Patent Publication Number: US-2023142094-A1

Title: Plasmid addiction system to drive desired gene expression

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Patent Applications No. 62/697,531, filed Jul. 13, 2018, entitled PLASMID ADDICTION SYSTEM TO DRIVE DESIRED GENE EXPRESSION; and No. 62/535,596, filed Jul. 21, 2017, entitled PLASMID ADDICTION SYSTEM TO DRIVE DESIRED GENE EXPRESSION, the disclosures of both of which are hereby incorporated by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     The field of the invention relates to methods and processes useful in maintaining extrachromosomal elements of interest in a microbial production strain using genes from the succinate pathway to ensure inclusion and expression of the elements in daughter cells. More specifically, it relates to the use of a plasmid addiction system that ensures that modified microbial cells will maintain plasmids carrying genes involved in producing desired expression products. 
     BACKGROUND OF THE INVENTION 
     The present invention is directed to a method of manipulating microbial cells in culture to maintain at least one extrachromosomal element of interest containing at least one gene of interest. Typically, this extrachromosomal element is a plasmid, though phages, prophages, phagemids, cosmids, bacterial artificial chromosomes (BACs) also contain extrachromosomal elements to contain transgenes of heterologous interest. Though naturally occurring in bacteria, not all wild type plasmids contain genetic information that is required to maintain the viability of the host cell in normal conditions. However, plasmids can contain genetic information that provides selective advantages to the host under specific environmental challenges such as antibiotic resistance or resistance to noxious compounds present in the environment. However, in those situations where adverse environmental conditions are not present, the presence of the plasmid is, in fact, a metabolic burden upon the cell (Nordstrom and Austin, 1989). In other words, the metabolic activity required to maintain plasmids exerts a small but real metabolic cost to the host cell relative to those cells not carrying the plasmid in question. This metabolic burden is why many daughter cells tend to ‘lose’ the plasmid of interest over time if they can continue to exist or reproduce without it. This process of loss or limited replication of the extrachromosomal element(s) also leads to diminished efficiency in those experiments that require the presence of a plasmid genetic component to produce a product of interest and therefore cultures with significant amounts of daughter cells that do not have the plasmid(s) of interest provide a reduced efficiency for the experiment being conducted. This is particularly acute in those fermentation experiments that rely upon economies of scale and consistent production of a molecule of interest to make their cost targets. Daughter cells deficient in the desired plasmids or extrachromosomal elements represent a media and energy sink in overall production and contribute to the economic benefits of fermentation costs. 
     In the biotechnology industry, plasmids and similar extrachromosomal elements have become very important tools in the genetic engineering of microbes and in the expression of proteins of interest and commercial synthetic biology. Such elements can be manipulated and designed to force the host cell to carry them forward or perish. (Balbas 2001: Baba 2006). In this sense, the cells become irreversibly ‘addicted’ to maintaining the extrachromosomal element in the cell despite the consequent metabolic burden (hence the term, Plasmid Addiction System or “PAS”). With such a system in hand the researcher can then focus on driving the host cell culture not just to maintain and express the PAS system genes, but to express all the genes contained on such an extrachromosomal element. According to the current invention, this can entail the expression of a number of genes and potential gene products of interest in microbial systems. 
     Plasmid Addiction Systems and Alternatives 
     Given the power of such techniques to drive the expression of proteins of interest, it is not surprising that a variety of approaches have been developed to ensure the stable maintenance of plasmids in cells (Nordstrom and Austin, 1989). This includes: (i) site-specific recombination systems functioning as plasmid maintenance systems for high-copy plasmid systems (Grindley et al., 2006); (ii) active partition systems (Funnell and Slavcev, 2004); and, as mentioned above, (iii) plasmid addiction systems (PAS), like the invention provided herein, that prevent the continuing survival/replication of cells not containing and expressing the genes of the plasmid of interest (Gerdes et al., 2005). 
     Site-Specific Recombination Control Systems 
     Site-specific recombination is a type of genetic recombination in which a DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology. In this system, a site-specific recombinase(s) (SSRs) performs rearrangements of DNA segments by recognizing and binding to short DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved and then rejoin the DNA strands. (Datsenko and Wanner, 2000). While in some site-specific recombination systems just a single recombinase enzyme and the corresponding recombination sites is enough to perform all these reactions, in other systems a number of accessory proteins and/or accessory sites are also needed—each addition adding to the complexity and thereby decreasing both the reliability and versatility of this system. (Baba et al., 2006). In addition, the constitutive expression of the required recombinases can also lead to undesired genotypic changes and the use of the system in terms of its initial development can be challenging in terms of the transfer of the recombinases genes to progeny. 
     Plasmid Instability 
     As mentioned above, microbes tend towards eliminating plasmids or limiting the reproduction of plasmids in cells due to the ongoing metabolic burden of both maintaining the plasmid itself and of expressing the gene(s) contained therein. (Rosano et al., 2014). Additionally, cells may not favor plasmid replication and expression when the plasmids in question may contain genes, that when expressed, produce toxic products in the cell or in its immediate environment of the cell. Of course, the interest to those utilizing such microbial systems is the maintenance of the engineered genetic changes and consequent expression of the inserted genes. In this sense, stable inheritance of the plasmid and host generally requires that: (1) the plasmid must replicate once each generation; (2) copy number deviations must be rapidly corrected before cell division, and, (3) upon cell division, the products of plasmid replication must be distributed to both daughter cells in a reliable and consistent manner. (Balbas et al., 1986). 
     In general, the stable maintenance of low-copy-number plasmids in bacteria is actively driven by partition mechanisms that are responsible for the positioning of plasmids inside the cell prior to replication. Various such partition systems are ubiquitous in the microbial world and are encoded by many bacterial chromosomes as well as plasmids. These systems, although different in sequence and mechanism, typically consist of two proteins and a DNA partition site or prokaryotic centromere on the plasmid in question. One protein binds to the centromere to form a partition complex, and the other protein uses the energy of nucleotide binding and hydrolysis to transport the plasmid as needed. For plasmids, this minimal cassette is sufficient to conduct appropriate segregation. In an optimal setting the strain selected to carry a plasmid of interest will have a partition system that provides or consistent and reliable plasmid reproduction. (Balbas et al., 1986; Rawlings 1999). 
     Engineered Plasmid Stabilization Systems 
     There are systems engineered to stably maintain the plasmids of interest. One particularly common system is the use of antibiotics as selection tools. In such systems, the antibiotic resistance gene in the plasmid of interest protects the cell carrying it, at the same time it effectively “forces” the cell to maintain it when the bacterial cell is grown in a media-enriched with the corresponding antibiotic. (Cranenburgh, R. M. et al., 2001). However, this method is subject to a number of difficulties and concerns. The antibiotic resistance approach is expensive, requiring the use of costly antibiotics and some may find it objectionable as a culture method in when used in industrial production methods could be a way that accelerates and/or spreads the development of bacterial antibiotic resistance that could affect human and/or animal populations negatively. Moreover, in large-scale production applications, the use of antibiotics may impose other limitations. With respect to commercial bioreactors, antibiotic resistance mechanisms can degrade the antibiotic itself and permit a substantial population of plasmid-less cells to persist in the culture. Such plasmid-less cells are unproductive and decrease the overall output of the bioreactor, thereby increasing cost and decreasing efficiency. (Balbas 2001; Baba 2006). 
     Segregational Plasmid Maintenance Functions 
     Stable lower copy number plasmids typically employ a partitioning function that actively distributes plasmid copies between daughter cells. Examples of partitioning mechanisms include: pSC101, F factor, P1 prophage, and IncFII drug resistance plasmids. Such functions act to physically segregate plasmids during replication. In terms of functionality many small plasmids rely on a high copy number, distributed throughout the cell, to ensure at least one copy is maintained by each daughter cell upon division. Many large, low-copy number plasmids, on the other hand, encode active segregation systems to avoid stochastic loss. A variety of partitioning systems exist, but most rely on three components: a centromeric DNA region, a cytomotive filament, and an adaptor protein linking the two. In type II segregation bacterial actin-like protein (ALP) filaments drive plasmid separation. (Balbas et al., 2001; Balbas 1986; Schumacher 2014). 
     Post-Segregational Killing (PSK) Functions 
     Naturally occurring PSK plasmid maintenance functions typically employ a two-component toxin-antitoxin system and generally operate as follows: The plasmid encodes both a toxin and an antitoxin. The antitoxins are less stable than the toxins, which tend to be quite stable. In a plasmid-less daughter cell, the toxins and anti-toxins are no longer being produced; however, the less stable antitoxins quickly degrade, thereby freeing the toxin to kill the cells in the surrounding area without the antitoxins being present. (Gerdes 1990). 
     The toxins are generally small proteins and the antitoxins are either small proteins or antisense RNAs which bind to the toxin-encoding mRNAs preventing their synthesis (EX: antisense systems such as hok-sok). In antisense maintenance systems, the antitoxins are antisense RNAs that inhibit translation of toxin-encoding mRNAs. Like the antitoxin peptides, the antisense RNAs are less stable than the toxin-encoding mRNA. Loss of the plasmid permits existing antitoxins to degrade, thereby permitting synthesis of the toxin which kills the host cell. A limitation of the hok-sok system is that a significant number of plasmid-less cells can arise when the hok-sok system is inactivated by mutations within the Hok open reading frame. (Gerdes 1990). 
     Balanced Lethal Systems 
     In a balanced-lethal system (a PSK function), a chromosomal gene encoding an essential structural protein or enzyme is deleted from the bacterial chromosome or is mutated such that the gene can no longer operate (Fu., 2000). The removed or damaged gene is then replaced by a plasmid comprising a fully operating gene. Loss of the plasmid results in an insufficiency of the essential protein and the death of the plasmid-less cell. Balanced-lethal systems based on catalytic enzyme production are subject to a number of deficiencies. In particular, since complementation of the chromosomal gene deletion requires only a single gene copy, it is inherently difficult to maintain more than a few copies of an expression plasmid. The plasmid less host strain must be grown on special media to chemically complement the existing metabolic deficiency. (Fu 2000). 
     Commercial Efforts &amp; Need 
     Biotechnical production processes often operate with plasmid-based expression systems in well-established prokaryotic and eukaryotic hosts such as  Escherichia coli  or  Saccharomyces cerevisiae , respectively. Genetically engineered organisms produce important chemicals, biopolymers, biofuels and high-value proteins like insulin. In those bioprocesses plasmids in recombinant hosts have an essential impact on productivity. (Kroll J., 2010). Plasmid-free cells lead to losses in the entire product recovery and decrease the profitability of the whole process (Table 1). Often, the use of antibiotics in industrial fermentations is not an available or desirable option to maintain plasmid stability. Especially in pharmaceutical or GMP-based fermentation processes, deployed antibiotics must be inactivated and removed. As stated above, they are also costly. Several plasmid addiction systems (PAS) have been described in the literature and referenced above. The current PAS provides a new method that is antibiotic free, remains absolutely necessary for cellular replication and homeostasis and allows multiple gene carrying plasmids, or the like, to be maintained efficiently in culture. 
     Given the above, there remains a need in the art for a new PAS that is reliant on a balanced lethal system, not requiring antibiotics is useful to industry and can drive the production of high volumes of compounds of interest in a commercially efficient way. 
     SUMMARY OF THE INVENTION 
     The present invention encompasses improved methods of devising a plasmid addiction system that can enhance the production of proteins of interest and do so at commercial scale. 
     According to the current invention, a biosynthetic method is provided for the production of one or more proteins of interest in a microbial system. 
     Recombinant plasmids carrying the gene of interest are obtained by cultivation of bacteria. For selecting bacterial transformants, and in order to ensure the maintenance of the plasmids in the bacterial host cell, an antibiotic resistance gene is traditionally included in the plasmid backbone. Selection for plasmids is achieved by growing the cells in a medium containing the respective antibiotic, in which only plasmid bearing cells are able to grow, often with a marker gene included. A number of plasmid addiction systems (PAS) already exist, mainly as toxin-antitoxin systems that limit the plasmids to single copy or aimed for use in open environments like bioremediation contexts. However, there are few examples of nutrition-based plasmid addiction systems, or ones exhibiting long-term stability in an industrial setting. The current invention provides both. 
     According to the current invention a plasmid addiction system utilizing the succinate pathway as the conditional mutant where key chromosomal genes have been removed and placed in the plasmids to be expressed and maintained in daughter cells. Such a system could be used for the production of specific amylases, pathway genes, lipases, proteases, vitamins or antibiotics, and according to the current invention could be forced to maintain up to four different plasmids. 
     According to the preferred embodiments of the invention, the applicants provide a plasmid addiction system based on the synthetic lethal deletion of either the double mutant sucAD or the quadruple mutant sucABCD, wherein the native mutations are complemented on one or more plasmids. The plasmid(s) of interest allows for near wild-type growth without supplementation of DAP or any other intermediate and is retained for many generations in the absence of selective markers. It is useful in a laboratory context, as transformants can be grown LB plates without any additional supplementation; the parent strains cannot grow without supplementation with DAP. It is useful in an industrial context wherein neither antibiotics nor their requisite selection marker genes are wanted or desired. Given the inclusion of up to four required genes this means that four plasmids of different compositions can be retained in a fermentation of interest and at low cost. That is, a single plasmid can be maintained with a single gene of interest or up to four different plasmid types, each with one of the four required genes, carrying other genes of interest can be provided in the current system efficiently and with low cost. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS.  1 A- 1 B . Show succinate and succinyl-CoA in context of central  E. coli  metabolism and cell wall biosynthesis ( FIGS.  1 A and  1 B ). 
         FIGS.  2 A- 2 C . Show the multiple deletions sucAD ( FIG.  2 B ) and sucABCD ( FIG.  2 C ), which are synthetic lethal in the  E. coli  chromosome. The genomic context for the native  E. coli  strain of the invention—BW25113 and its succinyl-CoA operon is shown in  FIG.  2 A ;  FIG.  2 B  provides a schematic of the genomic context of  E. coli  BW25113 ΔsucAD; and  FIG.  2 C  provides a schematic of the genomic context of  E. coli  BW25113 ΔsucABCD. 
         FIGS.  3 A- 3 E . Show plasmid maps of pDvS and pDvQ plasmids, cloning vectors designed to express sucAB and sucABCD complements rather than antibiotic resistance markers. pDvK-sucAD ( FIG.  3 A ); pDvK-sucABCD ( FIG.  3 B ); pDvS-Kan-dropout ( FIG.  3 C ); and pDvQ-Kan-dropout ( FIG.  3 D ); pDvK-sucBC ( FIG.  3 E ) 
         FIGS.  4 A- 4 B . Show succinate pathway knockout mutants, such as BW25113 ΔucAD ( FIG.  4 A ) and BW25113 ΔsucABCD ( FIG.  4 B ), cannot grow on rich fermentation media. 
         FIG.  5   . Growth curves of relevant cells on nonselective media. Shows differences between complementation of double- or quadruple knockouts 
         FIGS.  6 A- 6 B . Plasmid maps of succinate addiction vectors engineered to express GFP. dvp-a8-skb-sfgfp ( FIG.  6 A ); and pDvQ-GFP ( FIG.  6 B ). 
         FIG.  7   . Shows the production levels of GFP according to the transformed cellular system of the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The following abbreviations have designated meanings in the specification: 
     Explanation of Terms: 
     Cellular system is any cells that provide for the expression of ectopic proteins. It included bacteria, yeast, plant cells and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes the in vitro expression of proteins based on cellular components, such as ribosomes. 
     Growing the Cellular System. Growing includes providing an appropriate medium that would allow cells to multiply and divide given the changes to the succinate pathway. It also includes providing resources so that cells or cellular components can translate and make recombinant proteins. According to the current invention the cells grow on LB media. Such cells do not unless they are supplied with 120 μM DAP. 
     Protein Expression. Protein production can occur after requisite gene expression. It consists of the stages after DNA has been transcribed to messenger RNA (mRNA). The mRNA is then translated into polypeptide chains, which are ultimately folded into proteins. DNA is present in the cells through transfection—a process of deliberately introducing nucleic acids into cells. The term is often used for non-viral methods in eukaryotic cells. It may also refer to other methods and cell types, although other terms are preferred: “transformation” is more often used to describe non-viral DNA transfer in bacteria, non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated DNA transfer. Transformation, transduction, and viral infection are included under the definition of transfection for this application. 
     Acronyms:
         TCA—Tricarboxylic Acid   DAP—Diaminopimelic Acid   PAS—Plasmid addiction system   TB—Terrific Broth   LB—Luria Broth   Y(E)PD—Yeast Extract Peptone Dextrose (medium)   sucA— E. coli  gene encoding the E1 component of the 2-oxoglutarate dehydrogenase   enzyme   sucB— E. coli  gene encoding the E2 component of the 2-oxoglutarate dehydrogenase   enzyme   sucC— E. coli  gene encoding the 0 subunit of the succinyl-CoA synthetase enzyme   sucD— E. coli  gene encoding the a subunit of the succinyl-CoA synthetase enzyme       

     Alternative Marker Genes 
     If marker genes are required for one or more genes of the current invention examples include: genes encoding restriction nucleases (e.g. CviAII, a restriction endonuclease originating from  Chlorella  virus PBCV-1; Zhang et al., 1992), EcoRI (Torres et al., 2000), genes encoding toxins that interact with proteins, e.g. streptavidin or stv13 (a truncated, easy soluble streptavidin variant), as described by Szafransky et al., 1997: Kaplan et al., 1999; Sano et al., 1995, which act by deprivation of biotin, an essential protein in cell growth); genes encoding proteins that damage membranes (the E gene protein of φX174 (Ronchel et al., 1998; Haidinger et al., 2002), gef (Jensen et al., 1993; Klemm et al., 1995), relF (Knudsen et al., 1995); genes that encode other bacterial toxins, e.g. the ccdb gene (Bernard and Couturier, 1992) that encodes a potent cell killing protein from the F-plasmid trapping the DNA gyrase or sacB from  Bacillus subtilis  (Gay et al., 1983); or genes that encode eukaryotic toxins that are toxic to the bacterial host (e.g. FUS; Crozat et al., 1993). When using toxic genes, it is essential that their expression can be modulated by an inducible promoter. This promoter must not be active without an inductor, but provide expression upon induction, sufficient to inhibit cell growth. 
     In certain embodiments, the marker gene is selected from genes encoding restriction nucleases, streptavidin or genes that have an indirect toxic effect, e.g. sacB, as described above. 
     A repressor is a protein that binds to an operator located within the promoter of an operon, thereby down-regulation transcription of the gene(s) located within said operon. Examples for repressors suitable in the present invention are the tetracycline repressor (tet) protein TetR, which regulates transcription of a family of tetracycline resistance determinants in Gram-negative bacteria and binds to tetracycline (Williams, et al., 1998; Beck, et al., 1982; Postle et al., 1984), the tryptophan repressor (trp), which binds to the operator of the trp operon, which contains the tryptophan biosynthesis gene (Yanofski et al., 1987). 
     Examples for inducible promoters are promoters, where transcription starts upon addition of a substance, thus being regulatable by the environment, e.g. the lac promoter, which is inducible by IPTG (Jacob and Monod, 1961), the arabinose-promoter (pBAD), inducible by arabinose (Guzman et al., 1995), copper-inducible promoters (Rouch and Brown, 1997), and cumate-inducible promoters (Choi et al 2010). 
     Alternately, constitutive promoters may be used, wherein transcription of the desired transgene is always driven on, regardless of the growth phase or environmental variables. 
     In an alternative embodiment, one could monitor the expression of a single gene of interest through the use of a marker gene as a reporter gene. Genes that could be used to provide this functionality include genes encoding GFP (Green Fluorescent Protein), hSOD (human superoxide dismutase), lacZ (beta-glucosidase), CAT (chloramphenicol acetyltransferase), nptII (neomycin phosphotransferase) or luciferase. 
     A reporter gene is useful in cultivation processes whenever information on the presence or absence of a plasmid in a host cell or on plasmid copy number is needed. Such information is particularly useful when fermentation processes are to be optimized with regard to control of plasmid copy number. A reporter gene may also serve as a surrogate of a toxic marker gene and may thus be used in experimental settings that aim at proving the functionality of constructs to be employed for the gene-regulating or silencing and to determine their effect on a toxic marker gene. 
     In certain embodiments of the invention, the marker gene may be an endogenous host gene, which may be any gene of interest that is intended to be regulated. In this case, the host cell is engineered such that the sequence encoding the sequence is operably associated with the relevant host gene. 
     While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawing and will herein be described in detail. It should be understood, however, that the drawings and detailed description presented herein are not intended to limit the disclosure to the particular embodiment disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims. 
     Other features and advantages of this invention will become apparent in the following detailed description of preferred embodiments of this invention, taken with reference to the accompanying drawings. 
     The present invention relates to a system for an improved production method for proteins of interest in a microbial system that does not require markers, antibiotics and can produce proteins of interest at a high-level. 
     Bacterial Strains and Growth Conditions 
     BW25113 and the deletions for ΔsucA::KanR and ΔsucD::KanR were obtained from the  E. coli  Genetic Stock Center (CGSC). Cells were typically grown in Luria Broth (LB), but experiments were also performed in TB, YPD, YEPD, Nutrient Broth with corn steep liquor, and other rich media (Miller, 1972). Diaminopimelic acid (Sigma D1377) was used at 120 μM to aid in screening as the ΔsucAD double deletion is synthetic lethal (Mattozzi et al., 2013; Yu et al., 2006). 
     Construction of Strains with Chromosomal Mutations 
     Plvir transduction (Miller, 1972) was used to create kanamycin-resistant double knockout strains of  E. coli  BW25113 and screened with 120 μM DAP on LB kanamycin plates. These were screened for deletions of ΔsucA and ΔsucD via colony PCR. This KanR donor strain was also used to create double knockouts of  E. coli  strains BL21, BL21(DE3), MG1655, MG1655(DE3) ΔlacY, and W3110. Plasmid pCP20 was used to remove the kanamycin resistance markers using its FLP/FRT-based recombinase (Baba et al., 2006; Datsenko and Wanner, 2000). Since sucA and sucD are separated by only 6 kb, Kan sensitive cells exhibiting the quadruple deletion ΔsucABCD were usually isolated after the pCP20 FLP recombinase step (Datsenko and Wanner, 2000). 
     Construction of Recombinant Plasmids 
     Codon-optimized sequences encoding sucA, sutcB, sucC, and sucD were synthesized (Quintara Bioworks, Emeryville Calif.). CIDAR  E. coli  Modular cloning (Iverson et al., 2016), was used to generate versions of sucABCD natural operon and the sucAD synthetic operon. Both versions were based on the  E. coli  MG1655 native sequence, but with illegal BsaI and BpiI sites replaced in-frame so as not to affect protein sequences. Additional codon optimization was performed to minimize recombination effects. Operons sucABCD and sucAD were identical except that the sequence between the start codon of sucB and the stop codon of sucC were deleted. (Yu et al., 2005). 
     According to the current invention, plasmids were transformed into ΔsucAD and ΔsucABCD strains via electroporation and selected on LB plates without any additional supplementation; the parent strains cannot grow without supplementation with DAP. Clones were confirmed by sequence. 
     Cultivation of Plasmid-Addicted Strains 
     Plasmid-bearing  E. coli  strains were grown in LB without additional supplementation in 24-well plates and in a BioLector flower plates (Funke et al., 2009). 
     The present invention can be widely used in state-of-the-art fermentations, both for plasmid DNA production and for producing recombinant proteins. 
     Several approaches for fermentation of pDNA have been described that are useful for applying the present invention. The methods for plasmid DNA production differ with regard to the level of control imposed upon the cells and the numerous factors that influence fermentation. 
     To obtain higher quantities of plasmids, the cells can be cultivated in controlled fermenters in so-called “batch fermentations”, in which all nutrients are provided at the beginning and in which no nutrients are added during cultivation. (Reinikainen, P., et al; 1988). Cultivations of this type may be carried out with culture media containing so called “complex components” as carbon and nitrogen sources, as described e.g. by O&#39;Kennedy et al., 2003, and Lahijani et al., 1996, and in WO 96/40905, U.S. Pat. No. 5,487,986 and WO 02/064752. Alternatively, synthetic media may be used for pDNA production, e.g. defined culture media that are specifically designed for pDNA production (Wang et al., 2001; WO 02/064752). 
     The present invention may also be used in fed batch fermentations of  E. coli , in which one or more nutrients are supplied to the culture by feeding, typically by using a feed-back control algorithm by feeding nutrients in order to control a process parameter at a defined set point. Feed-back control is hence directly related to cell activities throughout fermentation. Control parameters which may be used for feed-back control of fermentations include pH value, on line measured cell density or dissolved oxygen tension (DOT). A feed-back algorithm for controlling the dissolved oxygen tension at a defined set point by the feeding rate was described in WO 99/61633. 
     Alternatively, the invention may be applied in a process for producing plasmid DNA, in which  E. coli  cells are first grown in a pre-culture and subsequently fermented in a main culture, the main culture being a fed-batch process comprising a batch phase and a feeding phase. The culture media of the batch phase and the culture medium added during the feeding phase are chemically defined, and the culture medium of the feeding phase contains a growth-limiting substrate and is added at a feeding rate that follows a pre-defined exponential function, thereby controlling the specific growth rate at a pre-defined value. 
     When the marker gene is under the control of an inducible promoter, the inducer may be added to the batch at the beginning and/or pulse-wise (both in a batch and in fed-batch cultivations). During the feed phase, the inducer may be added pulse-wise or continuously. 
     At the end of the fermentation process, the cells are harvested and the plasmid DNA is isolated and purified according to processes known in the art, e.g. by methods based on anion exchange and gel permeation chromatography, as described in U.S. Pat. No. 5,981,735 or by using two chromatographic steps, i.e. an anion exchange chromatography as the first step and reversed phase chromatography as the second step, as described in U.S. Pat. No. 6,197,553. Another suitable method for manufacturing plasmid DNA is described in WO 03/051483, which uses two different chromatographic steps, combined with a monolithic support. 
     In addition to applying the invention for plasmid production, e.g. for production of plasmids for gene therapy applications, it is also useful for recombinant protein production. (Rawlings 1999). 
     With regard to recombinant protein production, in principle, any method may be used that has proven useful for expressing a gene of interest in  E. coli , in particular from a ColE1 type plasmid (see, for review, e.g. Jonasson et al., 2002; Balbas, 2001). The protein may be obtained intracellularly (completely or partially soluble or as inclusion bodies) or by secretion (into the cell culture medium or the periplasmic space) from batch fermentations or, preferably, fed-batch cultivations, using complex, synthetic or semisynthetic media. 
     In plasmid DNA production, usually plasmid DNA for gene therapy applications, the gene of interest is not expressed in the bacterial host cell. In view of its application in mammals, preferably in humans, where it is to be ultimately expressed, the gene of interest is usually operably associated with a eukaryotic promoter. In contrast, for recombinant production of proteins in  E. coli , the gene of interest is to be expressed in the host cell therefore under the control of a prokaryotic promoter. 
     For recombinant protein production, the two promoters, i.e. the promoter controlling the marker gene and the promoter controlling the gene of interest, may be different or the same, as long as no interference occurs that disturbs expression of either one. 
     Advantageously, since their activity is independent of each other concerning time-point and level of transcription, the promoters are differently regulated. Preferably, the promoter controlling the marker gene is active at the start of the fermentation process and produces moderate amounts of mRNA, while the promoter of the gene of interest is rather strong and activated at a chosen time-point during fermentation. If inducible promoters are used for both the gene of interest and the marker gene, they are usually chosen such that they are turned on by different inducers. Alternatively, the marker gene may be under an inducible promoter and the gene of interest under a constitutive promoter, or vice versa. This applies both for methods in which the marker gene construct is integrated in the bacterial host genome and in which the marker gene construct is contained in a plasmid or phage, as described above. 
     With regard to induction of the promoter in the various phases of fermentation, the principle described above for plasmid DNA production applies. 
     The invention has the great advantage that all replicated plasmids are devoid of antibiotic resistance genes and are therefore, in addition to gene therapy applications, suitable for all applications for which the absence of antibiotic resistance genes is required or desirable, e.g. for the generation of recombinant yeast strains that are intended for human and animal food production or for the generation of recombinant plants. 
     Expression and Maintenance During Fermentation 
     Maintenance of heterologous DNA presents a major challenge in industrial systems. A number of systems already exist, but there are drawbacks to each of them. Integrating genes into the genome can be slow, require extensive screening, and is limited to a single copy per cell. Larger DNA loops like cosmids and bacterial artificial chromosomes (BACs) can be difficult to isolate from chromosomal DNA or cell debris pellets, and again are limited by copy number. Phages can be difficult to keep contained to the cell types of interest. They could become lytic unexpectedly, causing drastic consequences on a factory-scale. Thus, the most common way to introduce and maintain heterologous DNA into  E. coli  and other bacterial cultures is via plasmid, wherein the gene(s) of interest are maintained on a small loop of DNA containing sequences comprising an origin of replication and, typically, an antibiotic resistance marker. This marker can be problematic: antibiotics in the media can be expensive and can contaminate final small-molecule products with similar chemical properties. As well, the genes encoding these markers pose a biosafety issue: the antibiotics used in fermentation are the same or similar to the ones used in clinical settings. Though laboratory containment is usually good, large-scale use of antibiotic resistance genes could encourage the spread of dangerous resistant bacteria like methicillin-resistant  Staphylococcus aureus  (MRSA). 
     The principle of the invention, i.e. the metabolic context of the succinyl-CoA synthetic lethal deletions is shown in  FIG.  1   . 
     In embodiments of the invention, the following components are useful: 
     Host Cells 
     Since their replication depends on the host machinery, many plasmids are plasmids with a narrow host range. Replication is often limited to  E. coli  and related bacteria such as  Salmonella  and  Klebsiella  (Kues and Stahl, 1989). However, according to the current invention a great variety of functional hosts are available including eukaryotic systems. Other suitable hosts include: cells of the genera  Corynebacterium, Bacillus, Pseudomonas, Vibrio, Bulkholderia , and really any other bacterium that can stably maintain a heterologous plasmid and has a peptidoglycan cell wall. 
     Preferred genetic features of the host cell are mutations that improve plasmid stability and quality or recovery of intact recombinant protein. Examples of desirable genetic deletions are:
         sucA— E. coli  gene encoding the E1 component of the 2-oxoglutarate dehydrogenase enzyme   sucB— E. coli  gene encoding the E2 component of the 2-oxoglutarate dehydrogenase enzyme   sucC— E. coli  gene encoding the S subunit of the succinyl-CoA synthetase enzyme   sucD— E. coli  gene encoding the a subunit of the succinyl-CoA synthetase enzyme.       

     Each of the genes in this operon encodes part of a heterodimeric enzyme within the TCA cycle. Since sucAB and sucCD are synthetic lethal (Yu et al 2006), either sucAB OR sucCD pair may be deleted and still allow cell growth; albeit with reduced growth rates due to the inability of the cells to use oxygen as a terminal electron acceptor. This can eventually cause cell death, a reduced growth rate, low maximum cell density, and inefficient usage of carbon source. Deletion of at least three of the genes within the sucABCD cluster (or two from opposite conjugate pairs, e.g. ΔsucAD) creates a cell that is auxotrophic for succinyl-CoA. Because succinyl-CoA itself is unstable and expensive to procure commercially, it was discovered that supplementation of DAP in the medium can allow the cells to grow. This is because the external DAP can be incorporated into the cell walls, negating the need for the succinyl-CoA cofactor ( FIG.  1   ). The cells can still grow, but albeit with a growth defect due to their inability to fully utilize oxygen as a terminal electron acceptor. 
     Constructs for Engineering the Host Cells 
     The principle of a construct suitable for engineering the host cells is shown in  FIG.  2   : The host strains were generated via P1 transduction (above), and the plasmids were produced via Gibson assembly, cloning, Golden Gate and/or modular cloning. 
     Characteristics of Plasmids for the System 
     The plasmids are required to express the genes specifically deleted in the host strain. In this example, codon-optimized versions of  E. coli  sucAD and sucABCD are expressed on plasmids, complementing the deletions made to BW25113 ΔsucAD and ΔsucABCD respectively. 
     EXAMPLES 
     Two or four key genes expressing essential proteins for the tricarboxylic acid (TCA) cycle were deleted from the  E. coli  genome. Previously these genes have been shown to be synthetic lethal (Yu et al., 2006). These cells are thus auxotrophic for succinyl-CoA. The cells can make up the energetic needs of the TCA cycle simply through fermentative growth, but the lack of a complete TCA cycle causes inefficient growth, and accumulation of toxic fermentative byproducts ethanol and acetate because the cells are unable to effectively use oxygen as a terminal electron acceptor. This can eventually cause cell death, a reduced growth rate, low maximum cell density, and inefficient usage of carbon source. In addition to the TCA cycle, succinyl-CoA is also used as a cofactor in many metabolic pathways. Perhaps the most important is the lysine synthesis pathway, wherein succinyl-CoA is required as an essential cofactor for generating diaminopimelic acid (DAP). DAP is a key monomer in the murein or peptidoglycan cell wall and was thus required for growth. 
     Previously, we built a system taking advantage of this fact (Mattozzi et al., 2013), as a test of a carbon fixation system. However, the knockouts were only used as a proxy for cell metabolic processes from  Chloroflexus aurantiacus , not the ability of the cells to retain the plasmid or drive the production of proteins of interest. Double mutant ΔsucAD cells containing a plasmid expressing a succinyl-CoA:(S)-malyl-CoA transferase operon reduced but did not entirely remove the need for DAP in the system. 
     Bacterial Strains and Growth Conditions 
     BW25113 and the deletions for ΔsucA::KanR and ΔsucD::KanR were obtained from the  E. coli  Genetic Stock Center (CGSC) at Yale University. Cells were typically grown in Luria Broth (LB), but experiments were also performed in TB, YPD, YEPD, Nutrient Broth with corn steep liquor, and other rich media (Miller, 1972). Diaminopimelic acid (Sigma D1377) was used at 120 μM to aid in screening as the ΔsucA(B) Δsuc(C)D double deletion is synthetic lethal (Mattozzi et al., 2013; Yu et al., 2006). 
     Construction of Strains with Chromosomal Mutations 
     Plvir transduction (Miller, 1972) was used to create kanamycin-resistant double knockout strains of  E. coli  BW25113 and screened with 120 μM DAP on LB kanamycin plates. These were screened for deletions of ΔsucA and ΔsucD via colony PCR. This KanR donor strain was also used to create double knockouts of  E. coli  strains BL21, BL21(DE3), BL21*(DE3), MG1655, MG1655(DE3) ΔlacY, C41, and W3110. Plasmid pCP20 was used to remove the kanamycin resistance markers using its FLP/FRT-based recombinase (Baba et al., 2006; Datsenko and Wanner, 2000). Since sucA and sucD are separated by only 6 kb, Kan sensitive cells exhibiting the quadruple deletion ΔsucABCD were usually isolated after the pCP20 FLP recombinase step (Datsenko and Wanner, 2000). 
     Construction of Recombinant Plasmids 
     CIDAR  E. coli  Modular cloning (Iverson et al., 2016), a Golden Gate based technology, was used to generate versions of sucABCD natural operon and the sucAD synthetic operon. Both versions were based on the  E. coli  MG1655 native sequence, but with illegal BsaI and BpiI sites replaced in-frame so as not to affect protein sequences. Operons sucABCD and sucAD were identical except that the sequence between the start codon of sucB and the stop codon of sucC were deleted. Plasmids were transformed into sucAD and sucABCD strains via electroporation and selected on LB plates without any additional supplementation; the parent strains cannot grow without supplementation with DAP. Clones were confirmed by sequence. 
     Plasmids were transformed into ΔsucAD and ΔsucABCD strains via electroporation and selected on LB plates without any additional supplementation; the parent strains cannot grow without supplementation with DAP. Clones were confirmed by sequence. 
     In  FIG.  1 A , we see the general metabolic context of succinyl-CoA, diaminopimelic acid, and peptidoglycan on murein cell walls. Succinyl-CoA generated by the gene products of sucAB and sucCD is used to produce lysine and its immediate biochemical precursor, diaminopimelate (DAP), critically required for  E. coli  cell wall (peptidoglycan or murein) biosynthesis.  FIG.  1 B  provides the detailed metabolic context of the succinyl-CoA cofactor in diaminopimelate and lysine metabolism (Excerpted from Michel and Schomberg 2012). In  FIG.  2 A , the genomic context for the native  E. coli  strain of the invention—BW25113 and its succinyl-CoA operon are provided. According to the current invention the DNA sequence for this is (SEQ ID NO: 1). 
       FIG.  2 B  provides a schematic of the genomic context of  E. coli  BW25113 ΔsucAD. This is the result of a P1 transduction in the  E. coli  genome wherein ΔsucA::kanR was used as a donor. Recipient strain was  E. coli  BW25113 ΔsucD::kanS, generated by removing kanamycin resistance via pCP20-mediated FRT excision (thereby providing SEQ ID NO: 2).  FIG.  2 C  provides a schematic of the genomic context of  E. coli  BW25113 ΔsucABCD. The result is the removal of kanamycin resistance via pCP20-mediated FRT excision. Since sucA and sucD are within 6 kb, deletions of the entire sucABCD operon were isolated in the purification process (SEQ ID NO: 3). 
     In  FIG.  3 A , a map of plasmid pDvK-SucAD, according to the current invention is provided. It was used to test for plasmid retention in nonselective media, as hosted in ΔsucAD cells. In this case, the plasmid retains kanamycin resistance markers for later testing. Although promoters, RBS, and terminators are specifically enumerated here, the experiments have shown effectively no difference in expression upon varying these (SEQ ID NO: 4). In  FIG.  3 B , we provide a map of plasmid pDvK-SucABCD, used to test for plasmid retention in nonselective media, as hosted in ΔsucABCD cells. According to the current invention, the plasmid retains kanamycin resistance markers for later testing. Although promoters, RBS, and terminators are specifically enumerated here, the experiments have shown effectively no difference in expression upon varying these (SEQ ID NO: 5). In  FIG.  3 C , we see the plasmid map of pDvS-Kan of the invention, wherein the kanamycin resistance marker is easily removed by the gene of interest, and the genes sucAD can instead be used as a selection marker. Although promoters, RBS, and terminators are specifically enumerated here, the experiments have shown effectively no difference in expression upon varying these (SEQ ID NO: 6). In  FIG.  3 E , a map of plasmid pDvK-SucBC, according to the current invention is provided. It was used to test for plasmid retention in nonselective media, as hosted in ΔsucABCD cells in combination with pDvK-SucAD. In this case, the plasmid retains kanamycin resistance markers for later testing. Although promoters, RBS, and terminators are specifically enumerated here, the experiments have shown effectively no difference in expression upon varying these sequences (SEQ ID NO: 10). 
     In  FIG.  4 A , applicants show the succinate pathway knockout mutant BW25113 ΔsucAD cannot grow on rich fermentation media Luria Broth. However, supplanting the media with diaminopimelic acid (DAP) allows for an increase in growth rate, correlating to the concentration of DAP provided. According to the current invention, the plasmid map of pDvQ-Kan is provided in  FIG.  3 D , wherein the kanamycin resistance marker is easily removed by the gene of interest, and the genes sucABCD can instead be used as a selection marker. Although promoters, RBS, and terminators are specifically enumerated here, the experiments have shown effectively no difference in expression upon varying these (SEQ ID NO: 7). In  FIG.  4 B , applicants demonstrate that the succinate pathway knockout mutant BW25113 ΔsucABCD cannot grow on rich fermentation media Luria Broth. However, supplanting the media with diaminopimelic acid (DAP) allows for an increase in growth rate, correlating to the concentration of DAP provided. 
     In  FIG.  5   , we provide the rescue of growth phenotypes with plasmid-borne sucA(BC)D in artificial operons. Deletions of sucAD and sucABCD from  E. coli  BW25113 do not grow at all on rich fermentation media Luria Broth. However, supplying the cells with plasmids pDvK-SucAD and pDvK-SucABCD, respectively, allows the cells to reach densities of close to that of wild-type BW25113. In  FIG.  6 A , the plasmid map of pDvS-GFP contains a sequence encoding the green fluorescent protein cloned into the pDvS vector, wherein the kanamycin resistance marker is easily removed by the gene of interest. (SEQ ID NO: 8) is provided. In  FIG.  6 B , a plasmid map of pDvQ-GFP containing a sequence encoding the green fluorescent protein cloned into the pDvQ vector (SEQ ID NO: 9) is provided. In  FIG.  7   , we see the production levels of green fluorescent protein (GFP), normalized by cell density according to the transformed cellular system of the invention. The cells containing the deletions and corresponding complements (open symbols, solid lines) exhibit more GFP per unit cell density than those with wild-type backgrounds (filled symbols, dotted lines), or those without plasmids (open symbols, dashed lines). In Table 1 we see that over time in the absence of kanamycin selection, the cells lacking the deletions lose kanamycin resistance (borne on the plasmids) within a few days, whereas the deletion mutants retain their resistance and their plasmids over the entire course of the study. 
     In addition, in Table 1, Applicants demonstrate that the Fraction of colony forming units (cfu) that retains a KanR plasmid over days.  E. coli  BW25113 was transformed with three Kan resistant plasmids (pDvK-sucAD, pDvK-sucABCD, and pDvK, rows A-D).  E. coli  BW25113 deletions in sucAD and sucABCD were also transformed with complement plasmids (pDvK-sucAD, pDvK-sucABCD, respectively, rows E-F). 50-mL cultures were grown in LB without kanamycin as selective pressure. Aliquots of cells were plated on kanamycin and non-selective plates and cfu calculated daily. The fraction of KanR cfu over total cfu is reported. 
     Over time in the absence of kanamycin selection, the cells lacking the deletions lose kanamycin resistance (borne on the plasmids) within a few days, whereas the deletion mutants retain their resistance and their plasmids over the entire course of the study. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Table of growth characteristics for the retention of a single plasmid in the system. Fraction of cfu that retain kanamycin 
               
               
                 sensitivity (and thus maintain the plasmid expressing succinate pathway and kanamycin resistance genes) over time. 
               
            
           
           
               
               
            
               
                   
                 Day 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Strain 
                 Plasmid 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 BW25113 
                   
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 BW25113 
                   
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 ΔsucAD 
               
               
                 BW25113 
                   
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 ΔsucABCD 
               
               
                 BW25113 
                 pDvK-sucAD 
                 &lt;1.0 
                 1.35 ± 0.53 
                 0.75 ± 0.17 
                 0.48 ± 0.03 
                 0.09 ± 0.03 
                 0.08 ± 0.08 
                 ~0 
                 ~0 
               
               
                 BW25113 
                 pDvK-sucABCD 
                 &lt;1.0 
                 &lt;1.0 
                 0.04 ± 0.06 
                 0.04 ± 0.00 
                 0.16 ± 0.05 
                 ~0 
                 ~0 
                 ~0 
               
               
                 BW25113 
                 pDvK 
                 &lt;1.0 
                 0.96 ± 0.12 
                 0.63 ± 0.17 
                 0.55 ± 0.19 
                 0.44 ± 0.15 
                 0.50 ± 0.37 
                 0.08 ± 0.07 
                 0.12 ± 0 15 
               
               
                 BW25113 
                 pDvK-sucAD 
                 0.99 ± 0.23 
                 1.93 ± 0.12 
                 0.84 ± 0.17 
                 1.05 ± 0.42 
                 1.06 ± 0.04 
                 1.30 ± 0.61 
                 1.15 ± 0.30 
                 1.11 ± 0.19 
               
               
                 ΔsucAD 
               
               
                 BW25113 
                 pDvK-sucABCD 
                 0.86 ± 0.02 
                 0.99 ± 0.27 
                 0.98 ± 0.17 
                 1.26 ± 0.44 
                 1.02 ± 0.02 
                 0.94 ± 0.21 
                 1.23 ± 0.24 
                 1.19 ± 0.01 
               
               
                 ΔsucABCD 
               
               
                   
               
            
           
         
       
     
     Maintenance of Multiple Plasmids in the System 
     A similar experiment was performed to test the maintenance of multiple plasmids in the system. Cells of BW25113 ΔsucABCD should not be able to grow in LB without supplementation of DAP, unless at least two of the genes sucAB and sucCD are expressed on plasmids. Plasmids pDVK-sucAD and pDVK-sucBC, were constructed. Neither of these plasmids has a sufficient set of genes to allow growth of BW25113 ΔsucABCD without DAP supplementation, but they will in combination. Without supplementation with DAP, the cells retained their kanamycin resistance, and thus their ability to maintain both plasmids (Tables 2, 3). 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Retention of Two-Plasmids. Fraction of cfu that retain kanamycin 
               
               
                 sensitivity (and thus maintain the plasmids expressing succinate 
               
               
                 pathway and kanamycin resistance genes) over time). 
               
            
           
           
               
               
               
            
               
                 Strain 
                 Plasmid(s) 
                 Day 1 
               
               
                   
               
               
                 BW25113 
                 pDVK 
                 0.49 ± 0.27 
               
               
                 BW25113 ΔsucABCD 
                 pDVK-sucBC and pDVP-sucAD 
                 0.69 ± 0.37 
               
               
                   
               
            
           
         
       
     
     Retention of both plasmids utilized according to the current invention is shown in patch plates, wherein colonies of each strain/plasmid combination were struck on LB agar plates of different media conditions. Only with a complimentary and/or complete set of genes sucAB sucD can  E. coli  BW25113 ΔsucABCD grow without DAP supplementation. Kanamycin resistance shows maintenance of the plasmids, here two, as KanR is linked to the succinate operon genes. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Retention of Two-Plasmids. Patch growth of plasmids on different media. Cultures of each strain/plasmid 
               
               
                 were grown in LB + DAP overnight and diluted to OD 600  = 1.0. Ten μL of this dilution 
               
               
                 (and serial 50-fold dilutions) were plated onto the media conditions in each column. 
               
            
           
           
               
               
               
               
               
               
            
               
                 Strain 
                 Plasmid(s) 
                 LB 
                 LB + Kan 
                 LB + DAP 
                 LB + Kan 50  + DAP 
               
               
                   
               
               
                 BW25113 
                   
                 ++++ 
                 − 
                 ++++ 
                 − 
               
               
                 BW25113 ΔsucABCD 
                   
                 − 
                 − 
                 ++++ 
                 − 
               
               
                 BW25113 ΔsucABCD 
                 pDVK-sucAD 
                 + 
                 − 
                 ++++ 
                 ++++ 
               
               
                 BW25113 ΔsucABCD 
                 pDVK-sucBC 
                 − 
                 − 
                 ++++ 
                 ++++ 
               
               
                 BW25113 ΔsucABCD 
                 pDVK-sucAD and pDVK- 
                 +++ 
                 +++ 
                 ++++ 
                 +++ 
               
               
                   
                 sucBC 
               
               
                   
               
               
                 − = no growth. 
               
               
                 + = growth patch observed with OD 600  = 1.0 cells. 
               
               
                 ++ = growth patch observed from 50-fold serial dilution (OD 600  = 0.02). 
               
               
                 +++ = growth patch observed from 2500-fold serial dilution (OD 600  = 0.0004). 
               
               
                 ++++ = growth patch observed from 125,000-fold serial dilution (OD 600  = 0.000008). 
               
            
           
         
       
     
     Cultivation of Plasmid-Addicted Strains 
     Plasmid-bearing  E. coli  strains were grown in LB without additional supplementation in 24-well plates and in a BioLector flower plates (Funke et al., 2009). 
     To achieve tight regulation of toxic gene expression, a tightly regulable promoter like the arabinose-inducible PBAD promoter (Guzman et al., 1995) is preferably used, in particular in the case that the marker protein is per se toxic to the cells. 
     Another way to control expression of the marker gene is by using constitutive promoters in combination with a gene that is non-toxic (e.g. a reporter gene) or only toxic under defined conditions, e.g. the  Bacillus subtilis  sacB gene, which is only toxic to  E. coli  when sucrose is present. 
     The promoter is chosen in coordination with the effect of the marker gene product and the required efficiency of down-regulation or silencing effect. For example, for a construct containing a non-toxic or less toxic marker gene, a stronger promoter is desirable. 
     Additional Embodiments 
     As is evident from the foregoing description, certain aspects of the present disclosure are not limited by the particular details of the examples illustrated herein, and it is therefore contemplated that other modifications and applications, or equivalents thereof, will occur to those skilled in the art. It is accordingly intended that the claims shall cover all such modifications and applications that do not depart from the spirit and scope of the present disclosure. 
     Moreover, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials equivalent to or those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described above. 
     Although the foregoing invention has been described in some detail by way of illustration and example for purposes of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention, which is delineated by the appended claims. 
     Accordingly, it is to be understood that the embodiments of the invention herein providing for the production of specific molecules are merely illustrative of the application of the principles of the invention. It will be evident from the foregoing description that changes in the form, methods of use, and applications of the elements of the disclosed production methods and selected microbial strains may be resorted to without departing from the spirit of the invention, or the scope of the appended claims. 
     STATEMENT OF INDUSTRIAL APPLICABILITY/TECHNICAL FIELD 
     This disclosure has applicability in the commercial production of food ingredients, fragrances, medicines and pharmaceuticals. This disclosure relates generally to a method for enhanced and more precisely controlled biosynthetic production of desired end products via selected microbial strains. 
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         Vieira, J., et at;  The pUC Plasmids, an M 13 mp 7- Derived System for Insertion Mutagenesis and Sequencing with Synthetic Universal Primers ; G ENE  (1982) vol. 19 pp. 259-68. 
         Williams, S. G., et al.,  Repressor Titration: A Novel System for Selection and Stable Maintenance of Recombinant Plasmids , N UCLEIC  A CIDS  R ESEARCH  (1998) vol. 26, No. 9 pp. 2120-24. 
         Yu., B. J., et al.,  sucAB and sucCD are mutually essential genes in Escherichia coli ., FEMS M ICROBIOL . L ETT . (2005) 254, 245-50. 
         Yu, D., et al;  An Efficient Recombination System for Chromosome Engineering in Escherichia coli ; PNAS (2000) vol. 97, No. 11 pp. 5978-83. 
       
    
     
       
         
           
               
             
               
                   
               
               
                 Sequences of Interest: 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 SEQ ID NO: 1SE operon and genomic context sequence 
               
               
                 ccggtcaggcactgactgtgaatgagaaaggcgaagatgtggttgttccgggactgtttgccgttggtgaaatcgct 
               
               
                 tgtgtatcggtacacggcgctaaccgtctgggcggcaactcgctgctggacctggtggtctttggtcgcgcggcagg 
               
               
                 tctgcatctgcaagagtctatcgccgagcagggcgcactgcgcgatgccagcgagtctgatgttgaagcgtctctgg 
               
               
                 atcgcctgaaccgctggaacaataatcgtaacggtgaagatccggtggcgatccgtaaagcgctgcaagaatgtatg 
               
               
                 cagcataacttctcggtcttccgtgaaggtgatgcgatggcgaaagggcttgagcagttgaaagtgatccgcgagcg 
               
               
                 tctgaaaaatgcccgtctggatgacacttccagcgagttcaacacccagcgcgttgagtgcctggaactggataacc 
               
               
                 tgatggaaacggcgtatgcaacggctgtttctgccaacttccgtaccgaaagccgtggcgcgcatagccgcttcgac 
               
               
                 ttccaggatcgtgatgatgaaaactggctgtgccactccctgtatctgccagagtcggaatccatgacgcgccgaag 
               
               
                 cgtcaacatggaaccgaaactgcgcccggcattcccgccgaagattcgtacttactaatgcggagacaggaaaatga 
               
               
                 gactcgagttttcaatttatcgctataacccggatgttgatgatgctccgcgtatgcaggattacaccctggaagcg 
               
               
                 gatgaaggtcgcgacatgatgctgctggatgcgcttatccagctaaaagagaaagatcccagcctgtcgttccgccg 
               
               
                 ctcctgccgtgaaggtgtgtgcggttccgacggtctgaacatgaacggcaagaatggtctggcctgtattaccccga 
               
               
                 tttcggcactcaaccagccgggcaagaagattgtgattcgcccgctgccaggtttaccggtgatccgcgatttggtg 
               
               
                 gtagacatgggacaattctatgcgcaatatgagaaaattaagccttacctgttgaataatggacaaaatccgccagc 
               
               
                 tcgcgagcatttacagatgccagagcagcgcgaaaaactcgacgggctgtatgaatgtattctctgcgcatgttgtt 
               
               
                 caacctcttgtccgtctttctggtggaatcccgataagtttatcggcccggcaggcttgttagcggcatatcgtttc 
               
               
                 ctgattgatagccgtgataccgagactgacagccgcctcgacggtttgagtgatgcattcagcgtattccgctgtca 
               
               
                 cagcatcatgaactgcgtcagtgtatgtccgaaggggctgaacccgacgcgcgccatcggccatatcaagtcgatgt 
               
               
                 tgttgcaacgtaatgcgtaaaccgtaggcctgataagacgcgcaagcgtcgcatcaggcaaccagtgccggatgcgg 
               
               
                 cgtgaacgccttatccggcctacaagtcattacccgtaggcctgataagcgcagcgcatcaggcgtaacaaagaaat 
               
               
                 gcaggaaatctttaaaaactgcccctgacactaagacagtttttaaaggttccttcgcgagccactacgtagacaag 
               
               
                 agctcgcaagtgaaccccggcacgcacatcactgtgcgtggtagtatccacggcgaagtaagcataaaaaagatgct 
               
               
                 taagggatcacgatgcagaacagcgctttgaaagcctggttggactcttcttacctctctggcgcaaaccagagctg 
               
               
                 gatagaacagctctatgaagacttcttaaccgatcctgactcggttgacgctaactggcgttcgacgttccagcagt 
               
               
                 tacctggtacgggagtcaaaccggatcaattccactctcaaacgcgtgaatatttccgccgcctggcgaaagacgct 
               
               
                 tcacgttactcttcaacgatctccgaccctgacaccaatgtgaagcaggttaaagtcctgcagctcattaacgcata 
               
               
                 ccgcttccgtggtcaccagcatgcgaatctcgatccgctgggactgtggcagcaagataaagtggccgatctggatc 
               
               
                 cgtctttccacgatctgaccgaagcagacttccaggagaccttcaacgtcggttcatttgccagcggcaaagaaacc 
               
               
                 atgaaactcggcgagctgctggaagccctcaagcaaacctactgcggcccgattggtgccgagtatatgcacattac 
               
               
                 cagcaccgaagaaaaacgctggatccaacagcgtatcgagtctggtcgcgcgactttcaatagcgaagagaaaaaac 
               
               
                 gcttcttaagcgaactgaccgccgctgaaggtcttgaacgttacctcggcgcaaaattccctggcgcaaaacgcttc 
               
               
                 tcgctggaaggcggtgacgcgttaatcccgatgcttaaagagatgatccgccacgctggcaacagcggcacccgcga 
               
               
                 agtggttctcgggatggcgcaccgtggtcgtctgaacgtgctggtgaacgtgctgggtaaaaaaccgcaagacttgt 
               
               
                 tcgacgagttcgccggtaaacataaagaacacctcggcacgggtgacgtgaaataccacatgggcttctcgtctgac 
               
               
                 ttccagaccgatggcggcctggtgcacctggcgctggcgtttaacccgtctcaccttgagattgtaagcccggtagt 
               
               
                 tatcggttctgttcgtgcccgtctggacagacttgatgagccgagcagcaacaaagtgctgccaatcaccatccacg 
               
               
                 gtgacgccgcagtgaccgggcagggcgtggttcaggaaaccctgaacatgtcgaaagcgcgtggttatgaagttggc 
               
               
                 ggtacggtacgtatcgttatcaacaaccaggttggtttcaccacctctaatccgctggatgcccgttctacgccgta 
               
               
                 ctgtactgatatcggtaagatggttcaggccccgattttccacgttaacgcggacgatccggaagccgttgcctttg 
               
               
                 tgacccgtctggcgctcgatttccgtaacacctttaaacgtgatgtcttcatcgacctggtgtgctaccgccgtcac 
               
               
                 ggccacaacgaagccgacgagccgagcgcaacccagccgctgatgtatcagaaaatcaaaaaacatccgacaccgcg 
               
               
                 caaaatctacgctgacaagctggagcaggaaaaagtggcgacgctggaagatgccaccgagatggttaacctgtacc 
               
               
                 gcgatgcgctggatgctggcgattgcgtagtggcagagtggcgtccgatgaacatgcactctttcacctggtcgccg 
               
               
                 tacctcaaccacgaatgggacgaagagtacccgaacaaagttgagatgaagcgcctgcaggagctggcgaaacgcat 
               
               
                 cagcacggtgccggaagcagttgaaatgcagtctcgcgttgccaagatttatggcgatcgccaggcgatggctgccg 
               
               
                 gtgagaaactgttcgactggggcggtgcggaaaacctcgcttacgccacgctggttgatgaaggcattccggttcgc 
               
               
                 ctgtcgggtgaagactccggtcgcggtaccttcttccaccgccacgcggtgatccacaaccagtctaacggttccac 
               
               
                 ttacacgccgctgcaacatatccataacgggcagggcgcgttccgtgtctgggactccgtactgtctgaagaagcag 
               
               
                 tgctggcgtttgaatatggttatgccaccgcagaaccacgcactctgaccatctgggaagcgcagttcggtgacttc 
               
               
                 gccaacggtgcgcaggtggttatcgaccagttcatctcctctggcgaacagaaatggggccggatgtgtggtctggt 
               
               
                 gatgttgctgccgcacggttacgaagggcaggggccggagcactcctccgcgcgtctggaacgttatctgcaacttt 
               
               
                 gtgctgagcaaaacatgcaggtttgcgtaccgtctaccccggcacaggtttaccacatgctgcgtcgtcaggcgctg 
               
               
                 cgcgggatgcgtcgtccgctggtcgtgatgtcgccgaaatccctgctgcgtcatccgctggcggtttccagcctcga 
               
               
                 agaactggcgaacggcaccttcctgccagccatcggtgaaatcgacgagcttgatccgaagggcgtgaagcgcgtag 
               
               
                 tgatgtgttctggtaaggtttattacgacctgctggaacagcgtcgtaagaacaatcaacacgatgtcgccattgtg 
               
               
                 cgtatcgagcaactctacccgttcccgcataaagcgatgcaggaagtgttgcagcagtttgctcacgtcaaggattt 
               
               
                 tgtctggtgccaggaagagccgctcaaccagggcgcatggtactgcagccagcatcatttccgtgaagtgattccgt 
               
               
                 ttggggcttctctgcgttatgcaggccgcccggcctccgcctctccggcggtagggtatatgtccgttcaccagaaa 
               
               
                 cagcaacaagatctggttaatgacgcgctgaacgtcgaataaataaaggatacacaatgagtagcgtagatattctg 
               
               
                 gtccctgacctgcctgaatccgtagccgatgccaccgtcgcaacctggcataaaaaacccggcgacgcagtcgtacg 
               
               
                 tgatgaagtgctggtagaaatcgaaactgacaaagtggtactggaagtaccggcatcagcagacggcattctggatg 
               
               
                 cggttctggaagatgaaggtacaacggtaacgtctcgtcagatccttggtagcctgcgtgaaggcaacagcgccggt 
               
               
                 aaagaaaccagcgccaaatctgaagagaaagcgtccactccggcgcaacgccagcaggcgtctctggaagagcaaaa 
               
               
                 caacgatgcgttaagcccggcgatccgtcgcctgctggctgaacacaatctcgacgccagcgccattaaaggcaccg 
               
               
                 gtgtgggtggtcgtctgactcgtgaagatgtggaaaaacatctggcgaaagccccggcgaaagagtctgctccggca 
               
               
                 gcggctgctccggcggcgcaaccggctctggctgcacgtagtgaaaaacgtgtcccgatgactcgcctgcgcaagcg 
               
               
                 tgtggcagagcgtctgctggaagcgaaaaactccaccgccatgctgaccacgttcaacgaagtcaacatgaagccga 
               
               
                 ttatggatctgcgtaagcagtacggtgaagcgtttgaaaaacgccacggcatccgtctgggctttatgtccttctac 
               
               
                 gtgaaagcggtggttgaagccctgaaacgttacccggaagtgaacgcttctatcgacggcgatgacgtggtttacca 
               
               
                 caactatttcgacgtcagcatggcggtttctacgccgcgcggcctggtgacgccggttctgcgtgatgtcgataccc 
               
               
                 tcggcatggcagacatcgagaagaaaatcaaagagctggcagtcaaaggccgtgacggcaagctgaccgttgaagat 
               
               
                 ctgaccggtggtaacttcaccatcaccaacggtggtgtgttcggttccctgatgtctacgccgatcatcaacccgcc 
               
               
                 gcagagcgcaattctgggtatgcacgctatcaaagatcgtccgatggcggtgaatggtcaggttgagatcctgccga 
               
               
                 tgatgtacctggcgctgtcctacgatcaccgtctgatcgatggtcgcgaatccgtgggcttcctggtaacgatcaaa 
               
               
                 gagttgctggaagatccgacgcgtctgctgctggacgtgtagtagtttaagtttcacctgcactgtagaccggataa 
               
               
                 ggcattatcgccttctccggcaattgaagcctgatgcgacgctgacgcgtcttatcaggcctacgggaccaccaatg 
               
               
                 taggtcggataaggcgcaagcgccgcatccgacaagcgatgcctgatgtgacgtttaacgtgtcttatcaggcctac 
               
               
                 gggtgaccgacaatgcccggaagcgatacgaaatattcGGTCTACGGTTTAAAAGATAACGATTACTGAAGGATGGA 
               
               
                 CAGAACACatgaacttacatgaatatcaggcaaaacaactttttgcccgctatggcttaccagcaccggtgggttat 
               
               
                 gcctgtactactccgcgcgaagcagaagaagccgcttcaaaaatcggtgccggtccgtgggtagtgaaatgtcaggt 
               
               
                 tcacgctggtggccgcggtaaagcgggcggtgtgaaagttgtaaacagcaaagaagacatccgtgcttttgcagaaa 
               
               
                 actggctgggcaagcgtctggtaacgtatcaaacagatgccaatggccaaccggttaaccagattctggttgaagca 
               
               
                 gcgaccgatatcgctaaagagctgtatctcggtgccgttgttgaccgtagttcccgtcgtgtggtctttatggcctc 
               
               
                 caccgaaggcggcgtggaaatcgaaaaagtggcggaagaaactccgcacctgatccataaagttgcgcttgatccgc 
               
               
                 tgactggcccgatgccgtatcagggacgcgagctggcgttcaaactgggtctggaaggtaaactggttcagcagttc 
               
               
                 accaaaatcttcatgggcctggcgaccattttcctggagcgcgacctggcgttgatcgaaatcaacccgctggtcat 
               
               
                 caccaaacagggcgatctgatttgcctcgacggcaaactgggcgctgacggcaacgcactgttccgccagcctgatc 
               
               
                 tgcgcgaaatgcgtgaccagtcgcaggaagatccgcgtgaagcacaggctgcacagtgggaactgaactacgttgcg 
               
               
                 ctggacggtaacatcggttgtatggttaacggcgcaggtctggcgatgggtacgatggacatcgttaaactgcacgg 
               
               
                 cggcgaaccggctaacttccttgacgttggcggcggcgcaaccaaagaacgtgtaaccgaagcgttcaaaatcatcc 
               
               
                 tctctgacgacaaagtgaaagccgttctggttaacatcttcggcggtatcgttcgttgcgacctgatcgctgacggt 
               
               
                 atcatcggcgcggtagcagaagtgggtgttaacgtaccggtcgtggtacgtctggaaggtaacaacgccgaactcgg 
               
               
                 cgcgaagaaactggctgacagcggcctgaatattattgcagcaaaaggtctgacggatgcagctcagcaggttgttg 
               
               
                 ccgcagtggaggggaaataatgtccattttaatcgataaaaacaccaaggttatctgccagggctttaccggtagcc 
               
               
                 aggggactttccactcagaacaggccattgcatacggcactaaaatggttggcggcgtaaccccaggtaaaggcggc 
               
               
                 accacccacctcggcctgccggtgttcaacaccgtgcgtgaagccgttgctgccactggcgctaccgcttctgttat 
               
               
                 ctacgtaccagcaccgttctgcaaagactccattctggaagccatcgacgcaggcatcaaactgattatcaccatca 
               
               
                 ctgaaggcatcccgacgctggatatgctgaccgtgaaagtgaagctggatgaagcaggcgttcgtatgatcggcccg 
               
               
                 aactgcccaggcgttatcactccgggtgaatgcaaaatcggtatccagcctggtcacattcacaaaccgggtaaagt 
               
               
                 gggtatcgtttcccgttccggtacactgacctatgaagcggttaaacagaccacggattacggtttcggtcagtcga 
               
               
                 cctgtgtcggtatcggcggtgacccgatcccgggctctaactttatcgacattctcgaaatgttcgaaaaagatccg 
               
               
                 cagaccgaagcgatcgtgatgatcggtgagatcggcggtagcgctgaagaagaagcagctgcgtacatcaaagagca 
               
               
                 cgttaccaagccagttgtgggttacatcgctggtgtgactgcgccgaaaggcaaacgtatgggccacgcgggtgcca 
               
               
                 tcattgccggtgggaaagggactgcggatgagaaattcgctgctctggaagccgcaggcgtgaaaaccgttcgcagc 
               
               
                 ctggcggatatcggtgaagcactgaaaactgttctgaaataaatatctgtaataagaaatagccctcgccgcttccc 
               
               
                 tctacaggaatggcgaagggctgtcggtttcgacatggttggccatcgtatgatggccttttttgtgcttatcgcga 
               
               
                 tgattttcgctgcgctatcagggtaaatttatagtcatcggtattaaaagcgttgcggctatattcaaacacccgac 
               
               
                 catcaactaaatatccacgcgatactttttcaagaatcggctttgtctggctgatattaagcagacggctcatctct 
               
               
                 tcggttggcatcagaggaatgatttcctgttcgctacgatcgataaccattttcttcacttcttcgataaagtgata 
               
               
                 tttcgaattttccatgacctgccaggtgagatccgggaacaacgcaagcggcatccaggtttcttccagcgccattg 
               
               
                 gcttttgcttgcgatagcgcacgcgcttcacatgccacacacgatcctgcggggtgatttgtagctgttgctgaaga 
               
               
                 aaatcgtcagccggaatcacttcgaatatcagaacttcactgtgtgtatcgacgtgacggtccgacagtttttcatc 
               
               
                 aaaactggttaactgaaaaatatcgtaattgacccgctcttctttgacgtaagtcccgctgccctgaatgctttcga 
               
               
                 ggatctgctgctcgactagctggcgcaaagcctgacgcaccgtaacccggctgacgccaaactctgtttgtagcgct 
               
               
                 gattcagtgggtaacgcatcgccaggtttaagctcgccacgcgcaatttgttcacgaatgcgatcggcaatctgccg 
               
               
                 gtataagggcttgtgtcccatttttagtatctcattaatacgaatttaaccattatgcccgataaattcatcctgta 
               
               
                 aataatacaaatacaatacaaataatttcaatcaagtgaaattgatcacataatggtattgttttatcg 
               
               
                   
               
               
                 SEQ ID NO: 2. Sequence of the genomic context of  E .  coli  BW25113 ΔsucAD. 
               
               
                 ccggtcaggcactgactgtgaatgagaaaggcgaagatgtggttgttccgggactgtttgccgttggtgaaatcgct 
               
               
                 tgtgtatcggtacacggcgctaaccgtctgggcggcaactcgctgctggacctggtggtctttggtcgcgcggcagg 
               
               
                 tctgcatctgcaagagtctatcgccgagcagggcgcactgcgcgatgccagcgagtctgatgttgaagcgtctctgg 
               
               
                 atcgcctgaaccgctggaacaataatcgtaacggtgaagatccggtggcgatccgtaaagcgctgcaagaatgtatg 
               
               
                 cagcataacttctcggtcttccgtgaaggtgatgcgatggcgaaagggcttgagcagttgaaagtgatccgcgagcg 
               
               
                 tctgaaaaatgcccgtctggatgacacttccagcgagttcaacacccagcgcgttgagtgcctggaactggataacc 
               
               
                 tgatggaaacggcgtatgcaacggctgtttctgccaacttccgtaccgaaagccgtggcgcgcatagccgcttcgac 
               
               
                 ttcccggatcgtgatgatgaaaactggctgtgccactccctgtatctgccagagtcggaatccatgacgcgccgaag 
               
               
                 cgtcaacatggaaccgaaactgcgcccggcattcccgccgaagattcgtacttactaatgcggagacaggaaaatga 
               
               
                 gactcgagttttcaatttatcgctataacccggatgttgatgatgctccgcgtatgcaggattacaccctggaagcg 
               
               
                 gatgaaggtcgcgacatgatgctgctggatgcgcttatccagctaaaagagaaagatcccagcctgtcgttccgccg 
               
               
                 ctcctgccgtgaaggtgtgtgcggttccgacggtctgaacatgaacggcaagaatggtctggcctgtattaccccga 
               
               
                 tttcggcactcaaccagccgggcaagaagattgtgattcgcccgctgccaggtttaccggtgatccgcgatttggtg 
               
               
                 gtagacatgggacaattctatgcgcaatatgagaaaattaagccttacctgttgaataatggacaaaatccgccagc 
               
               
                 tcgcgagcatttacagatgccagagcagcgcgaaaaactcgacgggctgtatgaatgtattctctgcgcatgttgtt 
               
               
                 caacctcttgtccgtctttctggtggaatcccgataagtttatcggcccggcaggcttgttagcggcatatcgtttc 
               
               
                 ctgattgatagccgtgataccgagactgacagccgcctcgacggtttgagtgatgcattcagcgtattccgctgtca 
               
               
                 cagcatcatgaactgcgtcagtgtatgtccgaaggggctgaacccgacgcgcgccatcggccatatcaagtcgatgt 
               
               
                 tgttgcaacgtaatgcgtaaaccgtaggcctgataagacgcgcaagcgtcgcatcaggcaaccagtgccggatgcgg 
               
               
                 cgtgaacgccttatccggcctacaagtcattacccgtaggcctgataagcgcagcgcatcaggcgtaacaaagaaat 
               
               
                 gcaggaaatctttaaaaactgcccctgacactaagacagtttttaaaggttccttcgcgagccactacgtagacaag 
               
               
                 agctcgcaagtgaaccccggcacgcacatcactgtgcgtggtagtatccacggcgaagtaagcataaaaaagatgct 
               
               
                 taagggatcacgagtgtaggctggagctgcttcgaagttcctatactttctagagaataggaacttcggaataggaa 
               
               
                 cttcaagatccccttattagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgata 
               
               
                 ccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtc 
               
               
                 ctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcg 
               
               
                 gcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgcgcgccttgagcctggcgaacagttcg 
               
               
                 gctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcg 
               
               
                 ctcgatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcag 
               
               
                 ccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccggcacttcgcccaatagcagc 
               
               
                 cagtcccttcccgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccg 
               
               
                 cgctgcctcgtcctgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctg 
               
               
                 acagccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccaCccaa 
               
               
                 gcggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaacgatcctcatcctgtctcttgatcagatct 
               
               
                 tgatcccctgcgccatcagatccttggcggcaagaaagccatccagtttactttgcagggcttcccaaccttaccag 
               
               
                 agggcgccccagctggcaattccggttcgcttgctgtccataaaaccgcccagtctagctatcgccatgtaagccca 
               
               
                 ctgcaagctacctgctttctctttgcgcttgcgttttcccttgtccagatagcccagtagctgacattcatccgggg 
               
               
                 tcagcaccgtttctgcggactggctttctacgtgttccgcttcctttagcagcccttgcgccctgagtgcttgcggc 
               
               
                 agcgtgagcttcaaaagcgctctgaagttcctatactttctagagaataggaacttcgaactgcaggtcgacggatc 
               
               
                 cccggaattaattctcatgtttgacagaaaggatacacaatgagtagcgtagatattctggtccctgacctgcctga 
               
               
                 atccgtagccgatgccaccgtcgcaacctggcataaaaaacccggcgacgcagtcgtacgtgatgaagtgctggtag 
               
               
                 aaatcgaaactgacaaagtggtactggaagtaccggcatcagcagacggcattctggatgcggttctggaagatgaa 
               
               
                 ggtacaacggtaacgtctcgtcagatccttggtcgcctgcgtgaaggcaacagcgccggtaaagaaaccagcgccaa 
               
               
                 atctgaagagaaagcgtccactccggcgcaacgccagcaggcgtctctggaagagcaaaacaacgatgcgttaagcc 
               
               
                 cggcgatccgtcgcctgctggctgaacacaatctcgacgccagcgccattaaaggcaccggtgtgggtggtcgtctg 
               
               
                 actcgtgaagatgtggaaaaacatctggcgaaagccccggcgaaagagtctgctccggcagcggctgctccggcggc 
               
               
                 gcaaccggctctggctgcacgtagtgaaaaacgtgtcccgatgactcgcctgcgtaagcgtgtggcagagcgtctgc 
               
               
                 tggaagcgaaaaactccaccgccatgctgaccacgttcaacgaagtcaacatgaagccgattatggatctgcgtaag 
               
               
                 cagtacggtgaagcgtttgaaaaacgccacggcatccgtctgggctttatgtccttctacgtgaaagcggtggttga 
               
               
                 agccctgaaacgttacccggaagtgaacgcttctatcgacggcgatgacgtggtttaccacaactatttcgacgtca 
               
               
                 gcatggcggtttctacgccgcgcggcctggtgacgccggttctgcgtgatgtcgataccctcggcatggcagacatc 
               
               
                 gagaagaaaatcaaagagctggcagtcaaaggccgtgacggcaagctgaccgttgaagatctgaccggtggtaactt 
               
               
                 caccatcaccaacggtggtgtgttcggttccctgatgtctacgccgatcatcaacccgccgcagagcgcaattctgg 
               
               
                 gtatgcacgctatcaaagatcgtccgatggcggtgaatggtcaggttgagatcctgccgatgatgtacctggcgctg 
               
               
                 tcctacgatcaccgtctgatcgatggtcgcgaatccgtgggcttcctggtaacgatcaaagagttgctggaagatcc 
               
               
                 gacgcgtctgctgctggacgtgtagtagtttaagtttcacctgcactgtagaccggataaggcattatcgccttctc 
               
               
                 cggcaattgaagcctgatgcgacgctgacgcgtcttatcaggcctacgggaccaccaatgtaggtcggataaggcgc 
               
               
                 aagcgccgcatccgacaagcgatgcctgatgtgacgtttaacgtgtcttatcaggcctacgggtgaccgacaatgcc 
               
               
                 cggaagcgatacgaaatattcggtctacggtttaaaagataacgattactgaaggatggacagaacacatgaactta 
               
               
                 catgaatatcaggcaaaacaactttttgcccgctatggcttaccagcaccggtgggttatgcctgtactactccgcg 
               
               
                 cgaagcagaagaagccgcttcaaaaatcggtgccggtccgtgggtagtgaaatgtcaggttcacgctggtggccgcg 
               
               
                 gtaaagcgggcggtgtgaaagttgtaaacagcaaagaagacatccgtgcttttgcagaaaactggctgggcaagcgt 
               
               
                 ctggtaacgtatcaaacagatgccaatggccaaccggttaaccagattctggttgaagcagcgaccgatatcgctaa 
               
               
                 agagctgtatctcggtgccgttgttgaccgtagttcccgtcgtgtggtctttatggcctccaccgaaggcggcgtgg 
               
               
                 aaatcgaaaaagtggcggaagaaactccgcacctgatccataaagttgcgcttgatccgctgactggcccgatgccg 
               
               
                 tatcagggacgcgagctggcgttcaaactgggtctggaaggtaaactggttcagcagttcaccaaaatcttcatggg 
               
               
                 cctggcgaccattttcctggagcgcgacctggcgttgatcgaaatcaacccgctggtcatcaccaaacagggcgatc 
               
               
                 tgatttgcctcgacggcaaactgggcgctgacggcaacgcactgttccgccagcctgatctgcgcgaaatgcgtgac 
               
               
                 cagtcgcaggaagatccgcgtgaagcacaggctgcacagtgggaactgaactacgttgcgctggacggtaacatcgg 
               
               
                 ttgtatggttaacggcgcaggtctggcgatgggtacgatggacatcgttaaactgcacggcggcgaaccggctaact 
               
               
                 tccttgacgttggcggcggcgcaaccaaagaacgtgtaaccgaagcgttcaaaatcatcctctctgacgacaaagtg 
               
               
                 aaagccgttctggttaacatcttcggcggtatcgttcgttgcgacctgatcgctgacggtatcatcggcgcggtagc 
               
               
                 agaagtgggtgttaacgtaccggtcgtggtacgtctggaaggtaacaacgccgaactcggcgcgaagaaactggctg 
               
               
                 acagcggcctgaatattattgcagcaaaaggtctgacggatgcagctcagcaggttgttgccgcagtggaggggaaa 
               
               
                 taatgATTCCGGGGATCCGTCGACCTGCAGTTCGAAGTTCCTATCTAGAAAGTATAGGAACTTCGAAGCAGCTCCAG 
               
               
                 CCTACActgaaaactgttctgaaataaatatctgtaataagaaatagccctcgccgcttccctctacaggaatggcg 
               
               
                 aagggctgtcggtttcgacatggttggccatcgtatgatggccttttttgtgcttatcgcgatgattttcgctgcgc 
               
               
                 tatcagggtaaatttatagtcatcggtattaaaagcgttgcggctatattcaaacacccgaccatcaactaaatatc 
               
               
                 cacgcgatactttttcaagaatcggctttgtctggctgatattaagcagacggctcatctcttcggttggcatcaga 
               
               
                 ggaatgatttcctgttcgctacgatcgataaccattttcttcacttcttcgataaagtgatatttcgaattttccat 
               
               
                 gacctgccaggtgagatccgggaacaacgcaagcggcatccaggtttcttccagcgccattggcttttgcttgcgat 
               
               
                 agcgcacgcgcttcacatgccacacacgatcctgcggggtgatttgtagctgttgctgaagaaaatcgtcagccgga 
               
               
                 atcacttcgaatatcagaacttcactgtgtgtatcgacgtgacggtccgacagtttttcatcaaaactggttaactg 
               
               
                 aaaaatatcgtaattgacccgctcttctttgacgtaagtcccgctgccctgaatgctttcgaggatctgctgctcga 
               
               
                 ctagctggcgcaaagcctgacgcaccgtaacccggctgacgccaaactctgtttgtagcgctgattcagtgggtaac 
               
               
                 gcatcgccaggtttaagctcgccacgcgcaatttgttcacgaatgcgatcggcaatctgccggtataagggcttgtg 
               
               
                 tcccatttttagtatctcattaatacgaatttaaccattatgcccgataaattcatcctgtaaataatacaaataca 
               
               
                 atacaaataatttcaatcaagtgaaattgatcacataatggtattgttttatcg 
               
               
                   
               
               
                 SEQ ID NO: 3. Sequence of the genomic context of  E .  coli  BW25113 ΔsucABCD 
               
               
                 ccggtcaggcactgactgtgaatgagaaaggcgaagatgtggttgttccgggactgtttgccgttggtgaaatcgct 
               
               
                 tgtgtatcggtacacggcgctaaccgtctgggcggcaactcgctgctggacctggtggtctttggtcgcgcggcagg 
               
               
                 tctgcatctgcaagagtctatcgccgagcagggcgcactgcgcgatgccagcgagtctgatgttgaagcgtctctgg 
               
               
                 atcgcctgaaccgctggaacaataatcgtaacggtgaagatccggtggcgatccgtaaagcgctgcaagaatgtatg 
               
               
                 cagcataacttctcggtcttccgtgaaggtgatgcgatggcgaaagggcttgagcagttgaaagtgatccgcgagcg 
               
               
                 tctgaaaaatgcccgtctggatgacacttccagcgagttcaacacccagcgcgttgagtgcctggaactggataacc 
               
               
                 tgatggaaacggcgtatgcaacggctgtttctgccaacttccgtaccgaaagccgtggcgcgcatagccgcttcgac 
               
               
                 ttcccggatcgtgatgatgaaaactggctgtgccactccctgtatctgccagagtcggaatccatgacgcgccgaag 
               
               
                 cgtcaacatggaaccgaaactgcgcccggcattcccgccgaagattcgtacttactaatgcggagacaggaaaatga 
               
               
                 gactcgagttttcaatttatcgctataacccggatgttgatgatgctccgcgtatgcaggattacaccctggaagcg 
               
               
                 gatgaaggtcgcgacatgatgctgctggatgcgcttatccagctaaaagagaaagatcccagcctgtcgttccgccg 
               
               
                 ctcctgccgtgaaggtgtgtgcggttccgacggtctgaacatgaacggcaagaatggtctggcctgtattaccccga 
               
               
                 tttcggcactcaaccagccgggcaagaagattgtgattcgcccgctgccaggtttaccggtgatccgcgatttggtg 
               
               
                 gtagacatgggacaattctatgcgcaatatgagaaaattaagccttacctgttgaataatggacaaaatccgccagc 
               
               
                 tcgcgagcatttacagatgccagagcagcgcgaaaaactcgacgggctgtatgaatgtattctctgcgcatgttgtt 
               
               
                 caacctcttgtccgtctttctggtggaatcccgataagtttatcggcccggcaggcttgttagcggcatatcgtttc 
               
               
                 ctgattgatagccgtgataccgagactgacagccgcctcgacggtttgagtgatgcattcagcgtattccgctgtca 
               
               
                 cagcatcatgaactgcgtcagtgtatgtccgaaggggctgaacccgacgcgcgccatcggccatatcaagtcgatgt 
               
               
                 tgttgcaacgtaatgcgtaaaccgtaggcctgataagacgcgcaagcgtcgcatcaggcaaccagtgccggatgcgg 
               
               
                 cgtgaacgccttatccggcctacaagtcattacccgtaggcctgataagcgcagcgcatcaggcgtaacaaagaaat 
               
               
                 gcaggaaatctttaaaaactgcccctgacactaagacagtttttaaaggttccttcgcgagccactacgtagacaag 
               
               
                 agctcgcaagtgaaccccggcacgcacatcactgtgcgtggtagtatccacggcgaagtaagcataaaaaagatgAT 
               
               
                 TCCGGGGATCCGTCGACCTGCAGTTCGAAGTTCCTATCTAGAAAGTATAGGAACTTCGAAGCAGCTCCAGCCTACAc 
               
               
                 tgaaaactgttctgaaataaatatctgtaataagaaatagccctcgccgcttccctctacaggaatggcgaagggct 
               
               
                 gtcggtttcgacatggttggccatcgtatgatggccttttttgtgcttatcgcgatgattttcgctgcgctatcagg 
               
               
                 gtaaatttatagteatcggtattaaaagcgttgcggctatattcaaacacccgaccatcaactaaatatccacgcga 
               
               
                 tactttttcaagaatcggctttgtctggctgatattaagcagacggctcatctcttcggttggcatcagaggaatga 
               
               
                 tttcctgttcgctacgatcgataaccattttcttcacttcttcgataaagtgatatttcgaattttccatgacctgc 
               
               
                 caggtgagatccgggaacaacgcaagcggcatccaggtttcttccagcgccattggcttttgcttgcgatagcgcac 
               
               
                 gcgcttcacatgccacacacgatcctgcggggtgatttgtagctgttgctgaagaaaatcgtcagccggaatcactt 
               
               
                 cgaatatcagaacttcactgtgtgtatcgacgtgacggtccgacagtttttcatcaaaactggttaactgaaaaata 
               
               
                 tcgtaattgacccgctcttctttgacgtaagtcccgctgccctgaatgctttcgaggatctgctgctcgactagctg 
               
               
                 gcgcaaagcctgacgcaccgtaacccggctgacgccaaactctgtttgtagcgctgattcagtgggtaacgcatcgc 
               
               
                 caggtttaagctcgccacgcgcaatttgttcacgaatgcgatcggcaatctgccggtataagggcttgtgtcccatt 
               
               
                 tttagtatctcattaatacgaatttaaccattatgcccgataaattcatcctgtaaataatacaaatacaatacaaa 
               
               
                 taatttcaatcaagtgaaattgatcacataatggtattgttttatcg 
               
               
                   
               
               
                 SEQ ID NO: 4. Sequence of Plasmid pDVK-SucAD, used for testing plasmid retention in 
               
               
                 nonselective media 
               
               
                 ccacccatctgggtttgccggtatttaataccgtgcgtgaggcggttgccgcaaccggtgccacggcttcagttatc 
               
               
                 tatgttcctgccccattttgtaaagattcaattctggaagctattgatgcgggcatcaaattgattattacgattac 
               
               
                 cgaaggtatccctacgctggatatgttgacggttaaagtgaaacttgatgaagcgggggtacgcatgattggtccga 
               
               
                 attgtccgggcgttattactccaggtgagtgcaaaattggtattcagccgggtcatattcacaaacctgggaaagtc 
               
               
                 ggaattgtgtctcgttctggcactctgacgtatgaggcagttaaacagaccacagattatggctttgggcagagtac 
               
               
                 ctgtgtcggcatcggaggcgatcctattccggggagtaattttatcgatattctggaaatgtttgagaaagatccgc 
               
               
                 agaccgaggcaatcgtcatgattggcgagattggcggttccgcggaagaagaagctgcagcctatatcaaagaacat 
               
               
                 gtcacaaaaccggtagtgggctatatcgcgggagtcacggccccaaaaggtaaacgtatgggccatgccggagcgat 
               
               
                 catcgcgggcggcaaaggcactgcagatgaaaaatttgcagcccttgaggccgctggcgtaaaaacggtccgttccc 
               
               
                 ttgctgatattggtgaagcactgaaaaccgtgttgaaataaAGGTccaggcatcaaataaaacgaaaggctcagtcg 
               
               
                 aaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcggg 
               
               
                 tgggcctttctgcgtttatatgccatgtcttctactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtc 
               
               
                 accaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgctgc 
               
               
                 gctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggat 
               
               
                 aacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttttt 
               
               
                 ccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactat 
               
               
                 aaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctg 
               
               
                 tccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgt 
               
               
                 tcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttg 
               
               
                 agtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgta 
               
               
                 ggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctct 
               
               
                 gctgaagccagttaccttaggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtt 
               
               
                 tttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtct 
               
               
                 gacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcct 
               
               
                 tttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagctcgagtcccgtca 
               
               
                 agtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaa 
               
               
                 actgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactca 
               
               
                 ccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctat 
               
               
                 taatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggca 
               
               
                 aaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaacc 
               
               
                 aaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacagg 
               
               
                 aatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaata 
               
               
                 cctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatg 
               
               
                 gtaggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctaccttt 
               
               
                 gccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacat 
               
               
                 tatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctggagcaagacgtttcc 
               
               
                 cgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatt 
               
               
                 tttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagt 
               
               
                 tgaaggatcagctcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgta 
               
               
                 tcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctggaattcgcggccgcttcta 
               
               
                 gagactagtggaagacatCGCTttgacagctagctcagtcctaggtactgtgctagcTACTttaaactccccgagca 
               
               
                 atagtaatgcagaactcagcattgaaagcatggcctgatagcccctatttatcaggtgctaaccagagctqgatcga 
               
               
                 acagctgtatgaagattttctgacagatccggattcagtggatgcgaattggcgcagcacttttcagcagttgcctg 
               
               
                 gcaccggtgtaaaaccggatcagtttcattcccagacgcgggagtattttcgtcgtctggcgaaagatgcgagccgg 
               
               
                 tattcaagtacaatttctgatccggatacgaatgtaaaacaggtgaaagtgcttcagttaattaatgcgtatcgctt 
               
               
                 tagaggccatcagcatgcgaatctggatccgctgggcttatggcagcaggataaagtcgcggatctggatccaagtt 
               
               
                 ttcacgatttaacggaagctgattttcaggaaacctttaacgtcggctcattcgcaagtgggaaagaaacaatgaaa 
               
               
                 ctgggcgaacttcttgaggcgctgaaacagacttattgtggccctattggtgcggaatatatgcatattacctcaac 
               
               
                 tgaagagaaacgttggattcagcagagaatcgagagtggccgcgcgacttttaactccgaagaaaaaaaaagattcc 
               
               
                 tgtcagaactgacagccgcggaaggcttagagcggtatttgggtgccaaattcccaggagcaaaacggttcagcctg 
               
               
                 gagggcggtgatgcgctgatcccgatgctgaaagaaatgattcggcatgcgggaaatagcggaactcgggaagtggt 
               
               
                 gttaggaatggcacaccgcggccgtttgaatgtactggttaacgtattaggaaaaaaacctcaggatttatttgatg 
               
               
                 agttcgcgggaaaacataaagaacatctgggcactggtgatgtcaaatatcacatgggcttctcaagtgattttcag 
               
               
                 acggatggaggtctggttcacctggcactggcatttaatccttctcatctggaaatcgtaagtccggtcgttattgg 
               
               
                 ttccgtgcgcgctcgcttagatcggttagatgaacctagctcaaacaaagttttaccaatcacgatccatggggatg 
               
               
                 cagctgttaccggacagggtgttgtgcaggagactttgaatatgtccaaagcgcgcgggtatgaggtgggtggtacg 
               
               
                 gtgcgtattgttatcaataatcaggtgggttttacaaccagtaaccctctggatgctcgctctacgccgtattgcac 
               
               
                 tgatattggtaaaatggtgcaggcaccaatttttcacgtcaatgccgatgatccggaagctgttgcctttgttacgc 
               
               
                 gcctggctctggattttcgtaacactttcaaacgtgatgtatttatcgatttagtatgctatcgtcgtcatggtcat 
               
               
                 aatgaggctgatgaacctagcgctacccagccactgatgtatcagaaaattaaaaaacatcctacccctcgtaaaat 
               
               
                 ttatgcggataaactggagcaggaaaaagtggctactcttgaagatgctactgaaatggtcaatctttatcgggatg 
               
               
                 cattggatgcgggtgattgcgtggtcgcggaatggcgcccgatgaatatgcattcatttacttggtcaccgtattta 
               
               
                 aatcatgagtgggatgaggaatatccgaataaagtggagatgaaacgcctgcaggaattagcaaaacgtattagcac 
               
               
                 agtacctgaagcggttgagatgcagtctagagttgccaaaatctatggagatcgccaggccatggcagcaggggaaa 
               
               
                 aactttttgattgggggggagccgaaaacctggcatatgcgacgctggtagatgagggcattccggtgcgcctttct 
               
               
                 ggtgaagattctgggcgcggtactttttttcatcggcacgctgttattcataaccagtctaacggtagtacttatac 
               
               
                 tccgctgcagcacatccacaatggtcagggtgcgttccgtgtatgggattccgtgctgagtgaagaagcggttcttg 
               
               
                 cgtttgagtatgggtatgcaactgccgagccacgcacgctgacgatctgggaagcccagtttggcgattttgcaaat 
               
               
                 ggtgcccaggtggtaatcgatcagtttattagctccggcgaacagaaatgggggcggatgtgtggtttagttatgtt 
               
               
                 gttaccgcatggctatgaaggtcagggacctgagcacagctcagcgcgcctggaacgctatcttcagctgtgtgcgg 
               
               
                 aacagaacatgcaggtatgcgttccttccacgccggctcaggtttatcatatgttaagacgtcaggccttgcgcggt 
               
               
                 atgcggcgcccgttggtcgtgatgtccccgaaaagtttactgcgccatccgttagcagttagcagcctggaggaact 
               
               
                 ggcaaacggtacgttcttgccagctatcggcgaaatcgatgaactggatcctaaaggggtgaaacgcgttgttatgt 
               
               
                 gttctggtaaagtgtattatgatcttttggaacagcgtcgcaaaaataatcagcacgatgtagctattgtgcggatc 
               
               
                 gagcagctgtatccgttcccgcacaaagcaatgcaggaagtgctgcagcagttcgcacatgtcaaagattttgtctg 
               
               
                 gtgtcaggaggaaccgcttaatcagggggcctggtattgtagtcagcaccatttccgggaggtgatcccgtttgggg 
               
               
                 cgtccttacggtatgctggtcgccctgcctccgcaagtccggccgtgggatatatgagcgttcaccagaaacagcag 
               
               
                 caggatttggtgaatgatgctttgaatgtggaatgaatgtccatcctgatcgacaaaaacactaaagtaatttgtca 
               
               
                 gggctttaccggttcccagggcacatttcactcagagcaggccatcgcttatgggaccaaaatggtgggtggtgtaa 
               
               
                 cgcctggtaaaggaggca 
               
               
                   
               
               
                 SEQ ID NO: 5. Sequence of Plasmid pDVK-SucABCD, used for testing plasmid retention in 
               
               
                 nonselective media 
               
               
                 ccggcgaaagagtctgctccggcagcggctgctcggcggcgcaaccggctctggctgcacgtagtgaaaaacgtgt 
               
               
                 cccgatgactcgcctgcgtaagcgtgtggcagagcgtctgctggaagcgaaaaactccaccgccatgctgaccacgt 
               
               
                 tcaacgaagtcaacatgaagccgattatggatctgcgtaagcagtacggtgaagcgtttgaaaaacgccacggcatc 
               
               
                 cgtctgggctttatgtccttctacgtgaaagcggtggttgaagccctgaaacgttacccggaagtgaacgcttctat 
               
               
                 cgacggcgatgacgtggtttaccacaactatttcgacgtcagcatggcggtttctacgccgcgcggcctggtgacgc 
               
               
                 cggttctgcgtgatgtcgataccctcggcatggcagacatcgagaagaaaatcaaagagctggcagtcaaaggccgt 
               
               
                 gacggcaagctgaccgttgaagatctgaccggtggtaacttcaccatcaccaacggtggtgtgttcggttccctgat 
               
               
                 gtctacgccgatcatcaacccgccgcagagcgcaattctgggtatgcacgctatcaaagatcgtccgatggcggtga 
               
               
                 atggtcaggttgagatcctgccgatgatgtacctggcgctgtcctacgatcaccgtctgatcgatggtcgcgaatcc 
               
               
                 gtgggcttcctggtaacgatcaaagagttgctggaagatccgacgcgtctgctgctggacgtgtagtagtttaagtt 
               
               
                 tcacctgcactgtagaccggataaggcattatcgccttctccggcaattgaagcctgatgcgacgctgacgcgtctt 
               
               
                 atcaggcctacgggaccaccaatgtaggtcggataaggcgcaagcgccgcatccgacaagcgatgcctgatgtgacg 
               
               
                 tttaacgtgtcttatcaggcctacgggtgaccgacaatgcccggaagcgatacgaaatattcGGTCTACGGTTTAAA 
               
               
                 AGATAACGATTACTGAAGGATGGACAGAACACatgaacttacatgaatatcaggcaaaacaactttttgcccgctat 
               
               
                 ggcttaccagcaccggtgggttatgcctgtactactccgcgcgaagcagaagaagccgcttcaaaaatcggtgccgg 
               
               
                 tccgtgggtagtgaaatgtcaggttcacgctggtggccgcggtaaagcgggcggtgtgaaagttgtaaacagcaaaG 
               
               
                 AGgacatccgtgcttttgcagaaaactggctgggcaagcgtctggtaacgtatcaaacagatgccaatggccaaccg 
               
               
                 gttaaccagattctggttgaagcagcgaccgatatcgctaaagagctgtatctcggtgccgttgttgaccgtagttc 
               
               
                 ccgtcgtgtggtctttatggcctccaccgaaggcggcgtggaaatcgaaaaagtggcggaagaaactccgcacctga 
               
               
                 tccataaagttgcgcttgatccgctgactggcccgatgccgtatcagggacgcgagctggcgttcaaactgggtctg 
               
               
                 gaaggtaaactggttcagcagttcaccaaaatcttcatgggcctggcgaccattttcctggagcgcgacctggcgtt 
               
               
                 gatcgaaatcaacccgctggtcatcaccaaacagggcgatctgatttgcctcgacggcaaactgggcgctgacggca 
               
               
                 acgcactgttccgccagcctgatctgcgcgaaatgcgtgaccagtcgcaggaagatccgcgtgaagcacaggctgca 
               
               
                 cagtgggaactgaactacgttgcgctggacggtaacatcggttgtatggttaacggcgcaggtctggcgatgggtac 
               
               
                 gatggacatcgttaaactgcacggcggcgaaccggctaacttccttgacgttggcggcggcgcaaccaaagaacgtg 
               
               
                 taaccgaagcgttcaaaatcatcctctctgacgacaaagtgaaagccgttctggttaacatcttcggcggtatcgtt 
               
               
                 cgttgcgacctgatcgctgacggtatcatcggcgcggtagcagaagtgggtgttaacgtaccggtcgtggtacgtct 
               
               
                 ggaaggtaacaacgccgaactcggcgcgaagaaactggctgacagcggcctgaatattattgcagcaaaaggtctga 
               
               
                 cggatgcagctcagcaggttgttgccgcagtggaggggaaataatgtccattttaatcgataaaaacaccaaggtta 
               
               
                 tctgccagggctttaccggtagccaggggactttccactcagaacaggccattgcatacggcactaaaatggttggc 
               
               
                 ggcgtaaccccaggtaaaggcggcaccacccacctcggcctgccggtgttaaacaccgtgcgtgaagccgttgctgc 
               
               
                 cactggcgctaccgcttctgttatctacgtaccagcaccgttctgcaaagactccattctggaagccatcgacgcag 
               
               
                 gcatcaaactgattatcaccatcactgaaggcatcccgacgctggatatgctgaccgtgaaagtgaagctggatgaa 
               
               
                 gcaggcgttcgtatgatcggcccgaactgcccaggcgttatcactccgggtgaatgcaaaatcggtatccagcctgg 
               
               
                 tcacattcacaaaccgggtaaagtgggtatcgtttcccgttccggtacactgacctatgaagcggttaaacagacca 
               
               
                 cggattacggtttcggtcagtcgacctgtgtcggtatcggcggtgacccgatcccgggctctaactttatcgacatt 
               
               
                 ctcgaaatgttcgaaaaagatccgcagaccgaagcgatcgtgatgatcggtgagatcggcggtagcgctgaagaaga 
               
               
                 agcagctgcgtacatcaaagagcacgttaccaagccagttgtgggttacatcgctggtgtgactgcgccgaaaggca 
               
               
                 aacgtatgggccacgcgggtgccatcattgccggtgggaaagggactgcggatgagaaattcgctgctctggaagcc 
               
               
                 gcaggcgtgaaaaccgttcgcagcctggcggatatcggtgaagcactgaaaactgttctgaaataaaggtccaggca 
               
               
                 tcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctact 
               
               
                 agagtcacactggctcaccttcgggtgggcctttctgcgtttatatgccatgtcttctactagtagcggccgctgca 
               
               
                 gtccggcaaaaaagggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcag 
               
               
                 gcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaat 
               
               
                 acggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgta 
               
               
                 aaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcag 
               
               
                 aggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttcc 
               
               
                 gaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgta 
               
               
                 ggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgc 
               
               
                 gccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaa 
               
               
                 caggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaa 
               
               
                 gaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaa 
               
               
                 caaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaaga 
               
               
                 tcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattat 
               
               
                 caaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact 
               
               
                 tggtctgacagctcgagtcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattag 
               
               
                 aaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccg 
               
               
                 tttctgtaatgaaggagaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccga 
               
               
                 ctcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtg 
               
               
                 acgactgaatccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctc 
               
               
                 gtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgc 
               
               
                 tgttaaaaggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttca 
               
               
                 cctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatc 
               
               
                 aggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctg 
               
               
                 taacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatag 
               
               
                 attgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaa 
               
               
                 tcgcggcctggagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagaca 
               
               
                 gttttattgttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgt 
               
               
                 tgaataaatcgaacttttgctgagttgaaggatcagctcgagtgccacctgacgtctaagaaaccattattatcatg 
               
               
                 acattaacctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatga 
               
               
                 tttctggaattcgcggccgcttctagagactagtggaagacatcgctaccgtaggcctgataagacgcgcaagcgtc 
               
               
                 gcatcaggcaaccagtgccggatgcggcgtgaacgccttatccggcctacaagtcattacccgtaggcctgataagc 
               
               
                 gcagcgcatcaggcgtaacaaagaaatgcaggaaatctttaaaaactgcccctgacactaagacagtttttaaaggt 
               
               
                 tccttcgcgagccactacgtagacaagagctcgcaagtgaaccccggcacgcacatcactgtgcgtggtagtatcca 
               
               
                 cggcgaagtaagcataaaaaagatgcttaagggatcacgAATGcagaacagcgctttgaaagcctggttggactctt 
               
               
                 cttacctctctggcgcaaaccagagctggatagaacagctctatgaaGATttcttaaccgatcctgactcggttgac 
               
               
                 gctaactggcgttcgacgttccagcagttacctggtacgggagtcaaaccggatcaattccactctcaaacgcgtga 
               
               
                 atatttccgccgcctggcgaaagacgcttcacgttactcttcaacgatctccgaccctgacaccaatgtgaagcagg 
               
               
                 ttaaagtcctgcagctcattaacgcataccgcttccgtggtcaccagcatgcgaatctcgatccgctgggactgtgg 
               
               
                 cagcaagataaagtggccgatctggatccgtctttccacgatctgaccgaagcagacttccaggagACTttcaacgt 
               
               
                 cggttcatttgccagcggcaaagaaaccatgaaactcggcgagctgctggaagccctcaagcaaacctactgcggcc 
               
               
                 cgattggtgccgagtatatgcacattaccagcaccgaagaaaaacgctggatccaacagcgtatcgagtctggtcgc 
               
               
                 gcgactttcaatagcgaagagaaaaaacgcttcttaagcgaactgaccgccgctgaaggtcttgaacgttacctcgg 
               
               
                 cgcaaaattccctggagcaaaacgcttctcgctggaaggcggtgacgcgttaatcccgatgcttaaagagatgatcc 
               
               
                 gccacgctggcaacagcggcacccgcgaagtggttctcgggatggcgcaccgtggtcgtctgaacgtgctggtgaac 
               
               
                 gtgctgggtaaaaaaccgcaagacttgttcgacgagttcgccggtaaacataaagaacacctcggcacgggtgacgt 
               
               
                 gaaataccacatgggcttctcgtctgacttccagaccgatggcggcctggtgcacctggcgctggcgtttaacccgt 
               
               
                 ctcaccttgagattgtaagcccggtagttatcggttctgttcgtgcccgtctggacagacttgatgagccgagcagc 
               
               
                 aacaaagtgctgccaatcaccatccacggtgacgccgcagtgaccgggcagggcgtggttcaggaaaccctgaacat 
               
               
                 gtcgaaagcgcgtggttatgaagttggcggtacggtacgtatcgttatcaacaaccaggttggtttcaccacctcta 
               
               
                 atccgctggatgcccgttctacgccgtactgtactgatatcggtaagatggttcaggccccgattttccacgttaac 
               
               
                 gcggacgatccggaagccgttgcctttgtgacccgtctggcgctcgatttccgtaacacctttaaacgtgatGTTtt 
               
               
                 catcgacctggtgtgctaccgccgtcacggccacaacgaagccgacgagccgagcgcaacccagccgctgatgtatc 
               
               
                 agaaaatcaaaaaacatccgacaccgcgcaaaatctacgctgacaagctggagcaggaaaaagtggcgacgctggaa 
               
               
                 gatgccaccgagatggttaacctgtaccgcgatgcgctggatgctggcgattgcgtagtggcagagtggcgtccgat 
               
               
                 gaacatgcactctttcacctggtcgccgtacctcaaccacgaatgggacgaagagtacccgaacaaagttgagatga 
               
               
                 agcgcctgcaggagctggcgaaacgcatcagcacggtgccggaagcagttgaaatgcagtctcgcgttgccaagatt 
               
               
                 tatggcgatcgccaggcgatggctgccggtgagaaactgttcgactggggcggtgcggaaaacctcgcttacgccac 
               
               
                 gctggttgatgaaggcattccggttcgcctgtcgggtGAGgactccggtcgcggtaccttcttccaccgccacgcgg 
               
               
                 tgatccacaaccagtctaacggttccacttacacgccgctgcaacatatccataacgggcagggcgcgttccgtgtc 
               
               
                 tgggactccgtactgtctgaagaagcagtgctggcgtttgaatatggttatgccaccgcagaaccacgcactctgac 
               
               
                 catctgggaagcgcagttcggtgacttcgccaacggtgcgcaggtggttatcgaccagttcatctcctctggcgaac 
               
               
                 agaaatggggccggatgtgtggtctggtgatgttgctgccgcacggttacgaagggcaggggccggagcactcctcc 
               
               
                 gcgcgtctggaacgttatctgcaactttgtgctgagcaaaacatgcaggtttgcgtaccgtctaccccggcacaggt 
               
               
                 ttaccacatgctgcgtcgtcaggcgctgcgcgggatgcgtcgtccgctggtcgtgatgtcgccgaaatccctgctgc 
               
               
                 gtcatccgctggcggtttccagcctcgaagaactggcgaacggcaccttcctgccagccatcggtgaaatcgacgag 
               
               
                 cttgatccgaagggcgtgaagcgcgtagtgatgtgttctggtaaggtttattacgacctgctggaacagcgtcgtaa 
               
               
                 gaacaatcaacacgatgtcgccattgtgcgtatcgagcaactctacccgttcccgcataaagcgatgcaggaagtgt 
               
               
                 tgcagcagtttgctcacgtcaaggattttgtctggtgccaggaagagccgctcaaccagggcgcatggtactgcagc 
               
               
                 cagcatcatttccgtgaagtgattccgtttggggcttctctgcgttatgcaggccgcccggcctccgcctctccggc 
               
               
                 ggtagggtatatgtccgttcaccagaaacagcaacaagatctggttaatgacgcgctgaacgtcgaataaataaagg 
               
               
                 atacacaatgagtagcgtagatattctggtccctgacctgcctgaatccgtagccgatgccaccgtcgcaacctggc 
               
               
                 ataaaaaacccggcgacgcagtcgtacgtgatgaagtgctggtagaaatcgaaactgacaaagtggtactggaagta 
               
               
                 ccggcatcagcagacggcattctggatgcggttctggaagatgaaggtacaacggtaacgtctcgtcagatccttgg 
               
               
                 tcgcctgcgtgaaggcaacagcgccggtaaagaaaccagcgccaaatctgaagagaaagcgtccactccggcgcaac 
               
               
                 gccagcaggcgtctctggaagagcaaaacaacgatgcgttaagcccggcgatccgtcgcctgctggctgaacacaat 
               
               
                 ctcgacgccagcgccattaaaggcaccggtgtgggtggtcgtctgactcgtgaagatgtggaaaaacatctggcgaa 
               
               
                 agcc 
               
               
                   
               
               
                 SEQ ID NO: 6. Sequence of the pDvS vector, designed for facile cloning with a modular 
               
               
                 cloning system. It contains the sucAD gene pair instead of an antibiotic resistance marker. 
               
               
                 cgcgttgctggcgtttttccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggc 
               
               
                 gaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctg 
               
               
                 ccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatct 
               
               
                 cagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttat 
               
               
                 ccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatt 
               
               
                 agcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt 
               
               
                 atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacca 
               
               
                 ccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttg 
               
               
                 atcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaag 
               
               
                 gatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctg 
               
               
                 acagctcgagtttacggctagctcagtcctaggtatagtgctagcTACTtgttagaaaagagaagcacgtaatgcag 
               
               
                 aactcagcattgaaagcatggcttgatagctcctatttatcaggtgctaaccagagctggattgaacagctgtatga 
               
               
                 agattttctgacagatccggattcagtggatgcgaattggcgcagcacttttcagcagttgcctggcaccggtgtaa 
               
               
                 aaccggatcagtttcattcccagacgcgggagtattttcgtcgtctggcgaaagatgcgagccggtattcaagtaca 
               
               
                 atttctgatccggatacgaatgtaaaacaggtgaaagtgcttcagttaattaatgcgtatcgctttagaggccatca 
               
               
                 gcatgcgaatctggatccgctgggcttatggcagcaggataaagtcgcggatctggatccaagttttcacgatttaa 
               
               
                 cggaagctgattttcaggaaacctttaacgtcggctcattcgcaagtgggaaagaaacaatgaaactgggcgaactt 
               
               
                 cttgaggcgctgaaacagacttattgtggccctattggtgcggaatatatgcatattacctcaactgaagagaaacg 
               
               
                 ttggattcagcagagaatcgagagtggccgcgcgacttttaactccgaagaaaaaaaaagattcctgtcagaactga 
               
               
                 cagccgcggaaggcttagagcggtatttgggtgccaaattcccaggagcaaaacggttcagcctggagggcggtgat 
               
               
                 gcgctgatcccgatgctgaaagaaatgattcggcatgcgggaaatagcggaactcgggaagtggtgttaggaatggc 
               
               
                 acaccgcggccgtttgaatgtactggttaacgtattaggaaaaaaacctcaggatttatttgatgagttcgcgggaa 
               
               
                 aacataaagaacatctgggcactggtgatgtcaaatatcacatgggcttctcaagtgattttcagacggatggaggt 
               
               
                 ctggttcacctggcactggcatttaatccttctcatctggaaatcgtaagtccggtcgttattggttccgtgcgcgc 
               
               
                 tcgcttagatcggttagatgaacctagctcaaacaaagttttaccaatcacgatccatggggatgcagctgttaccg 
               
               
                 gacagggtgttgtgcaggagactttgaatatgtccaaagcgcgcgggtatgaggtgggtggtacggtgcgtattgtt 
               
               
                 atcaataatcaggtgggttttacaaccagtaaccctctggatgctcgctctacgccgtattgcactgatattggtaa 
               
               
                 aatggtgcaggcaccaatttttcacgtcaatgccgatgatccggaagctgttgcctttgttacgcgcctggctctgg 
               
               
                 attttcgtaacactttcaaacgtgatgtatttatcgatttagtatgctatcgtcgtcatggtcataatgaggctgat 
               
               
                 gaacctagcgctacccagccactgatgtatcagaaaattaaaaaacatcctacccctcgtaaaatttatgcggataa 
               
               
                 actggagcaggaaaaagtggctactcttgaagatgctactgaaatggtcaatctttatcgggatgcattggatgcgg 
               
               
                 gtgattgcgtggtcgcggaatggcgcccgatgaatatgcattcatttacttggtcaccgtatttaaatcatgagtgg 
               
               
                 gatgaggaatatccgaataaagtggagatgaaacgcctgcaggaattagcaaaacgtattagcacagtacctgaagc 
               
               
                 ggttgagatgcagtctagagttgccaaaatctatggagatcgccaggccatggcagcaggggaaaaactttttgatt 
               
               
                 gggggggagccgaaaacctggcatatgcgacgctggtagatgagggcattccggtgcgcctttctggtgaagattct 
               
               
                 gggcgcggtactttttttcatcggcacgctgttattcataaccagtctaacggtagtacttatactccgctgcagca 
               
               
                 catccacaatggtcagggtgcgttccgtgtatgggattccgtgctgagtgaagaagcggttcttgcgtttgagtatg 
               
               
                 ggtatgcaactgccgagccacgcacgctgacgatctgggaagcccagtttggcgattttgcaaatggtgcccaggtg 
               
               
                 gtaatcgatcagtttattagctccggcgaacagaaatgggggcggatgtgtggtttagttatgttgttaccgcatgg 
               
               
                 ctatgaaggtcagggacctgagcacagctcagcgcgcctggaacgctatcttcagctgtgtgcggaacagaacatgc 
               
               
                 aggtatgcgttccttccacgccggctcaggtttatcatatgttaagacgtcaggccttgcgcggtatgcggcgcccg 
               
               
                 ttggtcgtgatgtccccgaaaagtttactgcgccatccgttagcagttagcagcctggaggaactggcaaacggtac 
               
               
                 gttcttgccagctatcggcgaaatcgatgaactggatcctaaaggggtgaaacgcgttgttatgtgttctggtaaag 
               
               
                 tgtattatgatcttttggaacagcgtcgcaaaaataatcagcacgatgtagctattgtgcggatcgagcagctgtat 
               
               
                 ccgttcccgcacaaagcaatgcaggaagtgctgcagcagttcgcacatgtcaaagattttgtctggtgtcaggagga 
               
               
                 accgcttaatcagggggcctggtattgtagtcagcaccatttccgggaggtgatcccgtttggggcgtccttacggt 
               
               
                 atgctggtcgccctgcctccgcaagtccggccgtgggatatatgagcgttcaccagaaacagcagcaggatttggtg 
               
               
                 aatgatgctttgaatgtggaatgaatgtccatcctgatcgacaaaaacactaaagtaatttgtcagggctttaccgg 
               
               
                 ttcccagggcacattccactcagagcaggccatcgcttatgggaccaaaatggtgggtggtgtaacgcctggtaaag 
               
               
                 gaggcaccacccatctgggtttgccggtatttaataccgtgcgtgaggcggttgccgcaaccggtgccacggcttca 
               
               
                 gttatctatgttcctgccccattttgtaaagattcaattctggaagctattgatgcgggcatcaaattgattattac 
               
               
                 gattaccgaaggtatccctacgctggatatgttgacggttaaagtgaaacttgatgaagcgggggtacgcatgattg 
               
               
                 gtccgaattgtccgggcgttattactccaggtgagtgcaaaattggtattcagccgggtcatattcacaaacctggg 
               
               
                 aaagtcggaattgtgtctcgttctggcactctgacgtatgaggcagttaaacagaccacagattatggctttgggca 
               
               
                 gagtacctgtgtcggcatcggaggcgatcctattccggggagtaattttatcgatattctggaaatgtttgagaaag 
               
               
                 atccgcagaccgaggcaatcgtcatgattggcgagattggcggttccgcggaagaagaagctgcagcctatatcaaa 
               
               
                 gaacatgtcacaaaaccggtagtgggctatatcgcgggagtcacggccccaaaaggtaaacgtatgggccatgccgg 
               
               
                 agcgatcatcgcgggcggcaaaggcactgcagatgaaaaatttgcagcccttgaggccgctggcgtaaaaacggtcc 
               
               
                 gttcccttgctgatattggtgaagcactgaaaaccgtgttgaaataaAGGTccaggcatcaaataaaacgaaaggct 
               
               
                 cagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcacc 
               
               
                 ttcgggtgggcctttctgcgtttatactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacct 
               
               
                 ataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctggaat 
               
               
                 tcgcggccgcttctagagactagtggaagacatcgctagagacctgcaccatatgcggtgtgaaataccgcacagat 
               
               
                 gcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgg 
               
               
                 gcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttc 
               
               
                 ccagtcacgacgttgtaaaacgacggccagtgaattcgagctcggtacccggggatcctctagagtcgacctgcagg 
               
               
                 catgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacat 
               
               
                 acgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcac 
               
               
                 tgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgggggtttataaaatc 
               
               
                 ccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatca 
               
               
                 aatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaa 
               
               
                 aactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaataca 
               
               
                 acctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgaga 
               
               
                 atggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgca 
               
               
                 tcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattaca 
               
               
                 aacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattctt 
               
               
                 ctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgc 
               
               
                 ttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgct 
               
               
                 acctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcc 
               
               
                 cgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctggagcaagac 
               
               
                 gtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatga 
               
               
                 tatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttg 
               
               
                 ctgagttgaaggatcagggtctcttgccatgtcttctactagtagcggccgctgcagtccggcaaaaaagggcaagg 
               
               
                 tgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcg 
               
               
                 ctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcagg 
               
               
                 ggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggc 
               
               
                   
               
               
                 SEQ ID NO: 7. Sequence of the pDvQ vector, designed for facile cloning with a modular 
               
               
                 cloning system. It contains the entire sucABCD operon including native 5’ UTR instead of an 
               
               
                 antibiotic resistance marker. 
               
               
                 cgcgttgctggcgtttttccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggc 
               
               
                 gaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctg 
               
               
                 ccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatct 
               
               
                 cagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttat 
               
               
                 ccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatt 
               
               
                 agcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt 
               
               
                 atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacca 
               
               
                 ccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttg 
               
               
                 atcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaag 
               
               
                 gatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctg 
               
               
                 acagctcgaggtaggcctgataagacgcgcaagcgtcgcatcaggcaaccagtgccggatgcggcgtgaacgcctta 
               
               
                 tccggcctacaagtcattacccgtaggcctgataagcgcagcgcatcaggcgtaacaaagaaatgcaggaaatcttt 
               
               
                 aaaaactgcccctgacactaagacagtttttaaaggttccttcgcgagccactacgtagacaagagctcgcaagtga 
               
               
                 accccggcacgcacatcactgtgcgtggtagtatccacggcgaagtaagcataaaaaagatgcttaagggatcacgA 
               
               
                 ATGcagaacagcgctttgaaagcctggttggactcttcttacctctctggcgcaaaccagagctggatagaacagct 
               
               
                 ctatgaaGATttcttaaccgatcctgactcggttgacgctaactggcgttcgacgttccagcagttacctggtacgg 
               
               
                 gagtcaaaccggatcaattccactctcaaacgcgtgaatatttccgccgcctggcgaaagacgcttcacgttactct 
               
               
                 tcaacgatctccgaccctgacaccaatgtgaagcaggttaaagtcctgcagctcattaacgcataccgcttccgtgg 
               
               
                 tcaccagcatgcgaatctcgatccgctgggactgtggcagcaagataaagtggccgatctggatccgtctttccacg 
               
               
                 atctgaccgaagcagacttccaggagACTttcaacgtcggttcatttgccagcggcaaagaaaccatgaaactcggc 
               
               
                 gagctgctggaagccctcaagcaaacctactgcggcccgattggtgccgagtatatgcacattaccagcaccgaaga 
               
               
                 aaaacgctggatccaacagcgtatcgagtctggtcgcgcgactttcaatagcgaagagaaaaaacgcttcttaagcg 
               
               
                 aactgaccgccgctgaaggtcttgaacgttacctcggcgcaaaattccctggcgcaaaacgcttctcgctggaaggc 
               
               
                 ggtgacgcgttaatcccgatgcttaaagagatgatccgccacgctggcaacagcggcacccgcgaagtggttctcgg 
               
               
                 gatggcgcaccgtggtcgtctgaacgtgctggtgaacgtgctgggtaaaaaaccgcaagacttgttcgacgagttcg 
               
               
                 ccggtaaacataaagaacacctcggcacgggtgacgtgaaataccacatgggcttctcgtctgacttccagaccgat 
               
               
                 ggcggcctggtgcacctggcgctggcgtttaacccgtctcaccttgagattgtaagcccggtagttatcggttctgt 
               
               
                 tcgtgcccgtctggacagacttgatgagccgagcagcaacaaagtgctgccaatcaccatccacggtgacgccgcag 
               
               
                 tgaccgggcagggcgtggttcaggaaaccctgaacatgtcgaaagcgcgtggttatgaagttggcggtacggtacgt 
               
               
                 atcgttatcaacaaccaggttggtttcaccacctctaatccgctggatgcccgttctacgccgtactgtactgatat 
               
               
                 cggtaagatggttcaggccccgattttccacgttaacgcggacgatccggaagccgttgcctttgtgacccgtctgg 
               
               
                 cgctcgatttccgtaacacctttaaacgtgatGTTttcatcgacctggtgtgctaccgccgtcacggccacaacgaa 
               
               
                 gccgacgagccgagcgcaacccagccgctgatgtatcagaaaatcaaaaaacatccgacaccgcgcaaaatctacgc 
               
               
                 tgacaagctggagcaggaaaaagtggcgacgctggaagatgccaccgagatggttaacctgtaccgcgatgcgctgg 
               
               
                 atgctggcgattgcgtagtggcagagtggcgtccgatgaacatgcactctttcacctggtcgccgtacctcaaccac 
               
               
                 gaatgggacgaagagtacccgaacaaagttgagatgaagcgcctgcaggagctggcgaaacgcatcagcacggtgcc 
               
               
                 ggaagcagttgaaatgcagtctcgcgttgccaagatttatggcgatcgccaggcgatggctgccggtgagaaactgt 
               
               
                 tcgactggggcggtgcggaaaacctcgcttacgccacgctggttgatgaaggcattccggttcgcctgtcgggtGAG 
               
               
                 gactccggtcgcggtaccttcttccaccgccacgcggtgatccacaaccagtctaacggttccacttacacgccgct 
               
               
                 gcaacatatccataacgggcagggcgcgttccgtgtctgggactccgtactgtctgaagaagcagtgctggcgtttg 
               
               
                 aatatggttatgccaccgcagaaccacgcactctgaccatctgggaagcgcagttcggtgacttcgccaacggtgcg 
               
               
                 caggtggttatcgaccagttcatctcctctggcgaacagaaatggggccggatgtgtggtctggtgatgttgctgcc 
               
               
                 gcacggttacgaagggcaggggccggagcactcctccgcgcgtctggaacgttatctgcaactttgtgctgagcaaa 
               
               
                 acatgcaggtttgcgtaccgtctaccccggcacaggtttaccacatgctgcgtcgtcaggcgctgcgcgggatgcgt 
               
               
                 cgtccgctggtcgtgatgtcgccgaaatccctgctgcgtcatccgctggcggtttccagcctcgaagaactggcgaa 
               
               
                 cggcaccttcctgccagccatcggtgaaatcgacgagcttgatccgaagggcgtgaagcgcgtagtgatgtgttctg 
               
               
                 gtaaggtttattacgacctgctggaacagcgtcgtaagaacaatcaacacgatgtcgccattgtgcgtatcgagcaa 
               
               
                 ctctacccgttcccgcataaagcgatgcaggaagtgttgcagcagtttgctcacgtcaaggattttgtctggtgcca 
               
               
                 ggaagagccgctcaaccagggcgcatggtactgcagccagcatcatttccgtgaagtgattccgtttggggcttctc 
               
               
                 tgcgttatgcaggccgcccggcctccgcctctccggcggtagggtatatgtccgttcaccagaaacagcaacaagat 
               
               
                 ctggttaatgacgcgctgaacgtcgaataaataaaggatacacaatgagtagcgtagatattctggtccctgacctg 
               
               
                 cctgaatccgtagccgatgccaccgtcgcaacctggcataaaaaacccggcgacgcagtcgtacgtgatgaagtgct 
               
               
                 ggtagaaatcgaaactgacaaagtggtactggaagtaccggcatcagcagacggcattctggatgcggttctggaag 
               
               
                 atgaaggtacaacggtaacgtctcgtcagatccttggtcgcctgcgtgaaggcaacagcgccggtaaagaaaccagc 
               
               
                 gccaaatctgaagagaaagcgtccactccggcgcaacgccagcaggcgtctctggaagagcaaaacaacgatgcgtt 
               
               
                 aagcccggcgatccgtcgcctgctggctgaacacaatctcgacgccagcgccattaaaggcaccggtgtgggtggtc 
               
               
                 gtctgactcgtgaagatgtggaaaaacatctggcgaaagccccggcgaaagagtctgctccggcagcggctgctccg 
               
               
                 gcggcgcaaccggctctggctgcacgtagtgaaaaacgtgtcccgatgactcgcctgcgtaagcgtgtggcagagcg 
               
               
                 tctgctggaagcgaaaaactccaccgccatgctgaccacgttcaacgaagtcaacatgaagccgattatggatctgc 
               
               
                 gtaagcagtacggtgaagcgtttgaaaaacgccacggcatccgtctgggctttatgtccttctacgtgaaagcggtg 
               
               
                 gttgaagccctgaaacgttacccggaagtgaacgcttctatcgacggcgatgacgtggtttaccacaactatttcga 
               
               
                 cgtcagcatggcggtttctacgccgcgcggcctggtgacgccggttctgcgtgatgtcgataccctcggcatggcag 
               
               
                 acatcgagaagaaaatcaaagagctggcagtcaaaggccgtgacggcaagctgaccgttgaagatctgaccggtggt 
               
               
                 aacttcaccatcaccaacggtggtgtgttcggttccctgatgtctacgccgatcatcaacccgccgcagagcgcaat 
               
               
                 tctgggtatgcacgctatcaaagatcgtccgatggcggtgaatggtcaggttgagatcctgccgatgatgtacctgg 
               
               
                 cgctgtcctacgatcaccgtctgatcgatggtcgcgaatccgtgggcttcctggtaacgatcaaagagttgctggaa 
               
               
                 gatccgacgcgtctgctgctggacgtgtagtagtttaagtttcacctgcactgtagaccggataaggcattatcgcc 
               
               
                 ttctccggcaattgaagcctgatgcgacgctgacgcgtcttatcaggcctacgggaccaccaatgtaggtcggataa 
               
               
                 ggcgcaagcgccgcatccgacaagcgatgcctgatgtgacgtttaacgtgtcttatcaggcctacgggtgaccgaca 
               
               
                 atgcccggaagcgatacgaaatattcGGTCTAGGGTTTAAAAGATAACGATTACTGAAGGATGGACAGAACACatga 
               
               
                 acttacatgaatatcaggcaaaacaactttttgcccgctatggcttaccagcaccggtgggttatgcctgtactact 
               
               
                 ccgcgcgaagcagaagaagccgcttcaaaaatcggtgccggtccgtgggtagtgaaatgtcaggttcacgctggtgg 
               
               
                 ccgcggtaaagcgggcggtgtgaaagttgtaaacagcaaaGAGgacatccgtgcttttgcagaaaactggctgggca 
               
               
                 agcgtctggtaacgtatcaaacagatgccaatggccaaccggttaaccagattctggttgaagcagcgaccgatatc 
               
               
                 gctaaagagctgtatctcggtgccgttgttgaccgtagttcccgtcgtgtggtctttatggcctccaccgaaggcgg 
               
               
                 cgtggaaatcgaaaaagtggcggaagaaactccgcacctgatccataaagttgcgcttgatccgctgactggcccga 
               
               
                 tgccgtatcagggacgcgagctggcgttcaaactgggtctggaaggtaaactggttcagcagttcaccaaaatcttc 
               
               
                 atgggcctggcgaccattttcctggagcgcgacctggcgttgatcgaaatcaacccgctggtcatcaccaaacaggg 
               
               
                 cgatctgatttgcctcgacggcaaactgggcgctgacggcaacgcactgttccgccagcctgatctgcgcgaaatgc 
               
               
                 gtgaccagtcgcaggaagatccgcgtgaagcacaggctgcacagtgggaactgaactacgttgcgctggacggtaac 
               
               
                 atcggttgtatggttaacggcgcaggtctggcgatgggtacgatggacatcgttaaactgcacggcggcgaaccggc 
               
               
                 taacttccttgacgttggcggcggcgcaaccaaagaacgtgtaaccgaagcgttcaaaatcatcctctctgacgaca 
               
               
                 aagtgaaagccgttctggttaacatcttcggcggtatcgttcgttgcgacctgatcgctgacggtatcatcggcgcg 
               
               
                 gtagcagaagtgggtgttaacgtaccggtcgtggtacgtctggaaggtaacaacgccgaactcggcgcgaagaaact 
               
               
                 ggctgacagcggcctgaatattattgcagcaaaaggtctgacggatgcagctcagcaggttgttgccgcagtggagg 
               
               
                 ggaaataatgtccattttaatcgataaaaacaccaaggttatctgccagggctttaccggtagccaggggactttcc 
               
               
                 actcagaacaggccattgcatacggcactaaaatggttggcggcgtaaccccaggtaaaggcggcaccacccacctc 
               
               
                 ggcctgccggtgttcaacaccgtgcgtgaagccgttgctgccactggcgctaccgcttctgttatctacgtaccagc 
               
               
                 accgttctgcaaagactccattctggaagccatcgacgcaggcatcaaactgattatcaccatcactgaaggcatcc 
               
               
                 cgacgctggatatgctgaccgtgaaagtgaagctggatgaagcaggcgttcgtatgatcggcccgaactgcccaggc 
               
               
                 gttatcactccgggtgaatgcaaaatcggtatccagcctggtcacattcacaaaccgggtaaagtgggtatcgtttc 
               
               
                 ccgttccggtacactgacctatgaagcggttaaacagaccacggattacggtttcggtcagtcgacctgtgtcggta 
               
               
                 tcggcggtgacccgatcccgggctctaactttatcgacattctcgaaatgttcgaaaaagatccgcagaccgaagcg 
               
               
                 atcgtgatgatcggtgagatcggcggtagcgctgaagaagaagcagctgcgtacatcaaagagcacgttaccaagcc 
               
               
                 agttgtgggttacatcgctggtgtgactgcgccgaaaggcaaacgtatgggccacgcgggtgccatcattgccggtg 
               
               
                 ggaaagggactgcggatgagaaattcgctgctctggaagccgcaggcgtgaaaaccgttcgcagcctggcggatatc 
               
               
                 ggtgaagcactgaaaactgttctgaaataaaggtccaggcatcaaataaaacgaaaggctcagtcgaaagactgggc 
               
               
                 ctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttct 
               
               
                 gcgtttatactcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatc 
               
               
                 acgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctggaattcgcggccgcttctaga 
               
               
                 gactagtggaagacatcgctagagacctgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaatac 
               
               
                 cgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacg 
               
               
                 ccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgta 
               
               
                 aaacgacggccagtgaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttggcgta 
               
               
                 atcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataa 
               
               
                 agtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcg 
               
               
                 ggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgggggtttataaaatcccgtcaagtcagcgtaa 
               
               
                 tgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaattta 
               
               
                 ttcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagtt 
               
               
                 ccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccct 
               
               
                 cgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgc 
               
               
                 atttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttatt 
               
               
                 cattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcgaatgca 
               
               
                 accggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgct 
               
               
                 gttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagagg 
               
               
                 cataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctacctttgccatgtttca 
               
               
                 gaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcccgacattatcgcgagcc 
               
               
                 catttatacccatataaatcagcatccatgttggaatttaatcgcggcctggagcaagacgtttcccgttgaatatg 
               
               
                 gctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatgatgatatatttttatcttgtg 
               
               
                 caatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaacttttgctgagttgaaggatcag 
               
               
                 ggtctcttgccatgtcttctactagtagcggccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgccct 
               
               
                 ttttctttaaaaccgaaaagattacttcgcgttatgcaggcttcctcgctcactgactcgctgcgctcggtcgttcg 
               
               
                 gctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaaga 
               
               
                 acatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggc 
               
               
                   
               
               
                 SEQ ID NO: 8. Sequence of Plasmid pDvS-GFP containing a sequence encoding the green 
               
               
                 fluorescent protein cloned into the pDvS vector 
               
               
                 tactgogccatccgttagcagttagcagcctggaggaactggcaaacggtacgttcttgccagctatoggcgaaatc 
               
               
                 gatgaactggatcctaaaggggtgaaacgcgttgttatgtgttctggtaaagtgtattatgatcttttggaacagcg 
               
               
                 tcgcaaaaataatcagcacgatgtagctattgtgcggatcgagcagctgtatccgttcccgcacaaagcaatgcagg 
               
               
                 aagtgctgcagcagttcgcacatgtcaaagattttgtctggtgtcaggaggaaccgcttaatcagggggcctggtat 
               
               
                 tgtagtcagcaccatttccgggaggtgatcccgtttggggcgtccttacggtatgctggtcgccctgcctccgcaag 
               
               
                 tccggccgtgggatatatgagcgttcaccagaaacagcagcaggatttggtgaatgatgctttgaatgtggaatgaa 
               
               
                 tgtccatcctgatcgacaaaaacactaaagtaatttgtcagggctttaccggttcccagggcacatttcactcagag 
               
               
                 caggccatcgcttatgggaccaaaatggtgggtggtgtaacgcctggtaaaggaggcaccacccatctgggtttgcc 
               
               
                 ggtatttaataccgtgcgtgaggcggttgccgcaaccggtgccacggcttcagttatctatgttcctgccccatttt 
               
               
                 gtaaagattcaattctggaagctattgatgcgggcatcaaattgattattacgattaccgaaggtatccctacgctg 
               
               
                 gatatgttgacggttaaagtgaaacttgatgaagcgggggtacgcatgattggtccgaattgtccgggcgttattac 
               
               
                 tccaggtgagtgcaaaattggtattcagccgggtcatattcacaaacctgggaaagtcggaattgtgtctcgttctg 
               
               
                 gcactctgacgtatgaggcagttaaacagaccacagattatggctttgggcagagtacctgtgtcggcatcggaggc 
               
               
                 gatcctattccggggagtaattttatcgatattctggaaatgtttgagaaagatccgcagaccgaggcaatcgtcat 
               
               
                 gattggcgagattggcggttccgcggaagaagaagctgcagcctatatcaaagaacatgtcacaaaaccggtagtgg 
               
               
                 gctatatcgcgggagtcacggccccaaaaggtaaacgtatgggccatgccggagcgatcatcgcgggcggcaaaggc 
               
               
                 actgcagatgaaaaatttgcagcccttgaggccgctggcgtaaaaacggtccgttcccttgctgatattggtgaagc 
               
               
                 actgaaaaccgtgttgaaataaAGGTccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgtt 
               
               
                 ttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttat 
               
               
                 atcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagca 
               
               
                 tcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaagga 
               
               
                 gaaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaat 
               
               
                 acaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtg 
               
               
                 agaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaaaatcactc 
               
               
                 gcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaaggacaatt 
               
               
                 acaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatatt 
               
               
                 cttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaa 
               
               
                 tgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaac 
               
               
                 gctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgatt 
               
               
                 gcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctggagcaa 
               
               
                 gacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttattgttcatga 
               
               
                 tgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaatcgaactt 
               
               
                 ttgctgagttgaaggatcagctcgagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaa 
               
               
                 ataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctggaattcgcgg 
               
               
                 ccgcttctagagactagtggaagacatcgctggaaagtgaaacgtgatttcatgcgtcattttgaacattttgtaaa 
               
               
                 tcttatttaataatgtgtgcggcaattcacatttaatttatgaatgttttcttaacatcgcggcaactcaagaaacg 
               
               
                 gcaggttcggatcttagctactagagaaagaggagaaatactagatgcgtaaaggcgaagagctgttcactggtgtc 
               
               
                 gtccctattctggtggaactggatggtgatgtcaacggtcataagttttccgtgcgtggcgagggtgaaggtgacgc 
               
               
                 aactaatggtaaactgacgctgaagttcatctgtactactggtaaactgccggttccttggccgactctggtaacga 
               
               
                 cgctgacttatggtgttcagtgctttgctcgttatccggaccatatgaagcagcatgacttcttcaagtccgccatg 
               
               
                 ccggaaggctatgtgcaggaacgcacgatttcctttaaggatgacggcacgtacaaaacgcgtgcggaagtgaaatt 
               
               
                 tgaaggcgataccctggtaaaccgcattgagctgaaaggcattgactttaaagaggacggcaatatcctgggccata 
               
               
                 agctggaatacaattttaacagccacaatgtttacatcaccgccgataaacaaaaaaatggcattaaagcgaatttt 
               
               
                 aaaattcgccacaacgtggaggatggcagcgtgcagctggctgatcactaccagcaaaacactccaatcggtgatgg 
               
               
                 tcctgttctgctgccagacaatcactatctgagcacgcaaagcgttctgtctaaagatccgaacgagaaacgcgatc 
               
               
                 atatggttctgctggagttcgtaaccgcagcgggcatcacgcatggtatggatgaactgtacaaatgaccagccatc 
               
               
                 aaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactag 
               
               
                 agtcacactggctcaccttcgggtgggcctttctgcgtttatacgtgccatgtcttctactagtagcggccgctgca 
               
               
                 gtccggcaaaaaagggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcgttatgcag 
               
               
                 gcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaat 
               
               
                 acggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgta 
               
               
                 aaaaggccgcgttgctggcgtttttccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcag 
               
               
                 aggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttcc 
               
               
                 gaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgta 
               
               
                 ggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgc 
               
               
                 gccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaa 
               
               
                 caggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaa 
               
               
                 gaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaa 
               
               
                 caaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaaga 
               
               
                 tcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattat 
               
               
                 caaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaact 
               
               
                 tggtctgacagctcgagtttacggctagctcagtcctaggtatagtgctagcTACTtgttagaaaagagaagcacgt 
               
               
                 aatgcagaactcagcattgaaagcatggcttgatagctcctatttatcaggtgctaaccagagctggattgaacagc 
               
               
                 tgtatgaagattttctgacagatccggattcagtggatgcgaattggcgcagcacttttcagcagttgcctggcacc 
               
               
                 ggtgtaaaaccggatcagtttcattcccagacgcgggagtattttcgtcgtctggcgaaagatgcgagccggtattc 
               
               
                 aagtacaatttctgatccggatacgaatgtaaaacaggtgaaagtgcttcagttaattaatgcgtatcgctttagag 
               
               
                 gccatcagcatgcgaatctggatccgctgggcttatggcagcaggataaagtcgcggatctggatccaagttttcac 
               
               
                 gatttaacggaagctgattttcaggaaacctttaacgtcggctcattcgcaagtgggaaagaaacaatgaaactggg 
               
               
                 cgaacttcttgaggcgctgaaacagacttattgtggccctattggtgcggaatatatgcatattacctcaactgaag 
               
               
                 agaaacgttggattcagcagagaatcgagagtggccgcgcgacttttaactccgaagaaaaaaaaagattcctgtca 
               
               
                 gaactgacagccgcggaaggcttagagcggtatttgggtgccaaattcccaggagcaaaacggttcagcctggaggg 
               
               
                 cggtgatgcgctgatcccgatgctgaaagaaatgattcggcatgcgggaaatagcggaactcgggaagtggtgttag 
               
               
                 gaatggcacaccgcggccgtttgaatgtactggttaacgtattaggaaaaaaacctcaggatttatttgatgagttc 
               
               
                 gcgggaaaacataaagaacatctgggcactggtgatgtcaaatatcacatgggcttctcaagtgattttcagacgga 
               
               
                 tggaggtctggttcacctggcactggcatttaatccttctcatctggaaatcgtaagtccggtcgttattggttccg 
               
               
                 tgcgcgctcgcttagatcggttagatgaacctagctcaaacaaagttttaccaatcacgatccatggggatgcagct 
               
               
                 gttaccggacagggtgttgtgcaggagactttgaatatgtccaaagcgcgcgggtatgaggtgggtggtacggtgcg 
               
               
                 tattgttatcaataatcaggtgggttttacaaccagtaaccctctggatgctcgctctacgccgtattgcactgata 
               
               
                 ttggtaaaatggtgcaggcaccaatttttcacgtcaatgccgatgatccggaagctgttgcctttgttacgcgcctg 
               
               
                 gctctggattttcgtaacactttcaaacgtgatgtatttatcgatttagtatgctatcgtcgtcatggtcataatga 
               
               
                 ggctgatgaacctagcgctacccagccactgatgtatcagaaaattaaaaaacatcctacccctcgtaaaatttatg 
               
               
                 cggataaactggagcaggaaaaagtggctactcttgaagatgctactgaaatggtcaatctttatcgggatgcattg 
               
               
                 gatgcgggtgattgcgtggtcgcggaatggcgcccgatgaatatgcattcatttacttggtcaccgtatttaaatca 
               
               
                 tgagtgggatgaggaatatccgaataaagtggagatgaaacgcctgcaggaattagcaaaacgtattagcacagtac 
               
               
                 ctgaagcggttgagatgcagtctagagttgccaaaatctatggagatcgccaggccatggcagcaggggaaaaactt 
               
               
                 tttgattgggggggagccgaaaacctggcatatgcgacgctggtagatgagggcattccggtgcgcctttctggtga 
               
               
                 agattctgggcgcggtactttttttcatcggcacgctgttattcataaccagtctaacggtagtacttatactcagc 
               
               
                 tgcagcacatccacaatggtcagggtgcgttccgtgtatgggattccgtgctgagtgaagaagcggttcttgcgttt 
               
               
                 gagtatgggtatgcaactgccgagccacgcacgctgacgatctgggaagcccagtttggcgattttgcaaatggtgc 
               
               
                 ccaggtggtaatcgatcagtttattagctccggcgaacagaaatgggggcggatgtgtggtttagttatgttgttac 
               
               
                 cgcatggctatgaaggtcagggacctgagcacagctcagcgcgcctggaacgctatcttcagctgtgtgcggaacag 
               
               
                 aacatgcaggtatgcgttccttccacgccggctcaggtttatcatatgttaagacgtcaggccttgcgcggtatgcg 
               
               
                 gcgcccgttggtcgtgatgtccccgaaaagtt 
               
               
                   
               
               
                 SEQ ID NO: 9. Sequence of Plasmid pDvQ-GFP containing a sequence encoding the green 
               
               
                 fluorescent protein cloned into the pDvQ vector 
               
               
                 cgcgttgctggcgtttttccacaggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggc 
               
               
                 gaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctg 
               
               
                 ccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatct 
               
               
                 cagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttat 
               
               
                 ccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggatt 
               
               
                 agcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagt 
               
               
                 atttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacca 
               
               
                 ccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttg 
               
               
                 atcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaag 
               
               
                 gatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctg 
               
               
                 acagctcgaggtaggcctgataagacgcgcaagcgtcgcatcaggcaaccagtgccggatgcggcgtgaacgcctta 
               
               
                 tccggcctacaagtcattacccgtaggcctgataagcgcagcgcatcaggcgtaacaaagaaatgcaggaaatcttt 
               
               
                 aaaaactgtccctgacactaagacagtttttadaggttccttcgcgagccactacgtagacaagagctcgcaagtga 
               
               
                 accccggcacgcacatcactgtgcgtggtagtatccacggcgaagtaagcataaaaaagatgcttaagggatcacgA 
               
               
                 ATGcagaacagcgctttgaaagcctggttggactcttcttacctctctggcgcaaaccagagctggatagaacagct 
               
               
                 ctatgaaGATttcttaaccgatcctgactcggttgacgctaactggcgttcgacgttccagcagttacctggtacgg 
               
               
                 gagtcaaaccggatcaattccactctcaaacgcgtgaatatttccgccgcctggcgaaagacgcttcacgttactct 
               
               
                 tcaacgatctccgaccctgacaccaatgtgaagcaggttaaagtcctgcagctcattaacgcataccgcttccgtgg 
               
               
                 tcaccagcatgcgaatctcgatccgctgggactgtggcagcaagataaagtggccgatctggatccgtctttccacg 
               
               
                 atctgaccgaagcagacttccaggagACTttcaacgtcggttcatttgccagcggcaaagaaaccatgaaactcggc 
               
               
                 gagctgctggaagccctcaagcaaacctactgcggcccgattggtgccgagtatatgcacattaccagcaccgaaga 
               
               
                 aaaacgctggatccaacagcgtatcgagtctggtcgcgcgactttcaatagcgaagagaaaaaacgcttcttaagcg 
               
               
                 aactgaccgccgctgaaggtcttgaacgttacctcggcgcaaaattccctggcgcaaaacgcttctcgctggaaggc 
               
               
                 ggtgacgcgttaatcccgatgcttaaagagatgatccgccacgctggcaacagcggcacccgcgaagtggttctcgg 
               
               
                 gatggcgcaccgtggtcgtctgaacgtgctggtgaacgtgctgggtaaaaaaccgcaagacttgttcgacgagttcg 
               
               
                 ccggtaaacataaagaacacctcggcacgggtgacgtgaaataccacatgggcttctcgtctgacttccagaccgat 
               
               
                 ggcggcctggtgcacctggcgctggcgtttaacccgtctcaccttgagattgtaagcccggtagttatcggttctgt 
               
               
                 tcgtgcccgtctggacagacttgatgagccgagcagcaacaaagtgctgccaatcaccatccacggtgacgccgcag 
               
               
                 tgaccgggcagggcgtggttcaggaaaccctgaacatgtcgaaagcgcgtggttatgaagttggcggtacggtacgt 
               
               
                 atcgttatcaacaaccaggttggtttcaccacctctaatccgctggatgcccgttctacgccgtactgtactgatat 
               
               
                 cggtaagatggttcaggccccgattttccacgttaacgcggacgatccggaagccgttgcctttgtgacccgtctgg 
               
               
                 cgctcgatttccgtaacacctttaaacgtgatGTTttcatcgacctggtgtgctaccgccgtcacggccacaacgaa 
               
               
                 gccgacgagccgagcgcaacccagccgctgatgtatcagaaaatcaaaaaacatccgacaccgcgcaaaatctacgc 
               
               
                 tgacaagctggagcaggaaaaagtggcgacgctggaagatgccaccgagatggttaacctgtaccgcgatgcgctgg 
               
               
                 atgctggcgattgcgtagtggcagagtggcgtccgatgaacatgcactctttcacctggtcgccgtacctcaaccac 
               
               
                 gaatgggacgaagagtacccgaacaaagttgagatgaagcgcctgcaggagctggcgaaacgcatcagcacggtgcc 
               
               
                 ggaagcagttgaaatgcagtctcgcgttgccaagatttatggcgatcgccaggcgatggctgccggtgagaaactgt 
               
               
                 tcgactggggcggtgcggaaaacctcgcttacgccacgctggttgatgaaggcattccggttcgcctgtcgggtGAG 
               
               
                 gactccggtcgcggtaccttcttccaccgccacgcggtgatccacaaccagtctaacggttccacttacacgccgct 
               
               
                 gcaacatatccataacgggcagggcgcgttccgtgtctgggactccgtactgtctgaagaagcagtgctggcgtttg 
               
               
                 aatatggttatgccaccgcagaaccacgcactctgaccatctgggaagcgcagttcggtgacttcgccaacggtgcg 
               
               
                 caggtggttatcgaccagttcatctcctctggcgaacagaaatggggccggatgtgtggtctggtgatgttgctgcc 
               
               
                 gcacggttacgaagggcaggggccggagcactcctccgcgcgtctggaacgttatctgcaactttgtgctgagcaaa 
               
               
                 acatgcaggtttgcgtaccgtctaccccggcacaggtttaccacatgctgcgtcgtcaggcgctgcgcgggatgcgt 
               
               
                 cgtccgctggtcgtgatgtcgccgaaatccctgctgcgtcatccgctggcggtttccagcctcgaagaactggcgaa 
               
               
                 cggcaccttcctgccagccatcggtgaaatcgacgagcttgatccgaagggcgtgaagcgcgtagtgatgtgttctg 
               
               
                 gtaaggtttattacgacctgctggaacagcgtcgtaagaacaatcaacacgatgtcgccattgtgcgtatcgagcaa 
               
               
                 ctctacccgttcccgcataaagcgatgcaggaagtgttgcagcagtttgctcacgtcaaggattttgtctggtgcca 
               
               
                 ggaagagccgctcaaccagggcgcatggtactgcagccagcatcatttccgtgaagtgattccgtttggggcttctc 
               
               
                 tgcgttatgcaggccgcccggcctccgcctctccggcggtagggtatatgtccgttcaccagaaacagcaacaagat 
               
               
                 ctggttaatgacgcgctgaacgtcgaataaataaaggatacacaatgagtagcgtagatattctggtccctgacctg 
               
               
                 cctgaatccgtagccgatgccaccgtcgcaacctggcataaaaaacccggcgacgcagtcgtacgtgatgaagtgct 
               
               
                 ggtagaaatcgaaactgacaaagtggtactggaagtaccggcatcagcagacggcattctggatgcggttctggaag 
               
               
                 atgaaggtacaacggtaacgtctcgtcagatccttggtcgcctgcgtgaaggcaacagcgccggtaaagaaaccagc 
               
               
                 gccaaatctgaagagaaagcgtccactccggcgcaacgccagcaggcgtctctggaagagcaaaacaacgatgcgtt 
               
               
                 aagcccggcgatccgtcgcctgctggctgaacacaatctcgacgccagcgccattaaaggcaccggtgtgggtggtc 
               
               
                 gtctgactcgtgaagatgtggaaaaacatctggcgaaagccccggcgaaagagtctgctccggcagcggctgctccg 
               
               
                 gcggcgcaaccggctctggctgcacgtagtgaaaaacgtgtcccgatgactcgcctgcgtaagcgtgtggcagagcg 
               
               
                 tctgctggaagcgaaaaactccaccgccatgctgaccacgttcaacgaagtcaacatgaagccgattatggatctgc 
               
               
                 gtaagcagtacggtgaagcgtttgaaaaacgccacggcatccgtctgggctttatgtccttctacgtgaaagcggtg 
               
               
                 gttgaagccctgaaacgttacccggaagtgaacgcttctatcgacggcgatgacgtggtttaccacaactatttcga 
               
               
                 cgtcagcatggcggtttctacgccgcgcggcctggtgacgccggttctgcgtgatgtcgataccctcggcatggcag 
               
               
                 acatcgagaagaaaatcaaagagctggcagtcaaaggccgtgacggcaagctgaccgttgaagatctgaccggtggt 
               
               
                 aacttcaccatcaccaacggtggtgtgttcggttccctgatgtctacgccgatcatcaacccgccgcagagcgcaat 
               
               
                 tctgggtatgcacgctatcaaagatcgtccgatggcggtgaatggtcaggttgagatcctgccgatgatgtacctgg 
               
               
                 cgctgtcctacgatcaccgtctgatcgatggtcgcgaatccgtgggcttcctggtaacgatcaaagagttgctggaa 
               
               
                 gatccgacgcgtctgctgctggacgtgtagtagtttaagtttcacctgcactgtagaccggataaggcattatcgcc 
               
               
                 ttctccggcaattgaagcctgatgcgacgctgacgcgtcttatcaggcctacgggaccaccaatgtaggtcggataa 
               
               
                 ggcgcaagcgccgcatccgacaagcgatgcctgatgtgacgtttaacgtgtcttatcaggcctacgggtgaccgaca 
               
               
                 atgcccggaagcgatacgaaatattcGGTCTACGGTTTAAAAGATAACGATTACTGAAGGATGGACAGAACACatga 
               
               
                 acttacatgaatatcaggcaaaacaactttttgcccgctatggcttaccagcaccggtgggttatgcctgtactact 
               
               
                 ccgcgcgaagcagaagaagccgcttcaaaaatcggtgccggtccgtgggtagtgaaatgtcaggttcacgctggtgg 
               
               
                 ccgcggtaaagcgggcggtgtgaaagttgtaaacagcaaaGAGgacatccgtgcttttgcagaaaactggctgggca 
               
               
                 agcgtctggtaacgtatcaaacagatgccaatggccaaccggttaaccagattctggttgaagcagcgaccgatatc 
               
               
                 gctaaagagctgtatctcggtgccgttgttgaccgtagttcccgtcgtgtggtctttatggcctccaccgaaggcgg 
               
               
                 cgtggaaatcgaaaaagtggcggaagaaactccgcacctgatccataaagttgcgcttgatccgctgactggcccga 
               
               
                 tgccgtatcagggacgcgagctggcgttcaaactgggtctggaaggtaaactggttcagcagttcaccaaaatcttc 
               
               
                 atgggcctggcgaccattttcctggagcgcgacctggcgttgatcgaaatcaacccgctggtcatcaccaaacaggg 
               
               
                 cgatctgatttgcctcgacggcaaactgggcgctgacggcaacgcactgttccgccagcctgatctgcgcgaaatgc 
               
               
                 gtgaccagtcgcaggaagatccgcgtgaagcacaggctgcacagtgggaactgaactacgttgcgctggacggtaac 
               
               
                 atcggttgtatggttaacggcgcaggtctggcgatgggtacgatggacatcgttaaactgcacggcggcgaaccggc 
               
               
                 taacttccttgacgttggcggcggcgcaaccaaagaacgtgtaaccgaagcgttcaaaatcatcctctctgacgaca 
               
               
                 aagtgaaagccgttctggttaacatcttcggcggtatcgttcgttgcgacctgatcgctgacggtatcatcggcgcg 
               
               
                 gtagcagaagtgggtgttaacgtaccggtcgtggtacgtctggaaggtaacaacgccgaactcggcgcgaagaaact 
               
               
                 ggctgacagcggcctgaatattattgcagcaaaaggtctgacggatgcagctcagcaggttgttgccgcagtggagg 
               
               
                 ggaaataatgtccattttaatcgataaaaacaccaaggttatctgccagggctttaccggtagccaggggactttcc 
               
               
                 actcagaacaggccattgcatacggcactaaaatggttggcggcgtaaccccaggtaaaggcggcaccacccacctc 
               
               
                 ggcctgccggtgttcaacaccgtgcgtgaagccgttgctgccactggcgctaccgcttctgttatctacgtaccagc 
               
               
                 accgttctgcaaagactccattctggaagccatcgacgcaggcatcaaactgattatcaccatcactgaaggcatcc 
               
               
                 cgacgctggatatgctgaccgtgaaagtgaagctggatgaagcaggcgttcgtatgatcggcccgaactgcccaggc 
               
               
                 gttatcactccgggtgaatgcaaaatcggtatccagcctggtcacattcacaaaccgggtaaagtgggtatcgtttc 
               
               
                 ccgttccggtacactgacctatgaagcggttaaacagaccacggattacggtttcggtcagtcgacctgtgtcggta 
               
               
                 tcggcggtgacccgatttcgggctctaactttatcgacattctcgcaatgttcgaaaaagatccgcagaccgaagcg 
               
               
                 atcgtgatgatcggtgagatcggcggtagcgctgaagaagaagcagctgcgtacatcaaagagcacgttaccaagcc 
               
               
                 agttgtgggttacatcgctggtgtgactgcgccgaaaggcaaacgtatgggccacgcgggtgccatcattgccggtg 
               
               
                 ggaaagggactgcggatgagaaattcgctgctctggaagccgcaggcgtgaaaaccgttcgcagcctggcggatatc 
               
               
                 ggtgaagcactgaaaactgttctgaaataaaggtccaggcatcaaataaaacgaaaggctcagtcgaaagactgggc 
               
               
                 ctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttct 
               
               
                 gcgtttatatcccgtcaagtcagcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactc 
               
               
                 atcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgta 
               
               
                 atgaaggagaaaactcaccgaggcagttccacaggatggcaagatcctggtatcggtctgcgattccgactcgtcca 
               
               
                 acatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgagaaatcaccatgagtgacgactga 
               
               
                 atccggtgagaatggcaaaagcttatgcatttctttccagacttgttcaacaggccagccattacgctcgtcatcaa 
               
               
                 aatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcgatcgctgttaaaa 
               
               
                 ggacaattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatc 
               
               
                 aggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaaccatgcatcatcaggagtac 
               
               
                 ggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatctcatctgtaacatca 
               
               
                 ttggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgc 
               
               
                 acctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcc 
               
               
                 tggagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacagttttatt 
               
               
                 gttcatgatgatatatttttatcttgtgcaatgtaacatcagagattttgagacacaacgtggctttgttgaataaa 
               
               
                 tcgaacttttgctgagttgaaggatcagctcgagtgccacctgacgtctaagaaaccattattatcatgacattaac 
               
               
                 ctataaaaataggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctgga 
               
               
                 attcgcggccgcttctagagactagtggaagacatcgctggaaagtgaaacgtgatttcatgcgtcattttgaacat 
               
               
                 tttgtaaatcttatttaataatgtgtgcggcaattcacatttaatttatgaatgttttcttaacatcgcggcaactc 
               
               
                 aagaaacggcaggttcggatcttagctactagagaaagaggagaaatactagatgcgtaaaggcgaagagctgttca 
               
               
                 ctggtgtcgtccctattctggtggaactggatggtgatgtcaacggtcataagttttccgtgcgtggcgagggtgaa 
               
               
                 ggtgacgcaactaatggtaaactgacgctgaagttcatctgtactactggtaaactgccggttccttggccgactct 
               
               
                 ggtaacgacgctgacttatggtgttcagtgctttgctcgttatccggaccatatgaagcagcatgacttcttcaagt 
               
               
                 ccgccatgccggaaggctatgtgcaggaacgcacgatttcctttaaggatgacggcacgtacaaaacgcgtgcggaa 
               
               
                 gtgaaatttgaaggcgataccctggtaaaccgcattgagctgaaaggcattgactttaaagaggacggcaatatcct 
               
               
                 gggccataagctggaatacaattttaacagccacaatgtttacatcaccgccgataaacaaaaaaatggcattaaag 
               
               
                 cgaattttaaaattcgccacaacgtggaggatggcagcgtgcagctggctgatcactaccagcaaaacactccaatc 
               
               
                 ggtgatggtcctgttctgctgccagacaatcactatctgagcacgcaaagcgttctgtctaaagatccgaacgagaa 
               
               
                 acgcgatcatatggttctgctggagttcgtaaccgcagcgggcatcacgcatggtatggatgaactgtacaaatgac 
               
               
                 caggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctc 
               
               
                 tctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatacgtgccatgtcttctactagtagcgg 
               
               
                 ccgctgcagtccggcaaaaaagggcaaggtgtcaccaccctgccctttttctttaaaaccgaaaagattacttcgcg 
               
               
                 ttatgcaggcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag 
               
               
                 gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccag 
               
               
                 gaaccgtaaaaaggc 
               
               
                   
               
               
                 SEQ ID NO: 10. Sequence of Plasmid pDvK-SucBC, used for testing plasmid retention 
               
               
                 in nonselective media 
               
               
                 GGATGGACAGAACACATGAACTTACATGAATATCAGGCAAAACAACTTTTTGCCCGCTATGGCTTACCAGCACCGGT 
               
               
                 GGGTTATGCCTGTACTACTCCGCGCGAAGCAGAAGAAGCCGCTTCAAAAATCGGTGCCGGTCCGTGGGTAGTGAAAT 
               
               
                 GTCAGGTTCACGCTGGTGGCCGCGGTAAAGCGGGCGGTGTGAAAGTTGTAAACAGCAAAGAGGACATCCGTGCTTTT 
               
               
                 GCAGAAAACTGGCTGGGCAAGCGTCTGGTAACGTATCAAACAGATGCCAATGGCCAACCGGTTAACCAGATTCTGGT 
               
               
                 TGAAGCAGCGACCGATATCGCTAAAGAGCTGTATCTCGGTGCCGTTGTTGACCGTAGTTCCCGTCGTGTGGTCTTTA 
               
               
                 TGGCCTCCACCGAAGGCGGCGTGGAAATCGAAAAAGTGGCGGAAGAAACTCCGCACCTGATCCATAAAGTTGCGCTT 
               
               
                 GATCCGCTGACTGGCCCGATGCCGTATCAGGGACGCGAGCTGGCGTTCAAACTGGGTCTGGAAGGTAAACTGGTTCA 
               
               
                 GCAGTTCACCAAAATCTTCATGGGCCTGGCGACCATTTTCCTGGAGCGCGACCTGGCGTTGATCGAAATCAACCCGC 
               
               
                 TGGTCATCACCAAACAGGGCGATCTGATTTGCCTCGACGGCAAACTGGGCGCTGACGGCAACGCACTGTTCCGCCAG 
               
               
                 CCTGATCTGCGCGAAATGCGTGACCAGTCGCAGGAAGATCCGCGTGAAGCACAGGCTGCACAGTGGGAACTGAACTA 
               
               
                 CGTTGCGCTGGACGGTAACATCGGTTGTATGGTTAACGGCGCAGGTCTGGCGATGGGTACGATGGACATCGTTAAAC 
               
               
                 TGCACGGCGGCGAACCGGCTAACTTCCTTGACGTTGGCGGCGGCGCAACCAAAGAACGTGTAACCGAAGCGTTCAAA 
               
               
                 ATCATCCTCTCTGACGACAAAGTGAAAGCCGTTCTGGTTAACATCTTCGGCGGTATCGTTCGTTGCGACCTGATCGC 
               
               
                 TGACGGTATCATCGGCGCGGTAGCAGAAGTGGGTGTTAACGTACCGGTCGTGGTACGTCTGGAAGGTAACAACGCCG 
               
               
                 AACTCGGCGCGAAGAAACTGGCTGACAGCGGCCTGAATATTATTGCAGCAAAAGGTCTGACGGATGCAGCTCAGCAG 
               
               
                 GTTGTTGCCGCAGTGGAGGGGAAATAAAGGTCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTT 
               
               
                 TCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCG 
               
               
                 TTTATAGCTTATGTCTTCTACTAGTAGCGGCCGCTGCAGTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTT 
               
               
                 TTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGG 
               
               
                 CTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAA 
               
               
                 CATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCACAGGCTCCGCC 
               
               
                 CCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCG 
               
               
                 TTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCC 
               
               
                 TTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGG 
               
               
                 GCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA 
               
               
                 AGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA 
               
               
                 GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTA 
               
               
                 CCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAG 
               
               
                 CAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAA 
               
               
                 CGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAAT 
               
               
                 GAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGCTCGAGTCCCGTCAAGTCAGCGTAATGC 
               
               
                 TCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTC 
               
               
                 ATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCA 
               
               
                 TAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGT 
               
               
                 CAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGCATT 
               
               
                 TCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCAT 
               
               
                 TCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACC 
               
               
                 GGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTT 
               
               
                 TTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCAT 
               
               
                 AAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAA 
               
               
                 ACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCAT 
               
               
                 TTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTGGAGCAAGACGTTTCCCGTTGAATATGGCT 
               
               
                 CATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAA 
               
               
                 TGTAACATCAGAGATTTTGAGACACAACGTGGCTTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGCTC 
               
               
                 GAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAAACTATAAAAATAGGCGTATCACGAGGCAGAAT 
               
               
                 TTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGCGGCCGCTTCTAGAGACTAGTGGAAG 
               
               
                 ACATGGAGTTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCTACTTGTTAGAAAAGAGAAGCACGTAATGAGTAG 
               
               
                 CGTAGATATTCTGGTCCCTGACCTGCCTGAATCCGTAGCCGATGCCACCGTCGCAACCTGGCATAAAAAACCCGGCG 
               
               
                 ACGCAGTCGTACGTGATGAAGTGCTGGTAGAAATCGAAACTGACAAAGTGGTACTGGAAGTACCGGCATCAGCAGAC 
               
               
                 GGCATTCTGGATGCGGTTCTGGAAGATGAAGGTACAACGGTAACGTCTCGTCAGATCCTTGGTCGCCTGCGTGAAGG 
               
               
                 CAACAGCGCCGGTAAAGAAACCAGCGCCAAATCTGAAGAGAAAGCGTCCACTCCGGCGCAACGCCAGCAGTCGTCTC 
               
               
                 TGGAAGAGCAAAACAACGATGCGTTAAGCCCGGCGATCCGTCGCCTGCTGGCTGAACACAATCTCGACGCCAGCGCC 
               
               
                 ATTAAAGGCACCGGTGTGGGTGGTCGTCTGACTCGTGAAGATGTGGAAAAACATCTGGCGAAAGCCCCGGCGAAAGA 
               
               
                 GTCTGCTCCGGCAGCGGCTGCTCCGGCGGCGCAACCGGCTCTGGCTGCACGTAGTGAAAAACGTGTCCCGATGACTC 
               
               
                 GCCTGCGTAAGCGTGTGGCAGAGCGTCTGCTGGAAGCGAAAAACTCCACCGCCATGCTGACCACGTTCAACGAAGTC 
               
               
                 AACTGCGTAAGCGTGTGGCAGAGCGTCTGCTGGAAGCGAAAAACTCCACCGCCATGCTGACCACGTTCAACGAAGTC 
               
               
                 TATATGAAGCCGATTATGGATCTGCGTAAGCAGTACGGTGAAGCGTTTGAAAAACGCCACGGCATCCGTCTGGGCTT 
               
               
                 ACGTGGTTTACCACAACTATTTCGACGTCAGCATGGCGGTTTCTACGCCGCGCGGCCTGGTGACGCCGGTTCTGCGT 
               
               
                 GATGTCGATACCCTCGGCATGGCAGACATCGAGAAGAAAATCAAAGAGCTGGCAGTCAAAGGCCGTGACGGCAAGCT 
               
               
                 GACCGTTGAAGATCTGACCGGTGGTAACTTCACCATCACCAACGGTGGTGTGTTCGGTTCCCTGATGTCTACGCCGA 
               
               
                 TCATCAACCCGCCGCAGAGCGCAATTCTGGGTATGCACGCTATCAAAGATCGTCCGATGGCGGTGAATGGTCAGGTT 
               
               
                 GAGATCCTGCCGATGATGTACCTGGCGCTGTCCTACGATCACCGTCTGATCGATGGTCGCGAATCCGTGGGCTTCCT 
               
               
                 GGTAACGATCAAAGAGTTGCTGGAAGATCCGACGCGTCTGCTGCTGGACGTGTAGTAGTTTAAGTTTCACCTGCACT 
               
               
                 GTAGACCGGATAAGGCATTATCGCCTTCTCCGGCAATTGAAGCCTGATGCGACGCTGACGCGTCTTATCAGGCCTAC 
               
               
                 GGGACCACCAATGTAGGTCGGATAAGGCGCAAGCGCCGCATCCGACAAGCGATGCCTGATGTGACGTTTAACGTGTC 
               
               
                 TTATCAGGCCTACGGGTGACCGACAATGCCCGGAAGCGATACGAAATATTCGGTCTACGGTTTAAAAGATAACGATT 
               
               
                 ACTGAA