents the divergent promoter driving both tetR and the gene, which is intended to be under inducible control, and the -10 and -35 boxes of the inducible promoter in the sense orientation are underlined. References: Perutka J, Wang W, Goerlitz D, Lambowitz AM. Use of computer-designed group II introns to disrupt Escherichia coli DExH / D-box protein and DNA helicase genes. J Mol Biol. 2004 Feb 13;336(2):421-39. doi: 10.1016 / j.jmb.2003.12.009. PMID: 14757055. Woods C, Humphreys CM, Rodrigues RM, Ingle P, Rowe P, Henstra AM, Köpke M, Simpson SD, Winzer K, Minton NP. A novel conjugal donor strain for improved DNA transfer into Clostridium spp. Anaerobe. 2019 Oct;59:184-191. doi: 10.1016 / j.anaerobe.2019.06.020. Epub 2019 Jun 30. PMID: 31269456; PMCID: PMC6866869. Annan FJ, Al-Sinawi B, Humphreys CM, Norman R, Winzer K, Köpke M, Simpson SD, Minton NP, Henstra AM. Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum. Appl Microbiol Biotechnol.2019 Jun;103(11):4633- 4648. doi: 10.1007 / s00253-019-09763-6. Epub 2019 Apr 10. PMID: 30972463; PMCID: PMC6505512. Examples of NegaTron (Forge Editing) Summary of exemplified in-frame deletion knockouts: Example Organism RAM Meganuclease Gene target number 1. Clostridium CatA I-PpoI Spo0A sporogenes 2. Clostridium ErmB I-SceI codA autoethanogenum 3. Clostridium PyrE N / A codA autoethanogenum Further examples: Example Organism RAM Meganuclease Gene target number 4. Clostridium ErmB I-PpoI pyrF sporogenes 5. Clostridium ErmB I-DmoI pyrF sporogenes 6. Clostridium ErmB I-NjaI pyrF sporogenes 7. Clostridium ErmB I-DirI pyrF sporogenes 8. Clostridium ErmB I-BmoI pyrF sporogenes 9. Clostridium ErmB I-PpoI pyrF botulinum 10. Clostridium ErmB I-DmoI pyrF botulinum 11. Clostridium ErmB I-NjaI pyrF botulinum 12. Clostridium ErmB I-PpoI mtlD difficile 13. Escherichia coli Kan I-PpoI pyrE Each of the features in the above tables may be used in the invention in their presented combinations or individually (e.g. one element may be selected and combined with any one other element, and individual elements may be swapped in the above combinations with another example). We have demonstrated the technique is capable of generating knockouts in additional Clostridial species (C. botulinum and C. difficile), and that it can also be applied to non- clostridial bacteria, as shown in the case of E. coli. We have expanded the number of examples of retrotransposition activated markers (RAMs) to four, and the number of meganuclease enzymes to six, demonstrating that it is broadly applicable to many combinations of markers and counter-selective genes / meganucleases. Therefore, it clearly works effectively with a wide variety of candidates and in a broad range of organisms, to provide the delivery of a counter-selectable element alongside a selectable marker by a retargetable intron. Examples 4-8: pyrF in-frame deletion in C. sporogenes using NegaTron containing an ErmRAM (driven by C. kluyveri fdx promoter) and pMTL8815-Ptet I-PpoI (Example 4), pMTL8815-Ptet I-DmoI (Example 5), pMTL8815-Ptet I-NjaI (Example 6), pMTL8815-Ptet I-DirI (Example 7), pMTL8815-Ptet I-BmoI (Example 8). In the following Examples 4-8, the group II intron targeting region was specific to the pyrF (CLSPO_c33410) gene in Clostridium sporogenes. PyrF was selected as the deletion target as it has previously been shown to be non-essential. The Perutka algorithm (Perutka et al, 2004) was used to select the target site within pyrF, which was between base pairs 595|596s, the ‘s’ denominating that the intron will insert in the sense orientation relative to the gene target. The Perutka algorithm assigned this target insertion site a score of 7.525. Vector design: The pMTL007C-E2::CspopyrF595s ClosTron plasmid supplied by ATUM was modified in the form of removal of the two FRT sites flanking the ErmRAM with promoter driving expression of the RAM was replaced with the C. kluyveri fdx promoter, and several meganuclease recognition sites (with a two base pair spacer included between each) introduced immediately downstream of ErmB. The modifications were incorporated on oligonucleotides used to amplify the ErmRAM, and the existing backbone of the vector amplified using overlapping oligonucleotides for subsequent construction using NEBuilder HiFi DNA Assembly. Two new vectors were constructed in this manner with the meganuclease recognition sites incorporated altered, and both plasmids sequence confirmed through Sanger sequencing. The first vector was termed pMTL007C-E2-MN- Cassette1::CspopyrF595s, with I-PpoI I-SceI, I-CreI, I-DmoI, I-NjaI recognition sites inserted immediately downstream of ErmB. The second vector, termed pMTL007C-E2- MN-Cassette2::CspopyrF595s, alternatively had I-PpoI, I-DirI, I-BmoI, I-LtrWI, I- TevI, I-HmuI and I-DdiI meganuclease recognition sites inserted immediately downstream of ErmB. pMTL8815-Ptet-I-PpoI (Example 4), pMTL8815-Ptet-I-DmoI (Example 5), pMTL8815-Ptet-I-NjaI (Example 6), pMTL8815-Ptet-I-DirI (Example 7) and pMTL8815-Ptet-I-BmoI (Example 8) vectors were all constructed via NEBuilder HiFi assembly through oligonucleotide-based amplification of modules in the following way. The backbone sequence was based on the modular vector pMTL88151. The homology arms which constitute the editing cassette were designed to be approximately 750 bp in length, and represent the sequence flanking pyrF natively, including a small amount of coding sequence of pyrF (in this case two codons at the 5’ end and three codons at the 3’ end, including the stop codon) to ensure that a small non-functional protein product is still produced following deletion of pyrF. The amino acid sequence for each meganuclease was taken from the host organism (I-PpoI; Physarum polycephalum, I- DmoI; Desulfurococcus mobilis, I-NjaI; Naegleria jamiesoni, I-DirI; Didymium iridis, I-BmoI; Bacillus mojavensis), codon optimised for clostridial use and synthesised by Entelechon (now acquired by Eurofins). The tetracycline inducible module driving expression of meganuclease coding sequence was taken from pMTLCW21 (Woods et al, 2022). A terminator from pMTL007C-E2 was included downstream of meganuclease to prevent read-through from the tetracycline inducible promoter into the Gram-positive replicon. Following assembly resultant colonies were screened using PCR and their sequences confirmed using Sanger sequencing. Strain generation: The shuttle vector pMTL007C-E2-MN-Cassette1::CspopyrF595s or pMTL007C-E2- MN-Cassette2::CspopyrF595s was conjugated into C. sporogenes using E. coli sExpress as the conjugal donor strain (Woods et al, 2019) using the established methods described therein. Recipient cells were isolated on TYG agar plates using 15 µg.ml-1 thiamphenicol to select for acquisition of the vector, and 250 µg.ml-1 D-cycloserine to counter-select the Gram-negative donor sExpress cells. Colonies were then re-streaked to selective TYG agar plates containing 10 µg.ml-1 erythromycin to isolate cells in which mobilisation and integration of the group II intron had occurred, with self-spliced excision of the group I intron leading to acquisition of resistance to erythromycin through restoration of function of the ErmB resistance marker. Resistant colonies were replica plated to selective media containing thiamphenicol to screen for loss of the vector by virtue of the pseudo-suicide Gram-positive replicon, and sensitive colonies subsequently amplified by PCR at the pyrF locus to confirm the insertion of the group II intron at the target site (Figure 13). The PCR product was also submitted for Sanger sequencing to confirm the presence of the ErmB marker and meganuclease sites, and the insertional strains were termed C. sporogenes-pyrF::ermB-PpoI-SceI-CreI-DmoI- NjaI and C. sporogenes-pyrF::ermB-PpoI-DirI-BmoI-LtrWI-TevI-HmuI-DdiI created the by pMTL007C-E2-MN-Cassette1::CspopyrF595s and pMTL007C-E2-MN- Cassette2::CspopyrF595s respectively. The vectors pMTL8815-Ptet-I-PpoI-CspopyrF (example 4), pMTL8815-Ptet-I-DmoI- CspopyrF (example 5), pMTL8815-Ptet-I-NjaI-CspopyrF (example 6) were independently conjugated into C. sporogenes-pyrF::ermB-PpoI-SceI-CreI-DmoI-NjaI and pMTL8815-Ptet-I-DirI-CspopyrF (example 7), pMTL8815-Ptet-I-BmoI-CspopyrF (example 8) independently conjugated into C. sporogenes-pyrF::ermB-PpoI-DirI- BmoI-LtrWI-TevI-HmuI-DdiI, all using E. coli sExpress as the conjugal donor strain. Recipients were selected on the basis of acquiring thiamphenicol resistance as previously described. Resistant colonies were then re-streaked to TYG agar plates containing 15 µg.ml-1 thiamphenicol and 100 ng.ml-1 anhydrotetracycline. Single colonies which arose were then screened by PCR at the pyrF locus using primers which bind to genomic regions outside of the design of the homologous editing cassette for confirmation of successful in-frame deletions (Cspo-pyrF-SCRF1 and Cspo-pyrF- SCRR1) (Figure 14). The PCR products that identified the deletion were submitted for Sanger sequencing, which confirmed the anticipated nature of the in-frame deletions. Examples 9-11: pyrF in-frame deletion in C. botulinum using NegaTron containing an ErmRAM (driven by C. kluyveri fdx promoter) and pMTL8815-Ptet I-PpoI (Example 9), pMTL8815-Ptet I-DmoI (Example 10), pMTL8815-Ptet I-NjaI (Example 11). In the following examples 9-11, the group II intron targeting region used to target the pyrF (CBO3238) gene in Clostridium botulinum was the same as used to target the pyrF in C. sporogenes, due to sufficient shared homology at this locus. Clostridium botulinum is an important bacterium of industrial relevance to the pharmaceutical sector and an infamous pathogen due the production of its extremely potent neurotoxin. PyrF was selected as the deletion target as it has previously been shown to be non-essential. The Perutka algorithm (Perutka et al, 2004) was used to select the target site within pyrF, which was between base pairs 595|596s, the ‘s’ denominating that the intron will insert in the sense orientation relative to the gene target. The Perutka algorithm assigned this target insertion site a score of 7.525. Vector design: The pMTL007C-E2::CspopyrF595s ClosTron plasmid supplied by ATUM was modified in the form of removal of the two FRT sites flanking the ErmRAM with promoter driving expression of the RAM was replaced with the C. kluyveri fdx promoter, and several meganuclease recognition sites (with a two base pair spacer included between each) introduced immediately downstream of ErmB. The modifications were incorporated on oligonucleotides used to amplify the ErmRAM, and the existing backbone of the vector amplified using overlapping oligonucleotides for subsequent construction using NEBuilder HiFi DNA Assembly. Two new vectors were constructed in this manner with the meganuclease recognition sites incorporated altered, and both plasmids sequence confirmed through Sanger sequencing. The first vector was termed pMTL007C-E2-MN- Cassette1::CspopyrF595s, with I-PpoI I-SceI, I-CreI, I-DmoI, I-NjaI recognition sites inserted immediately downstream of ErmB. pMTL8815-Ptet-I-PpoI (Example 9), pMTL8815-Ptet-I-DmoI (Example 10), pMTL8815-Ptet-I-NjaI (Example 11) vectors were all constructed via NEBuilder HiFi assembly through oligonucleotide-based amplification of modules in the following way. The backbone sequence was based on the modular vector pMTL88151. The homology arms which constitute the editing cassette were designed to be approximately 750 bp in length, and represent the sequence flanking pyrF natively, including a small amount of coding sequence of pyrF (in this case two codons at the 5’ end and three codons at the 3’ end, including the stop codon) to ensure that a small non-functional protein product is still produced following deletion of pyrF. The amino acid sequence for each meganuclease was taken from the host organism (I-PpoI; Physarum polycephalum, I- DmoI; Desulfurococcus mobilis, I-NjaI; Naegleria jamiesoni), codon optimised for clostridial use and synthesised by Entelechon (now acquired by Eurofins). The tetracycline inducible module driving expression of meganuclease coding sequence was taken from pMTLCW21 (Woods et al, 2022). A terminator from pMTL007C-E2 was included downstream of meganuclease to prevent read-through from the tetracycline inducible promoter into the Gram-positive replicon. Following assembly resultant colonies were screened using PCR and their sequences confirmed using Sanger sequencing. Strain generation: The shuttle vector pMTL007C-E2-MN-Cassette1::CspopyrF595s was conjugated into C. botulinum using E. coli sExpress as the conjugal donor strain (Woods et al, 2019) using the established methods described therein. Recipient cells were isolated on TYG agar plates using 15 µg.ml-1 thiamphenicol to select for acquisition of the vector, and 250 µg.ml-1 D-cycloserine to counter-select the Gram-negative donor sExpress cells. Colonies were then re-streaked to selective TYG agar plates containing 10 µg.ml-1 erythromycin to isolate cells in which mobilisation and integration of the group II intron had occurred, with self-spliced excision of the group I intron leading to acquisition of resistance to erythromycin through restoration of function of the ErmB resistance marker. Resistant colonies were replica plated to selective media containing thiamphenicol to screen for loss of the vector by virtue of the pseudo-suicide Gram- positive replicon, and sensitive colonies subsequently amplified by PCR at the pyrF locus to confirm the insertion of the group II intron at the target site (Figure 15). The PCR product was also submitted for Sanger sequencing to confirm the presence of the ErmB marker and meganuclease sites, and the insertional strains were termed C. botulinum-pyrF::ermB-PpoI-SceI-CreI-DmoI-NjaI. The vectors pMTL8815-Ptet-I-PpoI-CspopyrF (example 9), pMTL8815-Ptet-I-DmoI- CspopyrF (example 10), pMTL8815-Ptet-I-NjaI-CspopyrF (example 11) were independently conjugated into C. sporogenes-pyrF::ermB-PpoI-SceI-CreI-DmoI-NjaI using E. coli sExpress as the conjugal donor strain. Recipients were selected on the basis of acquiring thiamphenicol resistance as previously described. Resistant colonies were then re-streaked to TYG agar plates containing 15 µg.ml-1 thiamphenicol and 100 ng.ml-1 anhydrotetracycline. Single colonies which arose were then screened by PCR at the pyrF locus using primers which bind to genomic regions outside of the design of the homologous editing cassette for confirmation of successful in-frame deletions (Cbot- pyrF-SCRF1 and Cbot-pyrF-SCRR1) (Figure 16). The PCR products that identified the deletion were submitted for Sanger sequencing, which confirmed the anticipated nature of the in-frame deletions. Example 12: mtlD in-frame deletion in C. difficile using NegaTron containing an ErmRAM and pFE2-Clo-8815-DmA-CdimtlD In this example, the group II intron targeting region was specific to the mtlD (CD630_23310) gene in Clostridioides difficile, a medically relevant organism and a major cause of nosocomial diarrhoea. MtlD was selected as the deletion target as it has previously been shown to be non-essential. The Perutka algorithm (Perutka et al, 2004) was used to select the target site within mtlD, which was between base pairs 204|205s, the ‘s’ denominating that the intron will insert in the sense orientation relative to the gene target. The Perutka algorithm assigned this target insertion site a score of 8.181. Vector design: The pMTL007C-E2::CdimtlD204s ClosTron plasmid supplied by ATUM was modified in the form of removal of the two FRT sites flanking the ErmRAM, introduction of I- PpoI, I-SceI, I-CreI, I-DmoI and I-NjaI recognition sites immediately downstream of ErmB, and the promoter driving expression of the RAM was replaced with the C. kluyveri P fdx promoter. The modifications were incorporated on oligonucleotides used to amplify the ErmRAM, and the existing backbone of the vector amplified using overlapping oligonucleotides for subsequent construction using NEBuilder HiFi DNA Assembly. The new vector was termed pMTL007C-E2-MN-Cassette1::CdimtlD204s, and its sequence confirmed through Sanger sequencing. pMTL8815-Ptet-I-DmoI-CdimtlD was constructed via NEBuilder HiFi assembly through oligonucleotide-based amplification of modules in the following way. The backbone sequence was based on the modular vector pMTL88151. The homology arms which constitute the editing cassette were designed to be approximately 500 bp in length, and represent the sequence flanking mtlD natively, including a small amount of coding sequence of mtlD (in this case two codons at the 5’ end and three codons at the 3’ end, including the stop codon) to ensure that a small non-functional protein product is still produced following deletion of mtlD. The I-DmoI amino acid sequence was taken from Desulfurococcus mucosus and codon optimised for clostridial use, synthesised by Entelechon (now acquired by Eurofins). The tetracycline inducible module driving expression of I-DmoI was taken from pMTLCW21 (Woods et al, 2022). A terminator from pMTL007C-E2 was included downstream of I-DmoI to prevent read-through from the tetracycline inducible promoter into the Gram-positive replicon. Following assembly resultant colonies were screened using PCR and their sequences confirmed using Sanger sequencing. This vector was termed pMTL8815-Ptet-I-DmoI-CdimtlD. Strain generation: The shuttle vector pMTL007C-E2-MN-Cassette1::CdimtlD204s was conjugated into C. difficile 630Δerm* using E. coli sExpress as the conjugal donor strain (Woods et al, 2019) using the established methods described therein. Recipient cells were isolated on BHIS agar plates using 15 µg.ml-1 thiamphenicol to select for acquisition of the vector, and 250 µg.ml-1 D-cycloserine and 8 µg.ml-1 cefoxitin to counter-select the Gram- negative donor sExpress cells. Colonies were then re-streaked to selective BHIS agar plates containing 10 µg.ml-1 erythromycin to isolate cells in which mobilisation and integration of the group II intron had occurred, with self-spliced excision of the group I intron leading to acquisition of resistance to erythromycin through restoration of function of the ErmB resistance marker. Resistant colonies were replica plated to selective media containing thiamphenicol to screen for loss of the vector by virtue of the pseudo-suicide Gram-positive replicon, and sensitive colonies subsequently amplified by PCR at the mtlD locus using oligonucleotides EBS Universal and Cdif mtlD SCRR1 to confirm the insertion of the group II intron at the target site (Figure 17). The insertional strain was termed C. difficile 630Δerm*-mtlD::ermB-PpoI-SceI- CreI-DmoI-NjaI. The vector pMTL8815-Ptet-I-DmoI-CdimtlD was conjugated into C. difficile 630Δerm*-mtlD::ermB-PpoI-SceI-CreI-DmoI-NjaI using E. coli sExpress as the conjugal donor strain, and recipients selected for on the basis of acquired thiamphenicol resistance as previously described. Resistant colonies were then re-streaked to BHIS agar plates containing 15 µg.ml-1 thiamphenicol and 100 ng.ml-1 anhydrotetracycline. Single colonies which arose were then screened with primers Cdif mtlD SCRF1 and Cdif mtlD SCRR1 using PCR at the mtlD locus for confirmation of the intended deletion (Figure 18). Following PCR amplification of the mtlD locus using primers which bind to genomic regions outside of the design of the homologous editing cassette, it was observed that 100% of the screened colonies were in-frame deletions. Furthermore, it was observed that none were mixed colonies. Three of the PCR products amplified here were submitted for Sanger sequencing, which confirmed the anticipated nature of the in-frame deletions. Examples 13-14: pyrF in-frame deletion in E. coli using NegaTron containing a KanRAM and pMTL8815-PBAD-I-SceI (Example 13), pMTL8815-PBAD-I-DmoI (Example 14), In the following Examples 13 and 14, the group II intron targeting region was specific to the pyrE gene in E. coli. The Perutka algorithm (Perutka et al, 2004) was used to select the target site within pyrF, which was between base pairs 522|523a, the ‘a’ denominating that the intron will insert in the antisense orientation relative to the gene target. The Perutka algorithm assigned this target insertion site a score of 9.122. Vector design: The pMTL007C-E2::EcpyrE522a ClosTron plasmid supplied by ATUM was modified in the form of removal of the two FRT sites and ErmRAM, replacing it with a KanRAM amplified from pMTL20::lacZ-Targetron (SBRC CC#453) and introduction of I-PpoI, I-SceI, I-CreI, I-DmoI and I-NjaI recognition sites immediately downstream of Kan. The modifications were incorporated on oligonucleotides used to amplify the KanRAM (sequence of the KanRAM used is detailed below as SEQ ID NO: 89), and the existing backbone of the vector amplified using overlapping oligonucleotides for subsequent construction using NEBuilder HiFi DNA Assembly. The new vector was termed pMTL007C-K1-MN-Cassette1::EcpyrE522a, and its sequence confirmed through Sanger sequencing. The pMTL8815-PBAD-I-SceI (Example 13) and pMTL8815-PBAD-I-DmoI (Example 14), vectors were all constructed via NEBuilder HiFi assembly through oligonucleotide- based amplification of modules in the following way. The backbone sequence was based on the modular vector pMTL88151. The homology arms which constitute the editing cassette were designed to be approximately 750 bp in length, and represent the sequence flanking pyrE natively, including a small amount of coding sequence of pyrE (in this case two codons at the 5’ end and two codons at the 3’ end, including the stop codon) to ensure that a small non-functional protein product is still produced following deletion of pyrE. The amino acid sequence for each meganuclease was taken from the host organism (I-SceI; Saccharomyces cerevisiae, I-DmoI; Desulfurococcus mobilis) and codon optimised for E. coli use and synthesised by Entelechon (now acquired by Eurofins). The arabinose inducible promoter module driving expression of meganuclease coding sequence was amplified from RBV-Reco (SBRC CC#6404). A terminator from pMTL007C-E2 was included downstream of meganuclease to prevent read-through from the inducible promoter into the Gram-positive replicon. Following assembly resultant colonies were screened using PCR and their sequences confirmed using Sanger sequencing. Strain generation: The shuttle vector pMTL007C-K1-MN-Cassette1::EcpyrE522a was transformed into E. coli and recipient cells were isolated on LB agar plates using 25 µg.ml-1 chloramphenicol to select for acquisition of the vector. Colonies were then re-streaked to selective LB agar plates containing 50 µg.ml-1 kanamycin to isolate cells in which mobilisation and integration of the group II intron had occurred, with self-spliced excision of the group I intron leading to acquisition of resistance to kanamycin through restoration of function of the Kan resistance marker. Resistant colonies were replica plated to selective media containing chloramphenicol to screen for loss of the vector by segregational instability of the Gram-negative replicon, and sensitive colonies subsequently amplified by PCR at the pyrE locus to confirm the insertion of the group II intron at the target site (Figure 19) . The PCR product was also submitted for Sanger sequencing to confirm the presence of the Kan marker and meganuclease sites, and the insertional strains were termed E.coli -pyrE::Kan-PpoI-SceI-CreI-DmoI-NjaI. The vectors pMTL8815-PBAD-I-SceI (Example 13) and pMTL8815-PBAD-I-DmoI (Example 14) were independently transformed into E. coli-pyrE::Kan-PpoI-SceI-CreI- DmoI-NjaI. Recipients were selected on the basis of acquiring chloramphenicol resistance as previously described. Resistant colonies were then re-streaked to LB agar plates containing 25 µg.ml-1 thiamphenicol and 10 mM L-arabinose. Single colonies which arose were then screened by PCR at the pyrE locus using primers which bind to genomic regions outside of the design of the homologous editing cassette for confirmation of successful in-frame deletions (Ec-pyrE-SCRF1 and Ec-pyrE-SCRR1) (Figure 20). The PCR products that identified the deletion were submitted for Sanger sequencing, which confirmed the anticipated nature of the in-frame deletions. Supplementary information: The following specific sequences may be used for the invention for the named protein. Nucleotide sequence of codon optimised I-SceI: ATGCATCAGAAAAATCAAGTTATGAACTTAGGACCAAATAGTAAGTTACTTA AAGAATATAAGTCACAGCTTATAGAACTTAATATAGAGCAGTTTGAAGCAGG TATTGGACTTATATTAGGAGATGCTTACATAAGATCTAGGGATGAGGGTAAA ACATATTGTATGCAGTTTGAATGGAAGAATAAAGCATATATGGACCATGTATG TCTTTTATATGATCAGTGGGTTCTTAGTCCTCCACACAAAAAGGAAAGAGTAA ACCATCTTGGTAATTTAGTAATTACATGGGGAGCTCAGACATTTAAGCATCAA GCATTCAATAAATTAGCTAACCTTTTTATAGTAAATAACAAAAAGACAATTCC TAACAATTTAGTTGAGAATTATCTTACTCCAATGTCACTTGCATATTGGTTTAT GGATGACGGAGGTAAATGGGATTATAATAAGAATAGCACAAACAAGTCTATA GTTCTTAATACTCAAAGCTTCACTTTTGAAGAGGTTGAATATCTTGTAAAAGG ACTTAGAAATAAGTTTCAACTTAATTGCTATGTTAAAATAAATAAGAATAAGC CAATTATATATATAGATAGCATGTCTTATCTTATATTTTACAATCTTATAAAGC CATATTTAATACCTCAGATGATGTATAAACTTCCAAACACAATAAGTAGCGAA ACTTTTCTTAAGTAA (SEQ ID NO. 79) Nucleotide sequence of codon optimised I-PpoI: ATGGCTAAGAGCAATCAAGCAAGGGTGAACGGTGGATCTAACTACGACTCTC TAACTCCTTTAAATATGGCATTAACAAATGCTCAGATTCTTGCTGTGATAGAC TCTTGGGAGGAAACAGTAGGACAATTCCCTGTTATTACTCACCACGTACCTCT AGGTGGAGGATTACAAGGTACACTACACTGCTATGAAATTCCTCTTGCTGCAC CTTACGGAGTAGGTTTCGCAAAGAATGGACCTACAAGATGGCAATACAAGAG AACAATAAATCAAGTTGTTCATAGGTGGGGATCTCATACTGTACCTTTCCTTT TGGAACCTGACAATATAAATGGAAAGACTTGCACAGCTTCTCATTTATGTCAT AATACTAGGTGCCATAACCCATTGCACCTTTGTTGGGAGAGTCTTGACGACAA CAAAGGAAGAAACTGGTGTCCTGGTCCTAACGGTGGTTGTGTTCATGCAGTGG TGTGCTTGAGACAGGGTCCTCTATACGGACCTGGTGCTACTGTGGCTGGACCA CAGCAAAGAGGTTCTCACTTCGTTGTATAA (SEQ ID NO. 80) Nucleotide sequence of codon optimised I-DmoI: ATGCACAACAATGAGAACGTGAGTGGAATTTCAGCTTACTTATTGGGATTGAT AATAGGAGACGGTGGATTGTACAAATTAAAGTATAAGGGTAATAGGTCAGAA TACAGAGTTGTGATTACACAAAAAAGCGAAAACCTAATTAAACAACACATAG CACCACTTATGCAGTTCTTGATAGACGAATTGAATGTTAAAAGCAAAATTCAG ATTGTAAAAGGTGATACAAGATATGAATTGAGAGTATCTTCAAAGAAACTAT ATTATTATTTTGCTAACATGTTAGAGAGGATAAGGCTTTTCAACATGAGGGAA CAAATAGCTTTCATTAAAGGATTGTATGTGGCAGAAGGAGATAAGACACTAA AAAGGTTAAGAATTTGGAACAAAAACAAAGCTCTACTAGAGATTGTTAGCAG GTGGTTAAATAATTTGGGAGTGAGGAATACTATTCACTTGGACGATCACAGAC ATGGAGTATATGTGTTAAACATAAGTTTGAGGGATAGGATTAAATTTGTACAT ACTATTCTAAGTAGTCATCTTAATCCATTGCCTCCTGAGTAG (SEQ ID NO. 81) Nucleotide sequence of codon optimised I-NjaI: ATGGCTTCAATTAAGCAAAAGGTTACTAGGCTTAGGAATAAGTTAAGGTCTA GGTCTAGTAGAAATGGACCTAAGACTATAACTGTTAAAATTGACAGAAAAAG CTTCAAAAACGGTTATGATCCTTTAGTTGATACTATAGATTACGGATACAGCA AAATGGCTAAAATTACAGTTAACAAAAATGACCAGTTAGCAAAGTTAAAAAA CTGTAAGCAGGCAGTGAACATATTCAACGAATGGCTAAGTAACAGGAAAGGA GACAAAGGTAGGAGTGGAAAGCAAAAACCTTATTGCTTCGATGAACTTAAGA AGCTAGACGTTTGCAAACATGAGTTTGGAGAGTGTTTAATTGGAGCAGCATCT AAGACAAAGTCAGGTTTTAAGGTGAGATTCATGAATAATAAGGGAAGTGATT CTTATGTTCATCATGTAAGTGTGTTTGCAAATAGTACTTGTGAGAACTGTATTC ACTCAAGAAAAATGCTTGAGACAGTATCTTCAAGTAAGAAGGACCCAGATGC TAGGACAATTAGCCACTTGTGTGGAAACGGTGGTTGTGCAAGACCAGGACAC CTAAGGATTGAAAAGAAAAGTGTTAATGATGAAAGAACACATTGCCACTTCT TATTAAGGAGATCTCAGAGCGTGGCTCAATCAGAAATGATAAGATTGGCATG TCCTCACACTCCAAGGTGTTTCGTTAATTTATACAAAATAAACAAGCCTTATT AT (SEQ ID NO. 82) Nucleotide sequence of codon optimised I-DirI: ATGGTTAAGGGAACCGCAAGGAAGAAGCAAGATCATGGTCTTGTTGGTCTTA AGGATCTTCTAGATACTCTTGAGTATCATTGGTTGCAAACTAGCGATAGGCCT AGGAAGACAATTAAGAGGAGCCATACAGTGCTACATTTGAAGAGGAGGCTTG CACAACTTGCAGGACAAAGGGTTTCATGTTTCGGTTACTTGACAGCTTCAGTT TTGGGTATGTCTCTTGAATGTAAGGGTGGTTACCTTGCTAACTTGGTTAACAG CGGTCCCATGCCCACAGCAGAGTGGTTCCATGAGCAAGATGTTAAGCTTACTA AGGAATATGGGATGTGCTTGATGAGCCCTAGGCCACATCACCAAATGAGGAT TCAAACATCAAAGGGGAGGGCTGGTAGGTTTAAGATCCAAGCTAGCGCACTT CAAGCAGTTCTAGTTAACAACCCTAGCTCACACGATGAACTTGTTGAACAAGT TAAGGGTCTTATCGATAGGGAGACAACAACATTTCATTCATCACATCTTTGTA AGGGGGATGGTAGCTGCATGGAACTAAAGCACACATTGAGGGTTCCTGCACA AACTAACCTTGCAGATCATGAACTTTGTCCTGCATTCGTTGTTATTTACGGGA ACTTGGTTAACTTGTGCACTTGCAGCGCAAACGAAGGGAGGCAATGTTTGGTT CCTGGTAGGCGTTTTAACTTTGCAAACTGGGCACGTGTGTATGCTCCTTTGAT GACAACATTTCTAAAGCCTAAGGCAGGAACAGGTATTGTTAACAAGTAA (SEQ ID NO. 83) Nucleotide sequence of codon optimised I-BmoI: ATTAAGTCAGGTGTTTATAAGATCACTAACAAGAACACTGGGAAGTTTTATAT TGGTAGCTCAGAAGACTGCGAAAGCAGGCTGAAGGTTCATTTTAGGAACCTG AAGAACAACAGGCATATTAACAGGTATCTTAACAACAGCTTTAACAAGCATG GTGAGCAAGTGTTTATTGGTGAGGTGATTCATATTCTTCCCATCGAAGAGGCA ATCGCAAAGGAGCAATGGTATATTGATAACTTCTATGAAGAAATGTACAACA TCAGCAAGTCAGCATACCATGGTGGTGACCTTACTTCATATCATCCTGACAAG AGGAACATTATTCTAAAGAGGGCAGACAGCCTTAAGAAGGTTTATCTTAAGA TGACTTCAGAAGAAAAGGCAAAGAGGTGGCAATGCGTTCAAGGTGAAAACAA CCCAATGTTTGGTAGGAAGCATACTGAAACAACTAAGCTGAAGATTTCAAAC CATAACAAGCTTTATTACTCAACCCATAAGAACCCTTTTAAGGGGAAGAAGC ATAGCGAGGAGAGCAAGACCAAGCTTTCAGAATATGCATCACAAAGGGTTGG GGAAAAGAACCCATTCTATGGTAAGACTCATAGCGATGAATTTAAGACCTAT ATGTCAAAGAAGTTTAAGGGTAGGAAGCCTAAGAACTCAAGGCCTGTTATTA TCGATGGTACTGAATATGAAAGCGCAACTGAAGCATCAAGGCAACTTAACGT GGTTCCTGCAACCATTCTACATAGGATTAAGAGCAAGAACGAAAAGTACAGC GGTTACTTTTACAAGTGA (SEQ ID NO. 84) Nucleotide sequence of E. coli codon optimised I-SceI: ATGAAAAACATCAAAAAAAACCAGGTAATGAACCTGGGTCCGAACTCTAAAC TGCTGAAAGAATACAAATCCCAGCTGATCGAACTGAACATCGAACAGTTCGA AGCAGGTATCGGTCTGATCCTGGGTGATGCTTACATCCGTTCTCGTGATGAAG GTAAAACCTACTGTATGCAGTTCGAGTGGAAAAACAAAGCATACATGGACCA CGTATGTCTGCTGTACGATCAGTGGGTACTGTCCCCGCCGCACAAAAAAGAAC GTGTTAACCACCTGGGTAACCTGGTAATCACCTGGGGCGCCCAGACTTTCAAA CACCAAGCTTTCAACAAACTGGCTAACCTGTTCATCGTTAACAACAAAAAAAC CATCCCGAACAACCTGGTTGAAAACTACCTGACCCCGATGTCTCTGGCATACT GGTTCATGGATGATGGTGGTAAATGGGATTACAACAAAAACTCTACCAACAA ATCGATCGTACTGAACACCCAGTCTTTCACTTTCGAAGAAGTAGAATACCTGG TTAAGGGTCTGCGTAACAAATTCCAACTGAACTGTTACGTAAAAATCAACAA AAACAAACCGATCATCTACATCGATTCTATGTCTTACCTGATCTTCTACAACC TGATCAAACCGTACCTGATCCCGCAGATGATGTACAAACTGCCGAACACTATC TCCTCCGAAACTTTCCTGAAATAA (SEQ ID NO. 87) Nucleotide sequence of E. coli codon optimised I-DmoI: ATGCACAACAATGAGAATGTTTCGGGAATCTCAGCCTATCTGCTTGGATTGAT TATTGGGGATGGTGGACTGTATAAGCTTAAATATAAGGGCAATCGTAGCGAG TACCGTGTGGTGATTACCCAGAAGAGCGAAAACCTGATTAAACAACATTTCG CGCCGTTAATGCAGTTCTTGATCGACGAACTTAATGTCAAATCTAAAATTCAG ATCGTCAAAGGCGACACCCGTTACGAGTTGCGCGTCTCTAGTAAGAAGTTGTA TTACTATTTCGCGAACATGCAGGAGCGCATCCGCCTTTTCAATATGCGCGAAC AAATCGCGTTTATTAAGGGTCTGTACGTTGCCGAAGGTGACAAGACATTAAA GCGCTTGCGTATTTGGAATAAGAATAAGGCGCTGCTGGAGATCGTCAGCCGCT GGTTGAACAACCTTGGCGTACGCAACACCATTCATCTGGACGACCATCGTCAT GGAGTATACGTCTTAAACATTTCTTTACGCGACCGTATCAAGTTTGTCCATAC CATTTTGTCGTCACACTTGAACCCATTGCCCCCCGAGTAA (SEQ ID NO. 88) Nucleotide sequence of Kanamycin RAM used in Examples 13 and 14: 1188 bp - Group I underlined - Exons bolded ATGGCTAAAATGAGAATATCACCGGAATTGAAAAAACTGATCGAAAAATACC GCTGCGTAAAAGATACGGAAGGAATGTCTCCTGCTAAGGTATATAAGCTGGT GGGAGAAAATGAAAACCTATATTTAAAAATGACGGACAGCCGGTATAAAGGG ACCACCTATGATGTGGAACGGGAAAAGGACATGATGCTATGGCTGGAAGGAA AGCTGCCTGTTCCAAAGGTCCTGCACTTTGAACGGCATGATGGCTGGAGCAAT CTGCTCATGAGTGAGGCCGATGGCGTCCTTTGCTCGGAAGAGTATGAAGATG AACAAAGCCCTGAAAAGATTATCGAGCTGTATGCGGAGTGCATCAGGCTCTTT CACTCCATCGACATATCGGATTGTCCCTATACGAATAGCTTAGACAGCCGCTT AGCCGAATTGGATTACTTACTGAATAACGATCTGGCCGATGTGGATTGCGAAA ACTGGGAAGAAGACACTCCATTTAAAGCATTATGTTCAGATAAGGTCGTTAAT CTTACCCCGGAATTATATCCAGCTGCATGTCACCATGCAGAGCAGACTATATC TCCAACTTGTTAAAGCAAGTTGTCTATCGTTTCGAGTCACTTGACCCTACTCCC CAAAGGGATAGTCGTTAGGCATTTATGTAGAACCAATTCCATTTATCAGATTT TACACGATAAGTAACTAATCCAGACGAAATTTTCTCTAGAGAAAGTATTTTTA ATCTGATAAATTCCGCTTTTCATAAATACCTCTTTAAATATAGAAGTATTTATT AAAGGGCAGTCCTACAATTTAGCACGGGATTGTCTACTAGAGAGGTTCCCCGT TTAGATAGATTACAAGTATAAGTCACCTTATACTCAGGCCTCAATTAACCCAA GAGAGCTGTATGATTTTTTAAAGACGGAAAAGCCCGAAGAGGAACTTGTCTT TTCCCACGGCGACCTGGGAGACAGCAACATCTTTGTGAAAGATGGCAAAGTA AGTGGCTTTATTGATCTTGGGAGAAGCGGCAGGGCGGACAAGTGGTATGACA TTGCCTTCTGCGTCCGGTCGATCAGGGAGGATATCGGGGAAGAACAGTATGTC GAGCTATTTTTTGACTTACTGGGGATCAAGCCTGATTGGGAGAAAATAAAATA TTATATTTTACTGGATGAATTGTTTTAG (SEQ ID NO. 89)

Claims

CLAIMS 1. A nucleic acid molecule encoding a group II intron, wherein the group II intron encodes a retrotransposition-activated marker (RAM) and a counter-selection marker.

2. A nucleic acid molecule encoding: (a) a first promoter operably-linked to a group II intron, wherein the group II intron does not encode a protein comprising reverse transcriptase activity, and (b) a second promoter operably-linked to a nucleic acid sequence encoding an intron-encoded protein (IEP), wherein the group II intron encodes: (i) a retrotransposition-activated marker (RAM), (ii) a counter-selection marker, and (iii) a targeting sequence.

3. The nucleic acid according to claim 2, wherein, in use in a host cell, a transcript of the group II intron is inserted into a target site of the DNA of the host cell by the IEP and the targeting sequence of the group II intron.

4. A nucleic acid molecule according to any of claims 1-3, wherein the nucleic acid molecule is DNA; or wherein the nucleic acid molecule is a plasmid.

5. A nucleic acid molecule according to any one of the preceding claims, wherein the group II intron comprises LI.LtrB.

6. A nucleic acid molecule according to any one of the preceding claims, wherein the counter-selection marker is a nucleotide sequence that confers a selection disadvantage to cells that have been transformed with a nucleotide sequence compared to cells that have not been transformed with the nucleotide sequence or cells from which the nucleotide sequence has been removed.

7. A nucleic acid molecule according to any one of the preceding claims, wherein the counter-selection marker is a nuclease recognition site or a toxic marker gene.

8. A nucleic acid molecule according to claim 7, wherein the nuclease recognition site comprises a meganuclease recognition site or an endonuclease recognition site.

9. A nucleic acid molecule according to claim 8, wherein the meganuclease recognition site is selected from a I-SceI meganuclease recognition site; a PpoI meganuclease recognition site; a I-DmoI meganuclease recognition site; a I-NjaI meganuclease recognition site; a I-DirI meganuclease recognition site; and a I-BmoI meganuclease recognition site; or wherein the meganuclease recognition site comprises a nucleotide sequence substantially as set out in a sequence selected from any of SEQ ID NOs. 1, 2, or 64-66.

10. A nucleic acid molecule according to claim 7, wherein the toxic marker gene comprises pyrE or pyrF, optionally wherein pyrE comprises a nucleotide sequence substantially as set out in any one of SEQ ID NOs. 54, 56 or 58.

11. A nucleic acid molecule according to any one of the preceding claims, wherein the marker gene of the RAM encodes or encompasses a group I intron, optionally wherein the group I intron is flanked by exons that enable self-splicing of the group I intron.

12. A nucleic acid molecule according to any one of the preceding claims, wherein the marker gene of the RAM is a gene that codes for antibiotic resistance or is an auxotrophy marker gene.

13. A nucleic acid molecule according to any one of the preceding claims, wherein the RAM is a Cat-RAM, optionally wherein the Cat-RAM has a nucleotide sequence substantially as set out in SEQ ID NO.

46.

14. A nucleic acid molecule according to claim 12, wherein the auxotrophy marker gene is pyrE, optionally wherein the pyrE has a nucleotide sequence substantially as set out in SEQ ID NO. 54, 56, or 58.

15. A nucleic acid molecule according to any one of the preceding claims, wherein the IEP encoded by the nucleic acid molecule is LtrA, optionally wherein LtrA comprises a nucleotide sequence substantially as set out in SEQ ID NO. 60.

16. A nucleic acid molecule according to any one of claims 2-14, wherein the first promoter is the Pfdx promoter and / or wherein the second promoter is the PltrA promoter.

17. A host cell comprising a nucleic acid molecule according to any one of claims 1 to 16.

18. A kit comprising a first nucleic acid molecule; and a second nucleic acid molecule encoding a homologous recombination cassette, wherein the first nucleic acid molecule encodes: (a) a first promoter operably-linked to a group II intron, wherein the group II intron does not encode a protein comprising reverse transcriptase activity, and (b) a second promoter operably-linked to a nucleic acid sequence encoding an intron encoded protein (IEP), wherein the group II intron encodes: (i) a retrotransposition-activated genetic marker (RAM), (ii) a counter-selection marker, and (iii) a targeting sequence.

19. The kit according to claim 18, wherein, in use in a host cell, a transcript of the group II intron is inserted into a target site of the DNA of the host cell by the IEP and the targeting sequence of the group II intron.

20. A kit comprising a host cell; and a second nucleic acid molecule encoding a homologous recombination cassette, wherein the host cell comprises a nucleic acid molecule encoding: (a) a first promoter operably-linked to a group II intron, wherein the group II intron does not encode a protein comprising reverse transcriptase activity, and (b) a second promoter operably-linked to a nucleic acid sequence encoding an intron encoded protein (IEP), wherein the group II intron encodes: (i) a retrotransposition-activated marker (RAM), (ii) a counter-selection marker, and(iii) a targeting sequence.

21. The kit according to claim 20, wherein, in use in a host cell, a transcript of the group II intron is inserted into a target site of the DNA of the host cell by the IEP and the targeting sequence of the group II intron.

22. A method of introducing a heterologous polynucleotide into the DNA of a plurality of host cells, the method comprising: (i) providing a plurality of host cells that comprise a group II intron, wherein the group II intron does not encode a protein comprising reverse transcriptase activity, and wherein the group II intron comprises a nucleotide sequence encoding a counter- selection marker and optionally a positive selection marker; (ii) using homologous recombination to replace the group II intron with a heterologous polynucleotide in one or more of the plurality of host cells; and (iii) applying a counter-selection pressure to the plurality host cells such that only host cells in which the group II intron comprising the counter-selection marker has been replaced, survive.

23. A method of removing a polynucleotide sequence from DNA of a plurality of host cells, the method comprising: (i) providing a plurality of host cells that comprise a group II intron, wherein the group II intron does not encode a protein comprising reverse transcriptase activity, and wherein the group II intron comprises a nucleotide sequence encoding a counter- selection marker and optionally a positive selection marker; (ii) using homologous recombination to remove the group II intron from the one or more of the plurality of host cells; and (iii) applying a counter-selection pressure to the plurality of host cells such that only host cells in which the group II intron comprising the counter-selection marker has been removed survive.

24. A method of producing a cell comprising a heterologous polynucleotide, the method comprising:

1. transforming a plurality of host cells with a first nucleic acid molecule according to any to claims 1 to 16 such that the group II intron is retrotransposed;2. applying a positive selection pressure to the plurality host cells such that only host cells encoding a functional positive selection marker of the RAM survive; 3. using homologous recombination to replace the nucleotide sequence encoding the counter-selection marker with the heterologous polynucleotide in one or more of the plurality of host cells by transforming one or more of the plurality of host cells with a second nucleic acid molecule that encodes a homologous recombination cassette comprising a first and a second homology arm that flank the heterologous polynucleotide, wherein the first homology arm is homologous with the first homology region and the second homology arm is homologous with the second homology region, and wherein the first homology region and the second homology region flank a nucleotide sequence encoding the retrotransposed group II intron comprising the counter selection marker; and 4. applying a counter-selection pressure to the plurality of host cells such that only host cells in which the counter-selection marker has been replaced survive.

25. A nucleic acid molecule encoding a chloramphenicol acetyl transferase antibiotic resistance marker gene with a group I intron inserted therein (Cat-RAM).

26. A nucleic acid molecule encoding a group II intron comprising a Cat retrotransposition-activated marker (Cat-RAM) gene, wherein the Cat-RAM comprises a Cat antibiotic resistance marker gene comprising a group I intron inserted therein.

27. A nucleic acid molecule according to claim 25 or claim 26, wherein the CatA antibiotic resistance marker gene comprises a nucleotide sequence substantially as set out in SEQ ID NO.

46.

28. A nucleic acid molecule according to any one of claims 1-16, or 25-27, wherein the group II intron comprise a nucleotide sequence substantially as set out in SEQ ID NO.

61.

29. A host cell according to claim 17, a kit according to any of claims 18-21 or a method according to any of claims 22-24, wherein the host cell is a bacterium or yeast.

30. A cell produced by the method according to any of claims 22-24.

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