PL228024B1 - Expression vector set - Google Patents

Description

The invention relates to a nucleic acid molecule encoding a promoter or linker region, a set of expression vectors, and a method of producing recombinant proteins with the aid of expression vectors, allowing for easy optimization of the expression of recombinant proteins.

Nucleic acid molecules used in the production of recombinant proteins are described, in particular those encoding promoter regions for the regulation of recombinant protein expression, a polylinker region used in classical cloning in plasmid vectors, containing specific sequences recognized by restriction enzymes and linker regions used in the modular synthesis of expression vectors. The present invention also discloses expression vectors containing the described nucleic acid molecules, bacterial cells containing said expression vectors, a method of producing expression vectors based on the described molecules, a method of performing expression assays using the described vectors, a method of determining a recombinant protein produced with the described vectors, including, in particular, by means of fluorescent labels or a method of releasing a recombinant protein using any enzyme recognizing the disclosed sequence.

State of the art

At present, recombinant proteins are widely used in various fields of science, biotechnology and pharmacy. The more important ones include basic research aimed at understanding the structure and properties of proteins, and research determining possible interactions with chemical molecules, which is crucial in the process of developing new drugs. Recombinant proteins are also used as so-called biopharmaceuticals, such as hormones, interferons, interlueukins or hematopoietic factors (1). Considering the constantly growing potential of the life-science industry, one should expect an increasing demand for high-quality protein preparations. Despite the existence of more technically advanced methods, laboratory scale protein production is usually carried out in a prokaryotic system, especially in Escherichia coli Gram-negative bacteria. This system is widely regarded as the fastest, easiest and most economically advantageous (2). Consequently, the production of recombinant proteins in these bacteria is commonly used for the early stages of analysis, which do not require taking into account the glycosylation profile, and for small-scale production (3).

E. coli bacteria are characterized by a dynamic growth, in properly selected conditions, cell division takes place every 20 minutes. This means that a few or a dozen or so hours is sufficient to reach the stationary phase of breeding. Rapid production of biomass allows to significantly shorten the production time of recombinant proteins (4). An additional advantage of E. coli is the fast and efficient transformation procedure that allows the introduction of the desired genetic construct into the bacterial cell (5).

The main disadvantage of the recombinant protein production process in E. coli is its chimeric nature. The efficiency and quality of production depends on the breeding conditions, the genetic context and, above all, the nature of the produced protein. Factors such as the composition of the media, the temperature, the aeration conditions, the duration of the bacterial culture or the conditions for the induction of protein expression are important in this process. In turn, the efficiency and effectiveness dictated by genetic factors depends on the genotype of the bacteria and the properties of the expression vector. Currently, there are over a dozen different strains of E. coli bacteria available on the market, among those dedicated to the production of recombinant proteins, the DH10B, TOP10 or BL21 strains can be mentioned. In addition to the genotype of bacteria, an important role is played by the selection of genetic elements used in the expression vector.

In the case of E. coli, genetic transformation is carried out using plasmids, which are short, autonomous replication-capable DNA molecules, independent of the main bacterial chromosome. Under laboratory conditions, the production of recombinant proteins uses modified plasmids containing an origin of replication, a gene for resistance to a selected antibiotic, a promoter region often also containing fusion tags, and protease recognition sites. For technical reasons, a polylinker containing restriction enzyme recognition sites is also added, which facilitates the molecular cloning procedure.

Origin of replication is responsible for maintaining the appropriate number of plasmid copies in the bacterial cell. On the one hand, the high number of plasmid copies promotes protein overproduction

Recombinant production, on the other hand, can lead to a strain on the metabolism of the cell, which in turn has a negative impact on the production efficiency. Commercially available plasmids contain several types of origins of replication, one of which pMBI present in the commonly used series of pET vectors provides from 15 to 60 copies of the plasmid in the cell (6). The modified version of the pMBI region present in the pUC series vectors increases the plasmid copy number to 500-700 per cell (7). Apart from them, there are also sites of origin of replication occurring in the natural environment, as is the case in pQE vectors (Qiagen). If there is a need for two plasmids in the cell, it is possible to use the p15A site present in the trowel and pad vectors providing a copy number of 10-15 (8, 9).

Due to the necessity to perform selection in the expression vectors, modules ensuring resistance to antibiotics are used. The most commonly used selection markers providing resistance to ampicillin, chloramphenicol, kanamycin and tetracycline. The most commonly used selection markers used in expression plasmids.

Two types of promoters are commonly used in expression vectors, i.e. sequences recognized by RNA polymerases, enzymes necessary for transcription. In other words, their action is crucial for the efficient expression of a protein. Constitutive promoters conditioning the constant expression of proteins in bacterial cells. In contrast, the second group inducible promoters are commonly used in systems for heterologous production of recombinant proteins. Their characteristic feature is a low level of expression in the conditions of no induction and a high level of expression in the presence of an inducer. In their case, it is necessary to include elements that regulate expression. This is especially needed when the proteins produced have a toxic effect on bacterial cells. A commonly used expression control solution is the use of lactose operon elements. Expression of a given protein under the control of the lactose operon repressor is forced by the addition of an IPTG inducer (isopropyl-3-D-1-thiogalactopyranoside), a chemical molecule resembling lactose.

Suboptimal activity of the promoter causes low efficiency of the protein production process, at the same time too strong activity may slow down the metabolism or cause problems with proper folding and protein solubility. The promoters present in the commercially available include the inducible phage T5 and T7 promoters (10, 11), the synthetic Trc promoter (12), the bacterial lac promoter or its modified version of lacUV5 (13, 14).

In addition to regulatory components, it is important to take into account the elements improving the solubility of the produced proteins, their subsequent detection and purification of the protein by chromium affinity atography. This is done by adding fusion tags, i.e. protein molecules or peptides located at the C or N terminus of the recombinant protein. One of the most commonly used fusion tags for purification purposes are the poly-histidine tag and the glutathione S-transferase. The poly-histidine tag allows for quick purification of the protein by the affinity method on a nickel bed (15). Glutathione transferase, which is a protein with a length of 223 amino acids and a molecular weight of 26 kDa, is used to purify proteins on the basis of glutathione affinity (16). Other used fusion tags are polyarginine or FLAG polypeptides as well as small proteins such as maltose binding proteins (17) which additionally prevent the appearance of inclusion bodies.

During the purification step, the fusion tags may be cleaved using proteases (18), in which case the fusion tag molecule must be separated from the target protein by a short sequence of amino acids recognized by proteases such as TEV, thrombin, factor Xa or Clean-Cut protease.

Additionally, the methods of expressing recombinant protein in E. coli can be divided into three types. The first one involves the production of recombinant protein in a form soluble in the cytoplasm of E. coli cells. The second is the expression of proteins in an insoluble form in the form of inclusion bodies. It is also possible to produce proteins into the periplasmic space. Protein production in the periplasmic space requires taking into account the appropriate signal sequence.

Since expression of transformed E. coli protein depends on many factors, it is imperative to establish optimal culture conditions to maximize production efficiency. Due to its complexity, the production process of recombinant proteins should be preceded by expression tests aimed at selecting such conditions of bacterial culture that the protein expression is as high as possible. Assuming that two different times are considered in the expression tests

With the culture, two different temperatures, four culture media and two different inducer concentrations, it is necessary to compare 32 culture conditions. While the selection of such conditions is feasible, the optimization of the genetic context is a much more complicated process and thus often neglected. Standard expression assays are based on the determination of the level of recombinant protein in the soluble fraction and in the insoluble fraction by SDS-PAGE electrophoresis. The presence of the protein can also be determined by means of antibodies that recognize the fusion tags like western blots or by enzyme immunoassay ELISA.

The available expression systems dedicated to E. coli usually consist of an expression vector with the necessary elements necessary to perform molecular cloning. Such kits often include ligase, reaction buffer, positive control reagents, primers, competent bacteria and transformation media. The products available on the market include three-frame reading sets such as FLAG-Shift Expression Vectors (Sigma-Aldrich). Alternatively, kits are available to obtain a recombinant expression vector molecule based on a ligase independent cloning system. An example of such a system is aLICator Ligation Independent Cloning and Expression System offered by Thermo Scientific. A number of procedures are also available in the literature to enable the preparation of a recombinant expression vector molecule without the use of a ligase (19-23). Some of them are offered in the form of commercial kits such as EZ Cloning (GenScript).

The object of the invention is to enable the achievement of genetic constructs in a single cloning method that ensure different genetic conditions for the production of recombinant proteins, and thus allow for an easy optimization of the expression conditions. Surprisingly, the object of the invention thus defined has been achieved in the present invention.

The essence of the invention

The invention relates to a nucleic acid molecule which codes for a promoter region determined by a sequence selected from Sequence No. 1-3;

Another object of the invention is a nucleic acid molecule encoding a linker region determined by a sequence selected from the sequence No. 4-12, preferably a polylinker region having the sequence No. 4.

The subject of the invention is a set of expression vectors characterized in that it consists of various expression vectors, each of the vectors comprising:

- an operably linked promoter region defined by a sequence selected from sequences No. 1 to 3 and a linker region determined by a sequence selected from sequences No. 4 to 12,

- a replication origin region defined by a sequence selected from sequences Nos. 13 to 15, the vectors comprising the set comprising all combinations of said promoter region variants with said replication origin variants.

Preferably, the expression vector further comprises a nucleic acid sequence encoding a reporter molecule, preferably a green fluorescent protein or a pink fluorescent protein.

Preferably, the expression vector is a linear molecule.

Preferably, the expression vector encodes a region corresponding to the WELQ amino acids recognized by the Clean-Cut protease;

Preferably, the transcription initiation site is located downstream of the sequence corresponding to the amino acids of WELQ.

Preferably, there is a sequence corresponding to the amino acids MIHHHHHH upstream of the WELQ sequence.

Preferably, the kit according to the invention comprises vectors with sequence Nos. 16 to 24. Preferably, the kit according to the invention comprises vectors with sequences Nos. 25 to 33.

Another object of the invention is a method of producing recombinant proteins, characterized in that E. coli cells are transformed with a set of expression vectors according to the invention as defined above, containing a sequence coding for a recombinant protein, the obtained transformants are cultured under predetermined culture conditions, and the expression level obtained for different transformants, and then the final production culture of the transformant strain giving the highest expression level is run and the recombinant protein is isolated from the culture broth.

PL 228 024 B1

Preferably, a Clean-Cut protease is used to release the recombinant protein.

Preferably, a Clean-Cut protease is used in the process of cleaving the fusion tag and releasing the N-terminus of the recombinant protein and / or the process of arbitrarily shaping the N-terminus of the recombinant protein.

Detailed Description of the Invention

The described system includes a number of innovative expression vectors containing unique nucleotide sequences corresponding to the promoter regions (sequences: 1-3), polylinker (sequence: 4), linkers for joining fragments of DNA modules (sequence: 5-12) and sites recognized by the protease Clean-Cut, which corresponds to the WELQ amino acids. Reported vectors available in two conformations, circular containing a polylinker and linear ended with 15 bp linkers, in the English literature referred to as the so-called overhangs.

The pBCM plasmids available in circular form are intended for classical molecular cloning using restriction enzymes and ligase. In this case, the target DNA fragment is inserted into the vector between the restriction sites contained in the polylinker. Linear forms, otherwise known as open expression vectors, are dedicated to ligase-independent cloning. Unlike the circular forms, they do not contain a polylinker, its site is taken by a target DNA fragment containing 15 nucleotide linker regions corresponding to the ends of the open vector. The linker modules were designed to allow the target insert to be joined with the vector molecule, the second version is dedicated to the LIC (ligation independent cloning) method carried out with the use of T4 phage DNA polymerase.

Figure 1 shows a schematic representation of the polylinker highlighting the restriction sites along with the position of the Clean-Cut protease recognition site. The vectors were developed in a minimalist version. This means that there are no additional sequences between the modules necessary for the correct operation of the vector and for cloning. The only exception is the 15-nucleotide linker sequences used in the synthesis of the vectors. The modules contained in vectors are:

• Antibiotic resistance module allowing the selection of transformants. It comes in two versions to provide resistance to ampicillin and kanamycin.

• A repressor synthesis module of the lactose operon present in each vector.

• The replication initiation module comes in three versions with a plasmid copy number of 10 to 15, 15 to 50 and 500 to 700 plasmid copies per cell.

• The promoter module has three versions corresponding to the modified BCMT5, BCMT7 and BCMTrc promoters.

• The polylinker module is present only in vectors intended for classical cloning using restriction enzymes and ligase.

Vectors are available as a mixture containing equimolar ratios of nine vectors differing in the combination of origins of replication (3x) and promoter regions (3x). Modules responsible for antibiotic resistance and lactose operon repressor production are present in each vector molecule. As a result, in a single cloning method, genetic constructs can be created that provide different genetic conditions for the production of recombined proteins. Thanks to this, during the transformation process, plasmids that differ in the type of the origin of replication and the strength of the promoter can enter the bacterial cell. Alternatively, a mixture of modules is available that will form a series of expression vectors during the ligation-independent cloning procedure. In such an experiment, the linker regions responsible for the formation of the complete vector molecule play a key role. The use of modules is known as the combinatorial approach.

Additionally, pBCM vectors are available in versions containing glutathione transferase (GST) or green fluorescent protein (GFP) contained in the promoter module. These elements are located above the target protein and separated by a Clean-Cut protease recognition site. Thanks to this, the resulting recombinant protein can be released by the Clean-Cut protease from the glutathione transferase protein or green fluorescence protein located at its N terminus.

For a series of vectors containing the green fluorescence protein, expression tests will be performed by measuring the fluorescence at the excitation wavelength and emission wavelength specific to the GFP protein of the E. coli culture. Recombinant proteins are produced in the pBCM system

PL 228 024 Β1 is linked with an attached green fluorescent protein, therefore the level of fluorescence directly corresponds to the level of recombinant protein in culture.

For a series of vectors containing glutathione transferase, expression tests should be performed by SDS-PAGE electrophoregram analysis of the soluble or insoluble fraction obtained for E. coli culture or further by western blot analysis with glutathione transferase specific antibodies.

The nucleic acid molecules represented by the sequences Nos. 1-3, corresponding to the promoter regions, are disclosed.

Preferably, expression of the target protein is operably linked to the promoter regions and.

Preferably, the promoter regions provide for stable or transient expression.

In addition, a nucleic acid molecule with sequence No. 4, corresponding to a polylinker region, is disclosed.

Preferably it is used in restriction enzyme and ligase cloning.

A particular embodiment of the invention are the nucleic acid molecules defined by the sequences Nos. 5-12, corresponding to the linker regions.

Preferably they are used in cloning using ligase independent methods.

Preferably, they are used for the synthesis of vector molecules in a combinatorial approach.

Preferably they contain a Clean-Cut protease recognition site.

Preferably, the target protein coding sequence is operably linked.

Preferably, T4 DNA polymerase is used in the cloning procedure.

Another embodiment of the invention is a nucleic acid molecule corresponding to the WELQ amino acids recognized by the Clean-Cut protease.

Preferably, the gene encoding glutathione transferase is present above the nucleotides corresponding to the amino acids of WELQ.

Preferably, the upstream group of proteins or peptides are those allowing the purification of the expression product by affinity chromatography.

Preferably, the gene encoding the GFP green fluorescent protein is located above the nucleotides corresponding to the WELQ amino acids.

Preferably, the group of fluorescent proteins also comprises CFP, RFP or YFP proteins.

Preferably, the transcription initiation site is located above the nucleotides corresponding to the amino acids of WELQ.

Preferably, there are nucleotides encoding isoleucine between the transcription initiation site and the nucleotides corresponding to the amino acids of WELQ.

Exemplary vectors of the invention, shown in the accompanying sequence listing, are listed in the table below.

vector promoter fusion tag number of copies marker sequence no pBCMIML T5 HIS -500 AMP 16 pBCM2ML T7 HIS -500 AMR 17 pBCM3ML TRC HIS -500 AMP 18 pBCM4ML T5 HIS -20-30 AMP 19 pBCMSML T7 HIS -20-30 AMP twenty pBCMBML TRC HIS -20-30 AMP 21 pBCM7ML T5 HIS -10 AMP 22 pBCMSML T7 HIS -10 AMP 23 pBCM9ML TRC HIS -10 AMP 24 pBCMIMLG T5 HIS / GST -500 AMP 25 pBCM2MLG T7 HIS / GST -500 AMP 26 pBCM3MLG TRC HIS / GST -500 AMP 27 PBCM4MLG T5 HIS / GST -20-30 AMP 28 pBCM5MLG T7 HIS / GST -20-30 AMP 29 PBCM6MLG TRC HIS / GST -20-30 AMP thirty pBCM7MLG T5 HIS / GST -10 AMP 31 pBCM3MLG T7 HIS / GST -10 AMP 32 pBCM9MLG TRC HIS / GST -10 AMP 33

PL 228 024 B1

Another embodiment of the invention is a bacterial cell containing the plasmid vector as defined above.

A further embodiment of the invention is a method for obtaining a recombinant protein in E. coli bacteria, characterized in that the genetic material constituting the plasmid vector as defined above is introduced into the host bacterial cell.

A further embodiment of the invention is the use of the nucleic acid molecules defined above for the construction of a vector which ensures the production of a recombinant protein in an E. coli bacterial cell.

Preferably, the obtained recombinant protein, which is a fusion or non-fusion protein, shows increased production efficiency.

Another embodiment of the invention is a method for determining the concentration of recombinant protein present in bacterial cells based on the measurement of fluorescence.

Preferably, a green fluorescent protein is used for determination.

Another embodiment of the invention is a method for selecting an E. coli bacterial clone containing the plasmid vector described above for the production of recombinant proteins based on the results of fluorescence measurements or other expression assays.

Another embodiment of the invention is a method of releasing the recombinant protein using the Clean-Cut protease.

Preferably, Clean-Cut protease is used for any N-terminus shaping of the recombinant protein.

In order to better explain the essence of the invention, it is also illustrated by the following examples, which, however, should not be equated with the full scope of the invention.

The presented examples demonstrate the production of human alcohol dehydrogenase (ADH1; M 12963.1) using the pBCM expression vector system. Alcohol dehydrogenase is a protein selected to demonstrate the effects of the invention.

P r z k ł a d I

Preparation of ADH1: pBCM constructs using restriction enzymes and ligase

Plasmid DNA containing the desired sequence provided by GenScript was used to amplify the gene encoding the alcohol dehydrogenase (ADH1) by PCR. The primers were designed to allow for the duplication of the gene sequence and to introduce cleavage sites for the restriction enzymes BamHI and XhoI:

• ADH1F: CTCGGATCCGATGCACTTGAGCAGG • ADH1R: GGGCTCGAGAAGAAGGAAGGTTTATTGGC

A PCR reaction was prepared with the composition 1x KOD Hot Start Master Mix (Merck Millipore), 6 µM of primer mix, 20 ng of plasmid DNA. The reaction had the following thermal profile: 95 ° C - 2 min .; 35x (95 ° C - 20 sec; 62 ° C - 10 sec; 70 ° C - 25 sec). The products of the PCR reaction were separated on an agarose gel in order to verify the correct course of the reaction. After positive verification, the PCR reaction was purified on DNA Sequencing Clean-up Kit silica columns (Zymo Research) according to the manufacturer's procedure. Subsequently, the PCR reaction products were subjected to restriction digestion with the enzymes BamHI, XhoI and DpnI (New England Biolabs) at 37 ° C for 60 minutes, the reaction buffers selected according to the manufacturer's instructions. After digestion, the reactions were re-purified on silica columns and analyzed on an agarose gel.

Meanwhile, the pBCM vector mixture was digested with enzymes BamHI, XhoI and HindIII (New England Biolabs) at 37 ° C for 60 minutes, the reaction buffers selected according to the manufacturer's instructions. After digestion, the reactions were re-purified on silica columns and analyzed on an agarose gel. Ligation reactions were performed with a mixture of the ADH1 insert and pBCM vectors in a 3: 1 molar ratio. The reaction was performed using the Quick Ligation kit (New Engalnd Biolabs) according to the manufacturer's instructions. The ligation mixture was used to transform E. coli strain BL21 (New England Biolabs) bacteria using the heat shock method. The bacteria were plated on an agar medium containing the appropriate antibiotic and incubated overnight at 37 ° C.

The next step was the isolation of plasmid DNA from the obtained bacterial colonies previously grown in an overnight culture. Isolation was performed with the Plasmid DNA purification kit (Zymo Research) according to the manufacturer's protocol. The obtained plasmids were analyzed on an agarose gel, the DNA concentration was determined spectrophotometrically by

Measure the absorbance at 260 and 280 nm. Then, the obtained clones were subjected to DNA sequencing with the use of dideoxy-derived nucleotides.

P r z x l a d II

Preparation of ADH1: pBCM constructs by ligase independent methods

Plasmid DNA containing the desired sequence provided by GenScript was used to amplify the gene encoding the alcohol dehydrogenase (ADH1) by PCR. The primers were designed to allow for the amplification of the gene sequence and to introduce the 15 nucleotide linker sequences.

ADH1F: TGGGAACTGCAGATGGATGCACTTGAGCAGG

ADH1 R: TCGGGGAAATTTCCCAAGAAGGAAGGTTTATTGGC

A PCR reaction was prepared with the composition 1x KOD Hot Start Master Mix (Merck Millipore), 6 µM of primer mix, 20 ng of plasmid DNA. The reaction had the following thermal profile: 95 ° C - 2 min .; 35x (95 ° C - 20 sec; 62 ° C - 10 sec; 70 ° C - 25 sec). The products of the PCR reaction were separated on an agarose gel in order to verify the correct course of the reaction. Then the PCR reaction products were subjected to restriction digestion with DpnI enzymes (New England Biolabs) at 37 ° C for 60 minutes, the enzyme was added directly to the PCR reaction mixture.

The PIPE transformation mixture (20) was prepared by mixing equal volumes of the PCR reaction mixture and the pBCM open expression vector solution, the mixture was incubated at room temperature for 30 minutes. Then it was used to transform E. coli strain BL21 (New England Biolabs) bacteria using the heat shock method. The bacteria were plated on an agar medium containing the appropriate antibiotic and incubated overnight at 37 ° C.

The next step was the isolation of plasmid DNA from the obtained bacterial colonies previously grown in an overnight culture. Isolation was performed with the Plasmid DNA purification kit (Zymo Research) according to the manufacturer's protocol.

The obtained plasmids were analyzed on an agarose gel, the DNA concentration was determined spectrophotometrically by measuring the absorbance at 260 and 280 nm. Then, the obtained clones were sequenced using dideoxy-derivative nucleotides.

P r x l a d III

Expression assays based on GFP fluorescence

The subject of expression tests are E. coli bacterial colonies obtained by cloning with the pBCM system. Green fluorescent colonies on LB culture medium containing 1 mM IPTG are analyzed. The basis of the expression assays is the assumption that recombinant protein and green fluorescent protein are produced as a fusion protein, so the level of fluorescence is dependent on the level of the recombinant protein. In addition, individual colonies may differ from one another by vectors that force the expression of recombinant proteins. The following expression assay variants are available:

1. Analyzed colonies are resuspended in 100 μl of LB culture medium containing the appropriate antibiotic and 1 mM IPTG in a 96-well titer plate. The samples are then incubated at 37 ° C with 200 rpm shaking for 1 hour. After the incubation period, an optical density measurement is performed at a wavelength of 600 nm and a fluorescence measurement at a wavelength specific to the green fluorescence protein. The results of the fluorescence measurement are normalized to the optical density value. Meanwhile, PCR tests are performed to verify that the clones contain the desired insert. The further steps in recombinant protein production are carried out on the sample or trials giving the most favorable results in standardized fluorescence measurement.

2. Analyzed colonies are resuspended in 100 μl of LB culture medium containing the appropriate antibiotic and analyzed for the presence of the desired insert using the PCR method. Positively verified colonies are resuspended in 2 ml of culture medium and incubated in a 12-well titer plate at 37 ° C with 200 rpm shaking until an optical density at 600 nm of 0.6-0.8 is reached. Upon reaching this value, the IPTG inducer is added at a concentration determined by the experimenter (e.g. 1 mM IPTG). Reading of fluorescence and optical density is 6-12 hours after induction. Fluorescence measurement results

PL 228 024 B1 are normalized to the optical density values. The further steps in recombinant protein production are carried out on the sample or trials giving the most favorable results in standardized fluorescence measurement.

Literature

1. S. Mitragotri, P. A. Burke, R. Langer, Overcoming the challenges in administering biopharmaceuticals: formulation and delivery strategies. Nat. Rev. Drug Discov. 13, 655-672 (2014).

2. G. L. Rosano, E. A. Ceccarelli, Recombinant protein expression in Escherichia coli: advances and challenges. Front. Microbiol. 5 (2014), doi: 10.3389 / fmicb.2014.00172.

3. S. Sahdev, S. K. Khattar, K. S. Saini, Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Moth. Cell. Biochem. 307, 249-264 (2008).

4. G. Sezonov, D. Joseleau-Petit, R. D'Ari, Escherichia coli Physiology in Luria-Bertani Broth. J. Bacteriol. 189, 8746-8749 (2007).

5. S. N. Cohen, A. C. Y. Chang, L. Hsu, Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA. Proc. Natl. Acad. Sci. 69, 2110-2114 (1972).

6. F. Bolivar et al., Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 2, 95-113 (1977).

7. N. P. Minton, Improved plasmid vectors for the isolation of translational lac gene fusions. Gene. 31, 269-273 (1984).

8. A. C. Chang, S. N. Cohen, Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134, 1141-1156 (1978).

9. L. M. Guzman, D. Belin, M. J. Carson, J. Beckwith, Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177,4121-4130 (1995).

10. R. Gentz, H. Bujard, Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5. J. Bacteriol. 164, 70-77 (1985).

11. F. W. Studier, B. A. Moffatt, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-130 (1986).

12. J. Brosius, M. Erfle, J. Storella, Spacing of the -10 and -35 regions in the tac promoter. Effect on its in vivo activity. J. Biol. Chem. 260, 3539-3541 (1985).

13. B. Gronenborn, Overproduction of phage Lambda repressor under control of the lac promotor of Escherichia coli. Mol Gen. Genet. MGG. 148, 243-250 (1976).

14. B. L. Wanner, R. Kodaira, F. C. Neidhardt, Physiological regulation of a decontrolled lac operon. J. Bacteriol. 130, 212-222 (1977).

15. K. Terpe, Overview of tag protein fusions: from molecular and biochemical fundamentalste commercial systems. Appl. Microbiol. Biotechnol. 60, 523-533 (2003).

16. D. B. Smith, K. S. Johnson, Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 67, 31-40 (1988).

17. R. B. Kapust, D. S. Waugh, Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci. Publ. Protein Soc. 8, 1668-1674 (1999).

18. D. S. Waugh, An overview of enzymatic reagents for the removal of affinity tags. Protein Expr. Purif. 80, 283-293 (2011).

19. W. H. Eschenfeldt, L. Stols, C. S. Millard, A. Joachimiak, M. I. Donnelly, A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins. Methods Mol. Biol. Clifton NJ. 498, 105-115 (2009).

20. H. E. Klock, S. A. Lesley, in High Throughput Protein Expression and Purification, S. A. Doyle, Ed. (Humana Press, 2009; http://link.springer.com/protocol/10.1007/978-1-59745-196-3_6), Methods in Molecular Biology, pp. 91 -103.

PL 228 024 B1

21. A. Matsumoto, T. Q. Itoh, Self-assembly cloning: a rapid construction method for recombinant molecules from multiple fragments. Bio Techniques. 51, 55-56 (2011).

22. J. Quan, J. Tian, Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways. PLoS ONE. 4, e6441 (2009).

23. Y. Zhang, U. Werling, W. Edelmann, SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res. 40, e55-e55 (2012).

PL 228 024 Β1

SEQLTXT.txt SEQUENCE LISTING <110> Selvita S.A.

<120> Expression vector set <130> PK./3420/RW <160> 33 <17O> Patentin version 3.5 <2l0> 1 <211> 147 <212> DNA <213> artificial <22O>

<223> BCMTS promoter <400> 1

ctggagatcc tgataaaatc atgaaaaatt tatttgcttt gtgagcggat aacaattata 60 atatgtggaa ttgtgagcgc tcacaattcc acataaggag gtaaaaaaat gatacatcat 120 catcatcatc attgggaact gcagatg 147 <210> 2 <211> 120 <212> DNA <213> artificial <22O> <223> BCMT7 promoter <400> 2 ctggagatcc tgatataata cgactcacta taggggtgtg gaattgtgag cgctcacaat 60 tccacataag gaggtaaaaa aatgatacat catcatcatc atcattggga actgcagatg 120 <210> 3 <211> 143 <212> DNA <213> artificial <220> <223> BCMTrc promoter <400> 3 ctggagatcc tgataattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat 60 gtggaattgt gagcgctcac aattccacat aaggaggtaa aaaaatgata catcatcatc 120 atcatcattg ggaactgcag atg 143 <210> 4 <211> 163 <212> DNA <213> artificial <220> <223> polylinker <400> 4 tgggaactgc agatgaattc gagctcggat ccaagatctt ctagacatat gatcgataag 60

PL 228 024 Β1

SEQLTXT.txt cttgatatcg gtaccaccat ggtcgacctc gagcccgggg ctagcataac cccttggggc 120 ctctaaacgg gtcttgaggg gttttttggg gaaatttccc ega 163

<210> 5 <211> 15 <212> GOUT <213> arti fi ci al <220> <223> OKI OH <400> 5 gggaaatttc cccga <210> 6 <211> 15 <212> GOUT <213> arti fi ci al <220> <223> OH ferry <400> 6 ctggagatcc tgata <210> 7 <211> 15 <212> GOUT <213> arti fi bodies <220> <223> RES OH <400> 7 tttgegatag gtaaa <210> 8 <211> 15 <212> GOUT <213> artifi of bodies <220> <223> Res oh <400> 8 tgggaactgc agatg <210> 9 <211> 18 <212> GOUT <213> arti fi bodies <220> <223> Expr OH LIC <400> 9 aggaccaagc aageagat

<210> 10 <211> 18

PL 228 024 Β1

SEQLTXT.txt <212> DNA <213> artificial <22 0>

<223> Ori OH LIC <400> 10 ctgcttctct ggtgctta 18 <210> 11 <211> 18 <212> DNA <213> artificial <220>

<223> Prom OH LIC <400> 11 ttgcttgtcc tgtgcgta 18 <210> 12 <211> 18 <212> DNA <213> artificial <220>

<223> Res oh lic <400> 12 gaacggcaag agcacaat 18 <210> 13 <211> 819 <212> DNA <213> artificial <22O>

<223> pUC ori <400> 13 gggaaatttc cccgactcat gaccaaaatc ccttaacgtg agttacgcgc gcgtcgttcc 60 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 120 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 180 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 240 atactgttct tctagtgtag ccgtagttag cccaccactt caagaactct gtagcaccgc 300 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 360 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 420 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 480 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 540 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 600 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 660 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 720 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 780

PL 228 024 Β1

SEQLTXT.txt ataaccgtat taccgccttt gagtgagctg ataccgctc 819 <210> 14 <211> 620 <212> DNA <213> artificial <220>

<223> PMBL on '<400> 14 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 60 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 120 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 180 agttaggcca ccacttcaag aactctgtag caccgcctac ataccrcgct ctgctaatcc 240 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 300 gatagttacc ggataaggcg cagcggtcgg getgaacggg gggttcgtgc acacagccca 360 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 420 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 480 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 540 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 600 ggaaaaacgc cagcaacgcg 620 <210> 15 <211> 837 <212> DNA <213> artificial < 22O>

<223> pl5A ori <400> 15 ttaataagat gatcttcttg agatcgtttt ggtctgcgcg taatctcttg ctctgaaaac 60 gaaaaaaccg ccttgcaggg cggtttttcg aaggttctct gagctaccaa ctctttgaac 120 cgaggtaact ggcttggagg agcgcagtca ccaaaacttg tcctttcagt ttagccttaa 180 ccggcgcatg acttcaagac taactcctct aaatcaatta ccagtggctg ctgccagtgg 240 tgcttttgca tgtctttccg ggttggactc aagacgatag ttaccggata aggcgcagcg 300 gtcggactga acggggggtt cgtgcataca gtccagcttg gagcgaactg cctacccgga 360 actgagtgtc aggcgtggaa tgagacaaac gcggccataa cagcggaatg acaccggtaa 420 accgaaaggc aggaacagga gagcgcacga gggagccgcc agggggaaac gcctggtatc 480 tttatagtcc tgtcgggttt cgccaccact gatttgagcg tcagatttcg tgatgcttgt 540 caggggggcg gagectatgg aaaaacggct ttgccgcggc cctctcactt ccctgttaag 600 tatcttcctg gcatcttcca ggaaatctcc gccccgttcg taagccattt ccgctcgccg 660 cagtcgaacg accgagcgta gcgagtcagt gagcgaggaa gcggaatata tcctgtatca 720

PL 228 024 Β1

SEQLTXT.txt eatattctgc tgacgcaccg gtgcagcctt ttttctcctg ccacatgaag cacttcactg 780 acaccctcat cagtgccaac atagtaagcc agtatacact ccgctagcgc tgaggtc 837 <21O> <220> <211> artificial> DNA <211> <211>

<223> pBCMlML <400> 16

ctggagatcc tgataaaatc atgaaaaatt tatttgcttt gtgagcggat aacaattata 60 atatgtggaa ttgtgagcgc tcacaattcc acataaggag gtaaaaaaat gtggaattgt 120 gagcgctcac aattccacat aaggaggtaa aaaaatgata catcatcatc atcatcattg 180 ggaactgcag atgaattcga gctcggatcc aagatcttct agacatatga tcgataagct 240 tgatatcggt accaccatgg tcgacctcga gcccggggct agcataaccc cttggggcct 300 ctaaacgggt cttgaggggt tttttgggga aatttccccg agggaaattt ccccgactca 360 tgaccaaaat cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt cagaccccgt 420 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 480 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 540 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 600 gccgtagtta gcccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 660 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 720 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 780 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 840 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 900 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 960 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 1020 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 1080 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 1140 tgagtgagct gataccgctc tttgcgatag gtaaaggcgc gccatcgaat ggcgcaaaac 1200 ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatggtgg 1260 tgaatgtgaa accagtaacg ttatacgatg tcgcagta tgccggtgtc tcttatcaga 1320 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg 1380 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca 1440 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa 1500 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg 1560 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg 1620

PL 228 024 Β1

SEQLTXT.txt

tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg 1680 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta 1740 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc 1800 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg 1860 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg 1920 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc 1980 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg 2040 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca 2100 gctcatgtta tatcccgccg tcaaccacca tcaaacagga ttttcgcctg ctggggcaaa 2160 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt 2220 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc 2280 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg 2340 ggcagtgagc gcaactttgc gataggtaaa tttttctaaa tacattcaaa tatgtatccg 2400 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 2460 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 2520 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 2580 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 2640 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 2700 gacgccgggc aagagcaact cggrcgccgc atacactatt ctcagaatga cttggttgag 2760 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 2820 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 2880 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 2940 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 3000 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 3060 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 3120 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 3180 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 3240 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 3300 attaagcatt ggtaactgtc 3320

<210> 17 <211> 3293 <212> DNA <213> artificial <22O>

<223> PBCM2ML

PL 228 024 Β1

5EQLTXT.txt

<400> 17 ctggagatcc tgatataata cgactcacta taggggtgtg gaattgtgag cgctcacaar 60 tccacataag gaggtaaaaa aatgtggaat tgtgagcgct cacaattcca cataaggagg 120 taaaaaaatg atacatcatc atcatcatca ttgggaactg cagatgaatt cgagctcgga ISO tccaagatct tctagacata tgatcgataa gcttgatatc ggtaccacca tggtcgacct 240 cgagcccggg gctagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgg 300 ggaaatttcc ccgagggaaa tttccccgac tcatgaccaa aatcccttaa cgtgagttac 360 gcgcgcgtcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 420 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 480 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 540 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttagcccacc acttcaagaa 600 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 660 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttacegg ataaggcgca 720 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 780 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 840 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 900 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 960 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 1020 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 1080 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctctttgcga 1140 taggtaaagg cgcgccatcg aatggcgcaa aacctttcgc ggtatggcat gatagcgccc 1200 ggaagagagt caattcaggg tggtgaatgg tggtgaatgt gaaaccagta acgttatacg 1260 atgtcgcaga gtatgccggt gtctcttatc agaecgtttc ccgcgtggtg aaccaggcca 1320 gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag ctgaattaca 1380 ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt ggcgttgcca 1440 cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa tcrcgcgccg 1500 atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc gaagcctgta 1560 aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatr aactatccgc 1620 tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg gcgttatttc 1680 ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa gacggtacgc 1740 gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg ttagcgggcc 1800 cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat ctcactcgca 1860 atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc ggttttcaac 1920 aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt gccaacgatc 1980 agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt ggtgcggata 2040

PL 228 024 Β1

SEQLTXT.txt

tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg ccgtcaacca 2100 ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg ctgcaactct 2160 ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg aaaagaaaaa 2220 ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc 2280 agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaactt tgcgataggt 2340 aaatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 2400 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 2460 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 2520 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 2580 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 2640 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 2700 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 2760 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 2820 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 2880 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 2940 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 3000 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 3060 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 3120 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 3180 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 3240 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtc 3293

<210> 13 <211> 3316 <212> DNA <213> artificial <220>

<223> pBCM3ML <400> 18 ctggagatcc tgataattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat 60 gtggaattgt gagcgctcac aattccacat aaggaggtaa aaaaatgtgg aattgtgagc 120 gctcacaatt ccacataagg aggtaaaaaa atgatacatc atcatcatca tcattgggaa 180 ctgcagatga attcgagctc ggatccaaga tcttctagac atatgatcga taagcttgat 240 atcggtacca ccatggtcga cctcgagccc ggggctagca taaccccttg gggcctctaa 300 acgggtcttg aggggttttt tggggaaatt tccccgaggg aaatttcccc gactcatgac 360 caaaatccct taacgtgagt tacgcgcgcg tcgttccact gagcgtcaga ccccgtagaa 420 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 480

PL 228 024 Β1

SEQLTXT.txt

aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 540 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg 600 tagttagccc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 660 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 720 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 780 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 840 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 900 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 960 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 1020 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 1080 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 1140 tgagctgata ccgctctttg cgataggtaa aggcgcgcca tcgaatggcg caaaaccttt 1200 cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa tggtggtgaa 1260 tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt 1320 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc 1380 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca 1440 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt 1500 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga 1560 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag 1620 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg 1680 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat 1740 tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca 1800 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgrctgc gtctggctgg 1860 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg 1920 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac 1980 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc 2040 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagacagctc 2100 atgttatatc ccgccgtcaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag 2160 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc 2220 cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg 2280 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca 2340 gtgagcgcaa ctttgcgata ggtaaatttt tctaaataca ttcaaatatg tatccgctca 2400 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc 2460 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc 2520

PL 228 024 Β1

SEQLTXT.txt acceagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt 2580 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt 2640 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg 2700 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact 2760 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg 2820 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga 2880 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg 2940 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa 3000 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac 3060 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc 3120 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca 3180 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga 3240 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta 3300 agcattggta actgtc 3316 <210> 19 <211> 3121 <212> DMA <213> artifi body <220>

<223> pBCM4ML <400> 19 ctggagatcc tgataaaatc atgaaaaatt tatttgcttt gtgagcggat aacaattata 60 atatgtggaa ttgtgagcgc tcacaattcc acataaggag gtaaaaaaat gtggaattgt 120 gagcgctcac aattccacat aaggaggtaa aaaaatgata catcatcatc atcatcattg 180 ggaactgcag atgaattcga gctcggatcc aagatcttct agacatatga tcgataagct 240 tgatatcggt accaccatgg tcgacctcga gcccggggct agcataaccc cttggggcct 300 ctaaacgggt cttgaggggt tttttgggga aatttccccg aagatcaaag gatcttcttg 360 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 420 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 480 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 540 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 600 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga egatagttac cggataaggc 660 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 720 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag 780 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 84 0 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 900

PL 228 024 Β1

5EQLTXT.txt

gcgtcgartt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg CCagcaacgc 960 gtttgcgata ggtaaaggcg cgccatcgaa tggcgcaaaa cctttcgcgg tatggcatga 1020 tagcgcccgg aagagagtca attcagggtg gtgaatggtg gtgaatgtga aaccagtaac 1080 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttCCC gcgtggtgaa 1140 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct 1200 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg 1260 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc 1320 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga 1380 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa 1440 ctatccgctg gatgaccagg atgccattgc tgtggaagct gCCtgcacta atgttccggc 1500 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga 1560 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt 1620 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct 1680 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtctgg 1740 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc 1800 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg 1860 tgcggatatc tcggtagtgg garacgacga taccgaagac agctcatgtt atatcccgcc 1920 gtcaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct 1980 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa 2040 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 2100 artaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaactttg 2160 cgataggtaa atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 2220 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 2280 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 2340 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 2400 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 2460 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 2520 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 2580 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 2640 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 2700 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 2760 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 2820 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 2880 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 2940

PL 228 024 Β1

SEQLTXT.txt

ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc 3000 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 3060 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 3120 c 3121 <210> 20 <211> 3094 <212> DNA <213> artificial <220> <223> pBCMSML <400> 20 ctggagatcc tgatataata cgactcacta taggggtgtg gaattgtgag cgctcacaat 60 tccacataag gaggtaaaaa aatgtggaat tgtgagcgct cacaattcca cataaggagg 120 taaaaaaatg atacatcatc atcatcatca ttgggaactg cagatgaatt cgagctcgga 180 tccaagatct tctagacata tgatcgataa gcttgatatc ggtaccacca tggtcgacct 240 cgagcccggg gctagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgg 300 ggaaatttcc ccgaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 360 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct 420 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct 480 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 540 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgr gtcttaccgg 600 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc 660 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 720 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg 780 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 840 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 900 ggggcggagc ctatggaaaa acgccagcaa cgcgtttgcg ataggtaaag gcgcgccatc 960 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg 1020 gtggtgaatg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg 1080 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac 1140 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca 1200 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca 1260 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt 1320 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct 1380 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat 1440 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac 1500

PL 228 024 Β1

SEQLTXT.txt

acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct 1560 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc 1620 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc 1680 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgcrgaa 1740 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat 1800 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga 1860 cgataccgaa gacagctcat gttatatccc gccgtcaacc accatcaaac aggattttcg 1920 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa 1980 gggcaatcag Ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac 2040 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 2100 ccgactggaa agcgggcagt gagcgcaact ttgcgatagg taaatttttc taaatacatt 2160 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 2220 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcarttt 2280 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 2340 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 2400 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 2460 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 2520 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 2580 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 2640 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 2700 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 2760 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 2820 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 2880 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 2940 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 3000 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 3060 taggtgcctc actgattaag cattggtaac tgtc 3094

<210> 21 <211> 3117 <212> DNA <213> artificial <220>

<22 3> pBCMfiML <400> 21 ctggagatcc tgataattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat 60 gtggaattgt gagcgctcac aattccacat aaggaggtaa aaaaatgtgg aattgtgagc 120

PL 228 024 Β1

SEQLTXT.txt

gctcacaatt ccacataagg aggtaaaaaa atgatacatc atcatcatca tcattgggaa 180 ctgcagatga attcgagctc ggatccaaga tcttctagac atatgatcga taagcttgat 240 atcggtacca ccatggtcga cctcgagccc ggggctagca taaccccttg gggcctaa 300 acgggtcttg aggggttttt tggggaaatt tccccgaaga tcaaaggatc ttcttgagat 360 ccttrttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 420 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 480 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 540 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 600 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 660 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 720 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 7S0 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 840 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 900 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcgttt 960 gcgataggta aaggcgcgcc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc 1020 gcccggaaga gagtcaattc agggtggtga atggtggtga atgtgaaacc agtaacgtta 1080 tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag 1140 gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat 1200 tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt 1260 gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc 1320 gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc 1380 tgtaaagcgg cggtgcacaa tCttCtcgcg caacgcgtca gtgggctgat cattaactat 1440 ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta 1500 tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt 1560 acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg 1620 ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact 1680 cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt 1740 caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac 1800 gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg 1860 gatatctcgg tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtca 1920 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa 1980 ctctctcagg gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga 2040 aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 2100 atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca actttgcgat 2160

PL 228 024 Β1

SEQLTXT.txt

aggtaaattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 2220 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 2280 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 2340 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 2400 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 2460 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 2520 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 2580 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 2640 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 2700 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 2760 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 2820 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 2880 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 2940 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 3000 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 3060 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtc 3117

<210> 22 <211> 3338 <212> DNA <213> artificals <220>

<223> pBCM7ML <400> 22 ctggagatcc tgataaaatc atgaaaaatt tatttgcttt gtgagcggat aacaattata 60 atatgtggaa ttgtgagcgc tcacaattcc acataaggag gtaaaaaaat gtggaattgt 120 gagcgctcac aattccacat aaggaggtaa aaaaatgata catcatcatc atcatcattg 180 ggaactgcag atgaattcga gctcggatcc aagatcttct agacatatga tcgataagct 240 tgatatcggt accaccatgg tcgacctcga gcccggggct agcataaccc cttggggcct 300 ctaaacgggt cttgaggggt tttttgggga aatttccccg attaataaga tgatcttctt 360 gagatcgttt tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc gccttgcagg 420 gcggtttttc gaaggttctc tgagctacca actctttgaa ccgaggtaac tggcttggag 480 gagcgcagtc accaaaactt gtcctttcag tttagcctta accggcgcat gacttcaaga 540 ctaactcctc taaatcaatt accagtggct gctgccagtg gtgcttttgc atgtctttcc 600 gggttggact caagacgata gttaccggat aaggcgcagc ggtcggactg aacggggggt 660 tcgtgcatac agtccagctt ggagcgaact gcctacccgg aactgagtgt caggcgtgga 720 atgagacaaa cgcggccata acagcggaat gacaccggta aaccgaaagg caggaacagg 780

PL 228 024 Β1

SEQLTXT.txt

agagcgcacg agggagccgc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 840 tcgccaccac tgatttgagc gtcagatttc gtgatgcttg tcaggggggc ggagcctatg 900 gaaaaacggc tttgccgcgg ccctctcact tccctgttaa gtatcttcct ggcatcttcc 960 aggaaatctc Cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac gaccgagcgt 1020 agcgagtcag tgagcgagga agcggaatat atcctgtatc acatattctg ctgacgcacc 1080 ggtgcagcct tttttctcct gccacatgaa gcacttcact gacaccctca tcagtgccaa 1140 catagtaagc cagtatacac tccgctagcg ctgaggtctt tgcgataggt aaaggcgcgc 1200 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt 1260 cagggtggtg aatggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg 1320 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga 1380 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg 1440 cacaacact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc 1500 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca 1560 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca 1620 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg 1680 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc 1740 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc 1800 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct 1860 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga 1920 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc 1980 tgaatgaggg carcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg 2040 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat 2100 acgacgatac cgaagacagc tcatgttata tcccgccgtc aaccaccatc aaacaggatt 2160 ttcgcctget ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg 2220 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca 2280 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg 2340 tttcccgact ggaaagcggg cagtgagcgc aactttgcga taggtaaatt tttctaaata 2400 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 2460 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 2520 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 2580 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 2640 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 2700 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 2760 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 2820

PL 228 024 Β1

SEQLTXT.txt gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 2880 ctgacaacga tcggaggacc gaaggagcta accgcrtttt tgcacaacat gggggatcat 2940 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 3000 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 3060 ettactetag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 3120 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 3180 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 3240 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 3300 gagataggtg cctcactgat taagcattgg taactgtc 3338 <210> 23 <211> 3311 <2I2> <220>> artificial

<223> pBCM8ML <4Q0> 23

ctggagatcc tgatataata cgactcacta taggggtgtg gaattgtgag cgctcacaat 60 tccacataag gaggtaaaaa aatgtggaat tgtgagcgct cacaattcca cataaggagg 120 taaaaaaatg atacatcatc atcatcatca ttgggaactg cagatgaatt cgagctcgga 180 tccaagatct tctagacata tgatcgataa gcttgatatc ggtaccacca tggtcgacct 240 cgagcccggg gctagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgg 300 ggaaatttcc ccgattaata agatgatctt cttgagatcg ttttggtctg cgcgtaatct 360 cttgctctga aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt ctctgagcta 420 ccaactcttt gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa cttgtccttt 480 cagtttagcc ttaaccggcg catgacttca agactaactc ctctaaatca attaccagtg 540 gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg actcaagacg atagttaccg 600 gataaggcgc agcggtcgga ctgaaegggg ggttcgtgca tacagtccag cttggagcga 660 actgcctacc cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc ataacagcgg 720 aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc acgagggagc cgccaggggg 780 aaacgcctgg tatctttata gtcctgtcgg grttcgccac cactgatttg agcgtcagat 840 ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac ggctttgccg cggccctctc 900 acttccctgt taagtatctt cetggcatct tccaggaaat ctccgccccg ttcgtaagcc 960 atttccgctc gccgcagtcg aacgaccgag cgtagcgagt cagtgagcga ggaagcggaa 1020 tatatcctgt atcacatatt ctgctgacgc accggtgcag ccttttttct cctgccacat 1080 gaagcacttc actgacaccc tcatcagtgc caacatagta agccagtata cactccgcta 1140 gcgctgaggt ctttgcgata ggtaaaggcg cgccatcgaa tggcgcaaaa cctttcgcgg 1200

PL 228 024 Β1

SEQLTXT.txt

tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatggtg gtgaatgtga 1260 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc 1320 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga 1380 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt 1440 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg 1500 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa 1560 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc 1620 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta 1680 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct 1740 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa 1800 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc 1860 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg 1920 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga 1980 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc 2040 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt 2100 atatcccgcc gtcaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg 2160 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct 2220 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt 2280 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 2340 cgcaactttg cgataggtaa atttttctaa atacattcaa atatgtatcc gctcatgaga 2400 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 2460 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 2520 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 2580 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 2640 atgargagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 2700 caagagcaac tcggtcgccg Catacactat tctcagaatg acttggttga gtactcacca 2760 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 2820 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 2880 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 2940 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 3000 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 3060 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 3120 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 3180 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 3240

PL 228 024 Β1

5EQLTXT.txt gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 3300 tggtaactgt c 3311 <210> 24 <211> 3334 <212> DNA <213> artificial <220>

<223> PBCM9ML <400> 24

ctggagatcc tgataattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat 60 gtggaattgt gagcgctcac aattccacat aaggaggtaa aaaaatgtgg aattgtgagc 120 gctcacaatt ccacataagg aggtaaaaaa atgatacatc atcatcatca tcattgggaa 180 ctgcagatga attcgagetc ggatccaaga tcttctagac atatgatcga taagcttgat 240 atcggtacca ccatggtcga cctcgagccc ggggctagca taaccccttg gggcctaa 300 acgggtcttg aggggttttt tggggaaatt tccccgatta ataagatgat cttcttgaga 360 tcgttttggt ctgcgcgtaa tctcttgctc tgaaaacgaa aaaaccgcct tgcagggcgg 420 tttttcgaag gttctctgag ctaccaactc tttgaaccga ggtaactggc ttggaggagc 480 gcagtcacca aaacttgtcc tttcagttta gccttaaccg gcgcatgact tcaagactaa 540 ctcctaaa tcaattacca gtggctgctg ccagtggtgc ttttgcatgt ctttccgggt 600 tggactcaag acgatagtta ccggataagg cgcagcggtc ggactgaacg gggggttcgt 660 gcatacagtc cagcttggag cgaactgcct acccggaact gagtgtcagg cgtggaatga 720 gacaaacgcg gccataacag cggaatgaca ccggtaaacc gaaaggcagg aacaggagag 780 cgcacgaggg agccgccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 840 eaccactgat ttgagcgtca gatttcgtga tgcttgtcag gggggcggag cctatggaaa 900 aacggctttg ccgcggccct ctcacttccc tgttaagtat cttcctggca tcttccagga 960 aatctccgcc ccgttcgtaa gccatttccg ctcgccgcag tcgaacgacc gagcgtagcg 1020 agtcagtgag cgaggaagcg gaatatatcc tgtatcacat attctgctga cgcaccggtg 1080 cagccttttt tctcctgcca catgaagcac ttcactgaca ccctcatcag tgccaacata 1140 gtaagccagt atacactccg ctagcgctga ggtctttgcg ataggtaaag gcgcgccatc 1200 gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg 1260 gtggtgaatg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg 1320 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac 1380 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca 1440 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca 1500 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt 1560 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct 1620

PL 228 024 Β1

SEQLTXT.txt

tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat 1680 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac 1740 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct 1800 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc 1860 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc 1920 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa 1980 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat 2040 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga 2100 cgataccgaa gacagctcat gttatatccc gccgtcaacc accatcaaac aggattttcg 2160 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa 2220 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac 2280 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc 2340 ccgactggaa agcgggcagt gagcgcaact ttgcgatagg taaatttttc taaatacatt 2400 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 2460 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 2520 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 2580 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 2640 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 2700 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 2760 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 2820 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 2880 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 2940 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 3000 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 3060 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 3120 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 3180 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtategtag 3240 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 3300 taggtgcctc actgattaag cattggtaac tgtc 3334

<210> 25 <211> 4034 <212> DNA <213> artificial <220>

<223> pBCMlMLG

PL 228 024 Β1

SEQLTXT.tXt

<400> 25 ctggagatcc tgataaaatc atgaaaaatt tatttgcttt gtgagcggat aacaattata 60 atatgtggaa ttgtgagcgc tcacaartcc acataaggag gtaaaaaaat gtggaattgt 120 gagcgctcac aattccacat aaggaggtaa aaaaatgata catcarcatc atcatcatat ISO gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt 240 ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg 300 gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg 360 tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat 420 gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga 480 tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga 540 ttttcttagc aagctaccrg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac 600 atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt 660 tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa 720 acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg 780 gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct 840 ggttccgcgt ggatccccag gaattcccgg gtcgactcga gcggccgcat cgtgggaact 900 gcagatgaat tcgagctcgg atccaagatc ttctagacat atgatcgata agcttgatat 960 cggtaccacc atggtcgacc tcgagcccgg ggctagcata accccttggg gcctctaaac 1020 gggtcttgag gggttttttg gggaaatttc cccgagggaa atttccccga ctcatgacca 1080 aaatccctta acgtgagtta cgcgcgcgtc gttccactga gcgtcagacc ccgtagaaaa 1140 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 1200 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 1260 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 1320 gttagcccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 1380 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 1440 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 1500 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 1560 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 1620 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 1680 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 1740 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 1800 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 1860 agctgatacc gctctttgcg ataggtaaag gcgcgccatc gaatggcgca aaacctttcg 1920 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg gtggtgaatg 1980 tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt 2040

PL 228 024 Β1

SEQLTXT.txt

cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg 2100 cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt 2160 cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg 2220 cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac 2280 gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg 2340 ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca 2400 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt 2460 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc 2520 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct 2580 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga 2640 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg 2700 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg 2760 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat 2820 gttatatccc gccgtcaacc accatcaaac aggattttcg cctgctgggg caaaccagcg 2880 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg 2940 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg 3000 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt 3060 gagcgcaact trgcgatagg taaatttttc taaatacatt caaatatgta tccgctcatg 3120 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 3180 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 3240 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 3300 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 3360 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 3420 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 3480 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 3540 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 3600 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 3660 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 3720 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 3750 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 3840 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 3900 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 3960 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 4020

cattggtaac tgtc

4034

PL 228 024 Β1

SEQLTXT.tXt <210> 26 <211> 4007 <212> DNA <213> artificial <220>

<223> pBCM2MLG <400> 26

ctggagatcc tgatataata cgactcacta taggggtgtg gaattgtgag cgctcacaat 60 tccacataag gaggtaaaaa aatgtggaat tgtgagcgct cacaattcca cataaggagg 120 taaaaaaatg atacatcatc atcatcatca tatgtcccct atactaggtt attggaaaat 180 taagggcctt gtgcaaccca ctcgacttct tttggaatat cttgaagaaa aatatgaaga 240 gcatttgtat gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt 300 ggagtttccc aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc 360 catcatacgt tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc 420 agagatttca atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc 480 atatagtaaa gactttgaaa ctctcaaagt tgattttctt agcaagctac ctgaaatgct 540 gaaaatgttc gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca 600 tcctgacttc atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct 660 ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga 720 taagtacttg aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt 780 tggtggtggc gaccatcctc caaaatcgga tctggttccg cgtggatccc part ci a ci L L part 840 cgggtcgact cgagcggccg catcgtggga actgcagatg aattcgagct cggatccaag 900 atcttctaga catatgatcg ataagcttga tatcggtacc accatggtcg acctcgagcc 960 cggggctagc ataacccctt ggggccta aacgggtctt gaggggtttt ttggggaaat 1020 ttccccgagg gaaatttccc cgactcatga ccaaaatccc ttaacgtgag ttacgcgcgc 1080 gtcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 1140 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 1200 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 1260 gataccaaat actgttcttc tagtgtagcc gtagttagcc caccacttca agaactctgt 1320 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 1380 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 1440 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 1500 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 1560 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 1620 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 1680 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 1740

PL 228 024 Β1

SEQLTXT.txt

acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 1800 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcttt gcgataggta 1860 aaggcgcgcc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga 1920 gagtcaattc agggtggtga atggtggtga atgtgaaacc agtaacgtta tacgatgtcg 1980 cagagtatgc cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg 2040 tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca 2100 accgcgtggc acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca 2160 gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac 2220 tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg 2280 cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg 2340 accaggatgc cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg 2400 tctctgacca gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg 2460 gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa 2520 gttctgtctc ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa 2580 ttcagccgat agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca 2640 tgcaaatgct gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg 2700 cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gatatctcgg 2760 tagtgggata cgacgatacc gaagacagct catgttatat cccgccgtca accaccatca 2820 aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg 2880 gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc 2940 tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg 3000 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca actttgcgat aggtaaattt 3060 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 3120 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt 3180 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat 3240 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 3300 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 3360 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 3420 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat 3480 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 3540 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 3600 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 3660 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 3720 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa 3780

PL 228 024 Β1

SEQLTXT.txt gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct 3840 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc 3900 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 3960 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtc 4007 <210> 27 <211> 4030

<212> DNA <213> artificals <220> <223> pBCM3MLG <400> 27 ctggagatcc tgataattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat 60 gtggaattgt gagcgctcac aattccacat aaggaggtaa aaaaatgtgg aattgtgagc 120 gctcacaatt ccacataagg aggtaaaaaa atgatacatc atcatcatca tcatatgtcc 180 cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact tcttttggaa 240 tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga taaatggcga 300 aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat tgatggtgat 360 gttaaattaa cacagtctat ggccatcata cgttatatag ctgacaagca caacatgttg 420 ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt tttggatatt 480 agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa agttgatttt 540 cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca taaaacatat 600 ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct tgatgttgtt 660 ttatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt 720 attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat agcatggcct 780 ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc ggatctggtt 840 ccgcgtggat ccccaggaat tcccgggtcg actcgagcgg ccgcatcgtg ggaactgcag 900 atgaattcga gctcggatcc aagatcttct agacatatga tcgataagct tgatatcggt 960 accaccatgg tcgacctcga gcccggggct agcataaccc cttggggcct ctaaacgggt 1020 cttgaggggt tttttgggga aatttccccg agggaaattt ccccgactca tgaccaaaat 1080 cccttaacgt gagttacgcg cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc 1140 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 1200 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 1260 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 1320 gcccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 1380 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 1440 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 1500

PL 228 024 Β1

SEQLTXT.txt

gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 1560 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 1620 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 1680 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 1740 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 1800 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 1860 gataccgctc tttgcgatag gtaaaggcgc gccatcgaat ggcgcaaaac ctttcgcggt 1920 atggcatgat agcgcccgga agagagtcaa ttcagggtgg tgaatggtgg tgaatgtgaa 1980 accagtaacg ttatacgatg tcgcagta tgccggtgtc tcttatcaga ccgtttcccg 2040 cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat 2100 ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt 2160 gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc 2220 gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag 2280 cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct 2340 gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa 2400 tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc 2460 ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat 2520 cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca 2580 taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc 2640 catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat 2700 gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct 2760 gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca gctcatgtta 2820 tatcccgccg tcaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga 2880 ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc 2940 actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt 3000 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 3060 gcaactttgc gataggtaaa tttttctaaa tacattcaaa tatgtatccg ctcatgagac 3120 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 3180 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 3240 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 3300 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 3360 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 3420 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 3480 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 3540

PL 228 024 Β1

SEQLTXT.txt ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 3600 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 3660 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 3720 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 3780 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 3840 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 3900 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 3960 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 4020 ggtaactgtc 4030 <210> 28 <211> 3835 <212> DNA <213> artificial <22O>

<223> pBCM4MLG <400> 28 ctggagatcc tgataaaatc atgaaaaatt tatttgcttt gtgagcggat aacaattata 60 atatgtggaa ttgtgagcgc tcacaattcc acataaggag gtaaaaaaat gtggaattgt 120 gagcgctcac aattccacat aaggaggtaa aaaaatgata catcatcatc atcatcatat 180 gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt 240 ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg 300 gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg 360 tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat 420 gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga 480 tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga 540 ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac 600 atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt 660 tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa 720 acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg 780 gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct 8 40 ggttccgcgt ggatccccag gaattcccgg gtcgactcga gcggccgcat cgtgggaact 900 gcagatgaat tcgagctcgg atccaagatc ttctagacat atgatcgata agcttgatat 960 cggtaccacc atggtcgacc tcgagcccgg ggctagcata accccttggg gcctctaaac 1020 gggtcttgag gggttttttg gggaaatttc cccgaagatc aaaggatctt cttgagatcc 1080 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 1140 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 1200

PL 228 024 Β1

SEQLTXT.txt

gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 1260 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 1320 cgataagtcg tgtcttaccg ggttgaactc aagacgatag ttaccggata aggcgcagcg 1380 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 1440 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 1500 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 1560 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 1620 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcgtttgc 1680 gataggtaaa ggcgcgccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc 1740 ccggaagaga gtcaattcag ggtggtgaat ggtggtgaat gtgaaaccag taacgttata 1800 cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc 1860 cagccacgtt trtgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta 1920 cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc 1980 cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc 2040 cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg 2100 taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc 2160 gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt 2220 tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg aagacggtac 2280 gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg 2340 cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg 2400 caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca 2460 acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga 2520 tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga 2580 tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc cgccgtcaac 2640 caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact 2700 ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcaetgg tgaaaagaaa 2760 aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 2820 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac tttgcgatag 2880 gtaaattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 2940 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 3000 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 3060 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 3120 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 3180 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 3240

PL 228 024 Β1

5EQLTXT.txt

gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 3300 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 3360 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 3420 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 3480 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 3540 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 3600 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 3660 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 3720 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 3780 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtc 3835

<210> 29 <211> 3808 <212> DNA <213> artificial <220>

<223> PBCM5MLG <400> 29

ctggagatcc tgatataata cgactcacta taggggtgtg gaattgtgag cgctcacaat 60 tecacataag gaggtaaaaa aatgtggaat tgtgagcgct cacaattcca cataaggagg 120 taaaaaaatg atacatcatc atcatcatca tatgtcccct atactaggtt attggaaaat 180 taagggcctt gtgcaaccca ctcgacttct tttggaatat cttgaagaaa aatatgaaga 240 gcatttgtat gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt 300 ggagtttccc aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc 360 catcatacgt tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc 420 agagatttca atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc 480 atatagtaaa gactttgaaa ctctcaaagt tgattttctt agcaagctac ctgaaatgct 540 gaaaatgttc gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca 600 tcctgacttc atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct 660 ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga 720 taagtacttg aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt 780 tggtggtggc gaccatcctc caaaatcgga tctggttccg cgtggatccc caggaattcc 840 cgggtcgact cgagcggccg catcgtggga actgcagatg aattcgagct cggatccaag 900 atcttctaga catatgatcg ataagcttga tatcggtacc accatggtcg acctcgagcc 960 cggggctagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttggggaaat 1020 ttccccgaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 1080 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 1140

PL 228 024 Β1

SEQLTXT.txt

tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 1200 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 1260 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 1320 ctcaagacga tagttacegg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 1380 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 1440 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 1500 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 1560 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 1620 gagcctatgg aaaaacgcca gcaacgcgtt tgcgataggt aaaggcgcgc catcgaatgg 1680 cgeaaaacct ttcgcggtat ggeatgatag cgcccggaag agagtcaatt cagggtggtg 1740 aatggtggtg aatgtgaaac cagtaacgtt ataegatgtc gcagagtatg ccggtgtctc 1800 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga 1860 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacact 1920 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc 1980 gtcgcaaatt gtegeggega ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt 2040 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc 2100 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt 2160 ggaagctgcc tgcactaatg ttccggcgtt atttettgat gtctctgacc agacacccat 2220 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc 2280 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct 2340 gcgtctggct ggctggcata aatatctcac tcgcaatcaa atteageega tagcggaacg 2400 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg 2460 catcgttccc actgcgatgc tggttgccaa egateagatg gcgctgggcg caatgcgcgc 2520 cattaccgag tccgggctgc gcgttggtgc ggatateteg gtagtgggat acgacgatac 2580 cgaagacagc teatgttata tcccgccgtc aaccaccatc aaacaggatt ttcgcctgct 2640 ggggcaaacc agcgtggacc gettgetge actctctcag ggccaggcgg tgaagggcaa 2700 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac 2760 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 2820 ggaaagcggg cagtgagcgc aactttgcga taggtaaatt tttctaaata cattcaaata 2880 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 2940 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 3000 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 3060 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 3120 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 3180

PL 228 024 Β1

SEQLTXT.txt cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 3240 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 3300 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 3360 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 3420 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 3480 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 3540 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 3600 gctcggccct tccggctggc tggtttattg ctgataaate tggagccggt gagcgtgggt 3660 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 3720 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 3780 cctcactgat taagcattgg taactgtc 3808 <210> 30 <211> 3831 <212> DNA <213> arfificial <220>

<223> pBCM6MLG <400> 30

ctggagatcc tgataattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat 60 gtggaattgt gagcgctcac aattccacat aaggaggtaa aaaaatgtgg aattgtgagc 120 gctcacaatt ccacataagg aggtaaaaaa atgatacatc atcatcatca tcatatgtcc 180 cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact tcttttggaa 240 tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga taaatggcga 300 aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat tgatggtgat 360 gttaaattaa cacagtctat ggccatcata cgttatatag ctgacaagca caacatgttg 420 ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt tttggatatt 480 agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa agttgatttt 540 cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca taaaacatat 600 ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct tgatgttgtt 660 ttatacatgg acccaatgtg cctggatgcg ttcceaaaat tagtttgttt taaaaaacgt 720 attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat agcatggcct 780 ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc ggatctggtt 840 ccgcgtggat ccccaggaat tcccgggtcg actcgagcgg ccgcatcgtg ggaactgcag 900 atgaattcga gctcggatcc aagatcttct agacatatga tcgataagct tgatatcggt 960 accaccatgg tcgacctcga gcccggggct agcataaccc cttggggcct ctaaacgggt 1020 cttgaggggt tttttgggga aatttccccg aagatcaaag gatcttcttg agatcctttt 1080

PL 228 024 Β1

SEQLTXT.txt

tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 1140 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 1200 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 1260 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 1320 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 1380 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 1440 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 1500 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 1560 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 1620 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc gtttgcgata 1680 ggtaaaggcg cgccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg 1740 aagagagtca attcagggtg gtgaatggtg gtgaatgtga aaccagtaac gttatacgat 1800 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc 1860 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt 1920 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc 1980 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat 2040 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa 2100 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg 2160 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt 2220 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga 2280 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca 2340 ttaagttctg tctcggcgcg tctgcgtctg g ct g g ct g g c ataaatatct cactcgcaat 2400 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa 2460 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag 2520 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc 2580 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gtcaaccacc 2640 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct 2700 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc 2760 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 2820 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaactttg cgataggtaa 2880 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 2940 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 3000 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 3060 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 3120

PL 228 024 Β1

SEQLTXT.tXt

taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 3180 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 3240 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 3300 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 3360 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 3420 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 3480 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 3540 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 3600 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 3660 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 3720 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 3780 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt c 3831

<210> 31 <211> 4052 <212> DNA <213> artificial <220>

<223> pBCM7MLG <400> 31

ctggagatcc tgataaaatc atgaaaaatt tatttgcttt gtgagcggat aacaattata 60 atatgtggaa ttgtgagcgc tcacaattcc acataaggag gtaaaaaaat gtggaattgt 120 gagcgctcac aattccacat aaggaggtaa aaaaatgata catcatcatc atcatcatat 180 gtcccctata ctaggttatt ggaaaattaa gggccttgtg caacccactc gacttctttt 240 ggaatatctt gaagaaaaat atgaagagca tttgtatgag cgcgatgaag gtgataaatg 300 gcgaaacaaa aagtttgaat tgggtttgga gtttcccaat cttccttatt atattgatgg 360 tgatgttaaa ttaacacagt ctatggccat catacgttat atagctgaca agcacaacat 420 gttgggtggt tgtccaaaag agcgtgcaga gatttcaatg cttgaaggag cggttttgga 480 tattagatac ggtgtttcga gaattgcata tagtaaagac tttgaaactc tcaaagttga 540 ttttcttagc aagctacctg aaatgctgaa aatgttcgaa gatcgtttat gtcataaaac 600 atatttaaat ggtgatcatg taacccatcc tgacttcatg ttgtatgacg ctcttgatgt 660 tgttttatac atggacccaa tgtgcctgga tgcgttccca aaattagttt gttttaaaaa 720 acgtattgaa gctatcccac aaattgataa gtacttgaaa tccagcaagt atatagcatg 780 gcctttgcag ggctggcaag ccacgtttgg tggtggcgac catcctccaa aatcggatct 840 ggttccgcgt ggatccccag gaattcccgg gtcgactcga gcggccgcat cgtgggaact 900 gcagatgaat tcgagctcgg atccaagatc ttctagacat atgatcgata agcttgatat 960 cggtaccacc atggtcgacc tcgagcccgg ggctagcata accccttggg gcctctaaac 1020

PL 228 024 Β1

SEQLTXT.txt

gggtcttgag gggttttttg gggaaatttc cccgattaat aagatgatct tcttgagatc 1080 gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg cagggcggtt 1140 tttcgaaggt tctctgagct accaactctt tgaaccgagg taactggctt ggaggagcgc 1200 agtcaccaaa acttgtcctt tcagtttagc cttaaccggc gcatgacttc aagactaact 1260 cctctaaatc aattaccagt ggctgctgcc agtggtgctt ttgcatgtct ttccgggttg 1320 gactcaagac gatagttacc ggataaggcg cagcggtcgg actgaacggg gggttcgtgc 1380 atacagtcca gcttggagcg aactgcctac ccggaactga gtgtcaggcg tggaatgaga 1440 caaacgcggc cataacagcg gaatgacacc ggtaaaccga aaggcaggaa caggagagcg 1500 cacgagggag ccgccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 1560 ccactgattt gagcgtcaga tttcgtgatg cttgtcaggg gggcggagcc tatggaaaaa 1620 cggctttgcc gcggccctct cacttccctg ttaagtatct tcctggcatc ttccaggaaa 1680 tctccgcccc gttcgtaagc catttccgct cgccgcagtc gaacgaccga gcgtagcgag 1740 tcagtgagcg aggaagcgga atatatcctg tatcacatat tctgctgacg caccggtgca 1800 gccttttttc tcctgccaca tgaagcactt cactgacacc ctcatcagtg ccaacatagt 1860 aagccagtat acactccgct agcgctgagg tctttgcgat aggtaaaggc gcgccatcga 1920 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt 1980 ggtgaatggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg 2040 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc 2100 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac 2160 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg 2220 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg 2280 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc 2340 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg 2400 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac 2460 ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg gagcatctgg 2520 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc 2580 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg 2640 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg 2700 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc 2760 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg 2820 ataccgaaga cagctcatgt tatatcccgc cgtcaaccac catcaaacag gattttcgcc 2880 tgctggggca aaccagcgtg gaccgcttgc tgCaactctc tcagggccag gcggtgaagg 2940 gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg cccaatacgc 3000 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc 3060

PL 228 024 Β1

SEQLTXT.txt

gactggaaag cgggcagtga gcgcaacttt gcgataggta aatttttcta aatacattca 3120 aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 3180 aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 3240 cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 3300 ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 3360 cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 3420 ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 3480 gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 3 540 gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 3600 acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 3660 cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 3720 acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 3780 ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 3840 ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 3900 gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 3960 atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 4020 ggtgcctcac tgattaagca ttggtaactg tc 4052

<210> 32 <211> 4025 <212> DNA <213> artificals <220>

<223> pBCM8MLG <400> 32 ctggagatcc tgatataata cgactcacta taggggtgtg gaattgtgag cgctcacaat 60 tccacataag gaggtaaaaa aatgtggaat tgtgagcgct cacaattcca cataaggagg 120 taaaaaaatg atacatcatc atcatcatca tatgtcccct atactaggtt attggaaaat 180 taagggcctt gtgcaaccca ctcgacttct tttggaatat cttgaagaaa aatatgaaga 240 gcatttgtat gagcgcgatg aaggtgataa atggcgaaac aaaaagtttg aattgggttt 300 ggagtttccc aatcttcctt attatattga tggtgatgtt aaattaacac agtctatggc 360 catcatacgt tatatagctg acaagcacaa catgttgggt ggttgtccaa aagagcgtgc 420 agagatttca atgcttgaag gagcggtttt ggatattaga tacggtgttt cgagaattgc 480 atatagtaaa gactttgaaa ctctcaaagt tgatcttctt agcaagctac ctgaaatgct 540 gaaaatgttc gaagatcgtt tatgtcataa aacatattta aatggtgatc atgtaaccca 600 tcctgacttc atgttgtatg acgctcttga tgttgtttta tacatggacc caatgtgcct 660 ggatgcgttc ccaaaattag tttgttttaa aaaacgtatt gaagctatcc cacaaattga 720

PL 228 024 Β1

SEQLTXT.txt

taagtacttg aaatccagca agtatatagc atggcctttg cagggctggc aagccacgtt 780 tggtggtggc gaccatcctc caaaatcgga tctggttccg cgtggatccc caggaattcc 840 cgggtcgact cgagcggccg catcgtggga actgcagatg aattcgagct cggatccaag 900 atcttctaga catatgatcg ataagcttga tatcggtacc accatggtcg acctcgagcc 960 cggggctagc ataacccctt ggggccta aacgggtctt gaggggtttt ttggggaaat 1020 ttccccgatt aataagatga tcttcttgag atcgttttgg tctgcgcgta atctcttgct 1080 ctgaaaacga aaaaaccgcc ttgcagggcg gtttttcgaa ggttctctga gctaccaact 1140 ctttgaaccg aggtaactgg cttggaggag cgcagtcacc aaaacttgtc ctttcagttt 1200 agccttaacc ggcgcatgac ttcaagacta actcctctaa atcaattacc agtggctgct 1260 gccagtggtg cttttgcatg tctttccggg ttggactcaa gacgatagtt accggataag 1320 gcgcagcggt cggactgaac ggggggttcg tgcatacagt ccagcttgga gcgaactgcc 1380 tacccggaac tgagtgtcag gcgtggaatg agacaaacgc ggecataaca gcggaatgac 1440 accggtaaac cgaaaggcag gaacaggaga gcgcacgagg gagccgccag ggggaaacgc 1500 ctggtatctt tatagtcctg tcgggtttcg ccaccactga tttgagcgtc agatttcgtg 1560 atgcttgtca gagggscgga gcctatggaa aaacggcttt gccgcggccc tctcacttcc 1620 ctgttaagta tcttcctggc atcttccagg aaatctccgc cccgttcgta agccatttcc 1680 gctcgccgca gtcgaacgac cgagcgtagc gagtcagtga gcgaggaagc ggaatatatc 1740 ctgtatcaca tattctgctg acgcaccggt gcagcctttt ttctcctgcc acatgaagca 1800 cttcactgac accctcatca gtgccaacat agtaagccag tatacactcc gctagcgctg 1860 aggtctttgc gataggtaaa ggcgcgccat cgaatggcgc aaaacctttc gcggtatggc 1920 atgatagcgc ccggaagaga gtcaattcag ggtggtgaat ggtggtgaat gtgaaaccag 1980 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg 2040 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg 2100 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga 2160 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta 2220 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg 2280 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca 2340 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc 2400 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg 2460 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc 2520 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat 2580 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt 2640 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg 2700 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg 2760

PL 228 024 Β1

SEQLTXT.txt

ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca tgttatatcc 2820 cgccgtcaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct 2880 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg 2940 tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 3000 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 3060 tttgcgatag gtaaattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 3120 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 3180 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 3240 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 3300 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 3360 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag 3420 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 3480 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 3540 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 3600 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 3660 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 3720 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 3780 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 3840 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 3900 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 3960 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 4020

ctgtc 4025 <210> 33 <211> 4048 <212> DNA <213> artificial <220>

<223> pBCM9MLG <400> 33 ctggagatcc tgataattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat 60 gtggaattgt gagcgctcac aattccacat aaggaggtaa aaaaatgtgg aattgtgagc 120 gctcacaatt ccacataagg aggtaaaaaa atgatacatc atcatcatca tcatatgtcc 180 cctatactag gttattggaa aattaagggc cttgtgcaac ccactcgact tcttttggaa 240 tatcttgaag aaaaatatga agagcatttg tatgagcgcg atgaaggtga taaatggcga 300 aacaaaaagt ttgaattggg tttggagttt cccaatcttc cttattatat tgatggtgat 360 gttaaattaa cacagtctat ggccatcata cgttatatag ctgacaagca caacatgttg 420

PL 228 024 Β1

SEQLTXT.tXt

ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt tttggatatt 480 agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa agttgatttt 540 cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca taaaacatat SOO ttaaatggtg atcatgtaac ccatcctgac ttcatgttgt atgacgctct tgatgttgtt 660 ttatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt 720 attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat agcatggcct 780 ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc ggatctggtt 840 ccgcgtggat ccccaggaat tcccgggtcg actcgagcgg ccgcatcgtg ggaactgcag 900 atgaattcga gctcggatcc aagatcttct agacatatga tcgataagct tgatatcggt 960 accaccatgg tcgacctcga gcccggggct agcataaccc cttggggcct ctaaacgggt 1020 Cttgaggggt tttttgggga aatttccccg attaataaga tgatcttctt gagatcgttt 1080 tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc gccttgcagg gcggtttttc 1140 gaaggttctc tgagctacca actctttgaa ccgaggtaac tggcttggag gagcgcagtc 1200 accaaaactt gtcctttcag tttagcctta accggcgcat gacttcaaga ctaactcctc 1260 taaatcaatt accagtggct gctgccagtg gtgcttttgc atgtctttcc gggttggact 1320 caagacgata gttaccggat aaggcgcagc ggtcggactg aacggggggt tcgtgcatac 1380 agtccagctt ggagcgaact gcctacccgg aactgagtgt caggcgtgga atgagacaaa 1440 cgcggccata acagcggaat gacaccggta aaccgaaagg caggaacagg agagcgcacg 1500 agggagccgc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac 1560 tgatttgagc gtcagatttc gtgatgcttg tcaggggggc ggagcctatg gaaaaacggc 1620 tttgccgcgg ccctctcact tccctgttaa gtatcttcct ggcatcttcc aggaaatctc 1680 cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac gaccgagcgt agcgagtcag 1740 tgagcgagga agcggaatat atcctgtatc acatattctg ctgacgcacc ggtgcagcct 1800 tttttctcct gccacatgaa gcacttcact gacaccctca tcagtgccaa catagtaagc 1860 cagtatacac tccgctagcg ctgaggtctt tgcgataggt aaaggcgcgc catcgaatgg 1920 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg 1980 aatggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc 2040 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga 2100 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacact 2160 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc 2220 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt 2280 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc 2340 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt 2400 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat 2460

PL 228 024 Β1

SEQLTXT.txt

caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc 2520 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct 2580 gcgtctggct ggctggcata aatatetcac tcgcaatcaa attcagccga tagcggaacg 2640 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg 2700 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc 2760 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac 2820 cgaagacagc tcatgttata tcccgccgtc aaccaccatc aaacaggatt ttcgcctgct 2880 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa 2940 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac 3000 Cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 3060 ggaaagcggg cagtgagcgc aactttgcga taggtaaatt tttctaaata cattcaaata 3120 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 3180 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 3240 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 3300 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 3360 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 3420 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 3480 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 3540 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 3600 tcggaggacc gaaggagcta accgcttttt tg cacaacat gggggatcat gtaactcgcc 3660 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 3720 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 3780 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 3840 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 3900 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 3960 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 4020

cctcactgat taagcattgg taactgtc

4048

PL 228 024 Β1

SEQLTXT.tXt

ggtggttgtc caaaagagcg tgcagagatt tcaatgcttg aaggagcggt tttggatatt 480 agatacggtg tttcgagaat tgcatatagt aaagactttg aaactctcaa agttgatttt 540 cttagcaagc tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca taaaacatat 600 ttaaatggtg atcatgtaac ccatcctgac rtcatgttgt atgacgctct tgatgttgtt 660 ttatacatgg acccaatgtg cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt 720 attgaagcta tcccacaaat tgataagtac ttgaaatcca gcaagtatat agcatggcct 780 ttgcagggct ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc ggatctggtt 840 ccgcgtggat ccccaggaat tcccgggtcg actcgagcgg ccgcatcgtg ggaactgcag 900 atgaattcga gctcggatcc aagatcttct agacatatga tcgataagct tgatatcggt 960 accaccatgg tcgacctcga gcccggggct agcataaccc cttggggcct ctaaacgggt 1020 cttgaggggt tttttgggga aatttccccg attaataaga tgatcttctt gagatcgttt 1080 tggtctgcgc gtaatctctt gctctgaaaa cgaaaaaacc gccttgcagg gcggtttttc 1140 gaaggttctc tgagctacca actctttgaa ccgaggtaac tggcttggag gagcgcagtc 1200 accaaaactt gtcctttcag tttagcctta accggcgcat gacttcaaga ctaactcctc 1260 taaatcaatt accagtggct gctgccagtg gtgcttttgc atgtctttcc gggttggact 1320 caagacgata gttaccggat aaggcgcagc ggtcggactg aacggggggt tcgtgcatac 1380 agtccagctt ggagcgaact gcctacccgg aactgagtgt caggcgtgga atgagacaaa 1440 cgcggccata acagcggaat gacaccggta aaccgaaagg caggaacagg agagcgcacg 1500 agggagccgc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccaccac 1560 tgatttgagc gtcagatttc gtgatgcttg tcaggggggc ggagcctatg gaaaaacggc 1620 tttgccgcgg ccctctcact tccctgttaa gtatcttcct ggcatcttcc aggaaatctc 1680 cgccccgttc gtaagccatt tccgctcgcc gcagtcgaac gaccgagcgt agcgagtcag 1740 tgagcgagga agcggaatat atcctgtatc acatattctg ctgacgcacc ggtgcagcct 1800 tttttctcct gccacatgaa gcacttcact gacaccctca tcagtgccaa catagtaagc 1860 cagtatacac tccgctagcg ctgaggtctt tgcgataggt aaaggcgcgc catcgaatgg 1920 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg 1980 aatggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc 2040 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga 2100 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacact 2160 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc 2220 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt 2280 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc 2340 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt 2400 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat 2460

PL 228 024 Β1

SEQLTXT.txt

caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atetggtcgc 2520 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct 2580 gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg 2640 ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg 2700 catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc 2760 cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac 2820 cgaagacagc tcatgttata tcccgccgtc aaccaccatc aaacaggatt ttcgcctgct 2880 ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa 2940 tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac 3000 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 3060 ggaaagcggg cagtgagcgc aactttgcga taggtaaatt tttctaaata cattcaaata 3120 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 3180 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 3240 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 3300 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 3360 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 3420 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 3480 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 3540 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 3600 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 3660 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 3720 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 3780 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 3840 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 3900 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 3960 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 4020

cctcactgat taagcattgg taactgtc

Claims (13)

1. Nucleic acid molecule encoding a promoter region determined by a sequence selected from sequences No. 1-3; 2. A nucleic acid molecule encoding a linker region defined by a sequence selected from sequence No. 4-12, preferably a polylinker region having the sequence No. 4. 3. An expression vector set comprising different expression vectors, each vector comprising: - an operably linked promoter region defined by a sequence selected from among sequences Nos. 1 to 3 and a linker region defined by a sequence selected from sequences Nos. 4 to 12, - a replication origin region defined by a sequence selected from sequences Nos. 13 to 15, the vectors comprising the set comprising all combinations of said promoter region variants with said replication origin variants. 4. The kit according to p. The method of claim 3, characterized in that the expression vector further comprises a nucleic acid sequence encoding a reporter molecule, preferably a green fluorescent protein or a pink fluorescent protein. 5. The kit according to p. The method of claim 3, wherein the expression vector is a linear molecule. 6. The kit according to p. 3. The method of claim 3, wherein the expression vector encodes a region corresponding to WELQ amino acids recognized by Clean-Cut protease; The kit according to claim 6, characterized in that the transcription initiation site is located downstream of the sequence corresponding to the WELQ amino acids. 8. The kit according to p. The method of claim 6, characterized in that there is a sequence corresponding to the amino acids MIHHHHHH upstream of the WELQ sequence. 9. The kit according to p. 6. The method according to claim 6, characterized in that it comprises vectors with sequences No. 16 to 24. 10. The kit according to p. 6. The method according to claim 6, characterized in that it comprises vectors of sequence Nos. 25 to 33. 11. A method of producing recombinant proteins, characterized in that E. coli cells are transformed with the set of expression vectors as defined in claim 1. from 3 to 10 containing the recombinant protein coding sequence, the obtained transformants are cultured under established culture conditions, the expression level obtained for the various transformants is determined, then the final production culture of the transformant strain giving the highest expression level is carried out and the recombinant protein is isolated from the broth breeding. 12. The method according to claim 11, characterized in that Clean-Cut protease is used to release the recombinant protein. 13. Method according to claims 11-12, characterized in that a Clean-Cut protease is used for the process of cleaving the fusion tag and releasing the N-terminus of the recombinant protein and / or the process of arbitrary shaping the N-terminus of the recombinant protein.