RU2754229C1 - UNIVERSAL RECOMBINANT VECTOR pSVO AND PLASMID GENETIC CONSTRUCTS pSVO-Gluc AND pSVO-Vic, PROVIDING SYNTHESIS AND SECRETION OF TARGET PROTEINS IN YEAST CELLS OF THE KLUYVEROMYCES LACTIS SPECIES AND OBTAINED USING THIS UNIVERSAL VECTOR - Google Patents

Abstract

FIELD: molecular genetics.SUBSTANCE: invention relates to molecular genetics and biotechnology, namely to a universal recombinant vector, which is used to obtain plasmid genetic constructs that ensure the synthesis and secretion of target recombinant proteins in yeast cells of the Kluyveromyces lactis species. The niversal recombinant vector pSVO has the nucleotide sequence SEQ ID No. 1 and contains elements in accordance with the physical and genetic map shown in fig. 1g. The plasmid genetic construct pSVO-Gluc provides synthesis and secretion of the target luciferase protein from Gaussia princeps in yeast cells of the Kluyveromyces lactis species, has the nucleotide sequence SEQ ID No. 2 and contains elements in accordance with the physical and genetic map shown in fig. 2. The pSVO-Vic plasmid genetic construct provides synthesis and secretion of the target protein prochymosin alpaca in yeast cells of the Kluyveromyces lactis species, has a nucleotide sequence SEQ ID No. 3 and contains elements in accordance with the physical and genetic map shown in fig. 5.EFFECT: creation of a universal recombinant vector pSVO and plasmid genetic constructs pSVO-Gluc and pSVO-Vic, providing synthesis and secretion of target proteins in yeast cells of the Kluyveromyces lactis species and obtained using this universal vector.3 cl, 5 dwg, 2 tbl, 3 ex

Description

The invention relates to a universal recombinant vector, which is used to obtain plasmid genetic constructs that provide synthesis and secretion of target recombinant proteins in yeast cells of the species Kluyveromyces lactis and can be used in molecular genetics and biotechnology.

State of the art. For more than 30 years, the yeast Kluyveromyces lactis has been used for the production of heterologous proteins (Van Ooyen AJJ et al. Heterologous protein production in the yeast Kluyveromyces lactis. FEMS yeast research 2006; 6; 3: 381-392; Spohner SC et al. Kluyveromyces lactis: an emerging tool in biotechnology 2016. J Biotechnol 222: 104-116). K. lactis has many properties that make it attractive for protein production; it is easily amenable to genetic manipulation, has a sequenced genome (Dujon B. et al. Genome evolution in yeasts. Nature 2004; 430: 35-44; Chuzel L. et al. Complete genome sequence of Kluyveromyces lactis strain GG799, a common yeast host for heterologous protein expression.Genome Announc 2017; 5: e00623-17), has a negative Crabtree metabolism (Gonzalez-Siso M.I. et al. Respirofermentative metabolism in Kluyveromyces lactis: insights and perspectives. Enzyme Microb Technol 2000; 26: 699-705 ; Merico A. et al. The oxygen level determines the fermentation pattern in Kluyveromyces lactis. FEMS Yeast Res 2009; 9: 749-756). Kluyveromyces lactis can be cultured using simple culture media. To date, more than 100 heterologous proteins have been obtained using K. lactis (Van Ooyen AJJ et al. Heterologous protein production in the yeast Kluyveromyces lactis. FEMS yeast research 2006; 6; 3: 381-392; Spohner S. C. et al. Kluyveromyces lactis: an emerging tool in biotechnology 2016. J Biotechnol 222: 104-116). In addition, K. lactis has a long history of safe use in the food industry and is considered a safe (GRAS) microorganism (Coenen TM et al. Safety evaluation of a lactase enzyme preparation derived from Kluyveromyces lactis. Food Chem Toxicol 2000; 38: 671 -677).

Together, these features have made K. lactis a convenient yeast protein production system for both laboratory and industrial processes. Despite the long history of this organism as an expression system, only a small number of promoters have been used to express foreign genes in K. lactis (Van Ooyen AJJ et al. Heterologous protein production in the yeast Kluyveromyces lactis. FEMS yeast research 2006; 6; 3: 381 -392; Spohner SC et al. Kluyveromyces lactis: an emerging tool in biotechnology 2016. J Biotechnol 222: 104-116). In addition, for these yeasts, no regulated promoter has been described that functions with the same efficiency and tight regulation as the methanol-inducible AOX1 promoter (Tschopp JF et al. Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 1987; 15: 3859-3876) from Pichia pastoris. The LAC4 promoter (PLAC4), a strong promoter that regulates the expression of the endogenous galactosidase (Lac4p) gene in response to the addition of lactose or galactose, is most commonly used for the expression of foreign genes in K. lactis. Thus, there is still a need to develop a vector to provide efficient and regulated gene expression for the production of heterologous proteins at any scale.

The closest analogues. The commercial plasmid pKLAC2 is known. from NEB (USA), which is an expression vector capable of replication both in E. coli and stable integration into the genome of the yeast Kluyveromyces lactis. pKLAC2 contains a universal multiple cloning site (MCS). After transformation of competent K. lactis cells, pKLAC2 integrates into the K. lactis chromosome at the LAC4 locus. For the selection of clones, the selection marker acetamidase (amdS) is used, the synthesis of which is under the control of the yeast ADH1 promoter. Acetamidase synthesized by yeast upon integration of the pKLAC2 fragment into the chromosome allows transformed cells to use acetamide as a nitrogen source in a selective nutrient medium. The multiple cloning site (MCS) is located to allow translational fusion of the sex α-factor (α-MF) signal sequence to the N-terminus of the recombinant target protein. This addresses the chimeric protein in the general secretory pathway, but α-MF is cleaved in the Golgi apparatus by the Kex protease, which leads to the secretion of only the recombinant protein. The synthesis of the recombinant fusion protein is under the control of the K. lactis LAC4 promoter.

Despite the listed advantages, this vector has several disadvantages. In particular, as mentioned above, the integration of the target gene under the control of the yeast LAC4 promoter can lead to significant background synthesis of the target protein in the absence of the inducer, which can be critical in the case of the expression of genes of toxic proteins. In addition, the LAC4 promoter is not autoinducible, which introduces an additional step in the cultivation of the target protein. Another disadvantage of this vector is the use of the acetamidase gene as a selectable marker. The YEB culture medium used for the selection of K. lactis clones is expensive and does not allow the amount of the selective marker introduced into the medium to be regulated.

The closest analogue (prototype) is the pUC19 / Pr ₃₅₀ plasmid described in the article by Hassan Sakhtah and his colleagues (Sakhtah, H., Behler, J., AH-Reynolds, A., Causey, T. B., Vainauskas, S., & Taron, S. N. A novel regulated hybrid promoter that permits autoinduction of heterologous protein expression in Kluyveromyces lactis. 2019. Applied and environmental microbiology. 85 (14), e00542-19), engineered from the commercial pUC19 vector. This expression vector (pUC19 / Pr ₃₅₀ ) is capable of replication in E. coli and stable integration of various heterologous proteins into the genome of the yeast Kluyveromyces lactis. _{The hybrid P 350} promoter used to control the expression of recombinant genes provides autoinduction of protein synthesis in K. lactis.

However, in addition to the new promoter, the rest of the structural elements of the plasmid pUC19 / Pr ₃₅₀ do not differ from the commercial vector pKLAC2, which imposes the same restrictions on its use. Thus, the use of the sex α-factor (α-MF) as a signal sequence has a number of disadvantages. This secretion signal promotes post-translational translocation into the endoplasmic reticulum (ER); therefore, proteins concealed in the cytosol can be ineffectively translocated and, as a consequence, poorly secreted. In addition, if the protein is self-associating, the α-factor pro-region can potentially induce aggregation, thereby preventing export from the ER.

The technical result of the claimed invention is the creation of a universal recombinant vector pSVO and plasmid genetic constructs pSVO-Gluc and pSVO-Vic, providing the synthesis and secretion of target proteins in yeast cells of the species Kluyveromyces lactis and obtained using the specified universal vector, which are devoid of the disadvantages of the prototype.

This technical result is achieved by the fact that the universal recombinant vector pSVO for creating plasmid genetic constructs that provide synthesis and secretion of target proteins in yeast cells of the species Kluyveromyces lactis has the nucleotide sequence SEQ ID No. 1 and contains, in accordance with the physical and genetic map shown in FIG. 1d, the following elements:

- autoinducible hybrid promoter 341 to 1017, including the core sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the regulatory sequence of the isocitrate lyase (ICL) promoter;

- hybrid signal sequence (Ost2) to ensure the secretion of the protein into the extracellular space from 1018 to 1287;

- region of insertion of the target gene (MCS) from 1297 to 1353;

- terminator region of the S. cerevisiae gene (CYC) from 1360 to 1638;

- promoter of the alcohol dehydrogenase gene (ADH1 promoter) from 1647 to 2339;

- gene for resistance to zeocin (BleoR) from 2360 to 2734;

- 5 'sequence of the isocitrate lyase promoter gene (ICL-5') from 2735 to 3694;

- origin of replication (ori) from 4504 to 5092;

- gene for ampicillin resistance (AmpR) from 5263 to 6122.

This technical result is also achieved by the fact that the plasmid genetic construct pSVO-Gluc provides the synthesis and secretion of the target luciferase protein from Gaussia princeps in yeast cells of the species Kluyveromyces lactis, has the nucleotide sequence SEQ ID No. 2 and contains, in accordance with the physical and genetic map shown in FIG. 2, the following items:

- autoinducible hybrid promoter 341 to 1017, including the core sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the regulatory sequence of the isocitrate lyase (ICL) promoter;

- hybrid signal sequence (Ost2) to ensure the secretion of the protein into the extracellular space from 1018 to 1287;

- the sequence of the luciferase gene from Gaussia princeps (Glue) from 1300 to 1803;

- the terminator region of the S. cerevisiae gene (CYC) from 1813 to 2091;

- promoter of the alcohol dehydrogenase gene (ADH1 promoter) from 2100 to 2792;

- gene for resistance to zeocin (BleoR) from 2813 to 3187;

- 5 'sequence of the isocitrate lyase promoter gene (ICL-5') from 3188 to 4147;

- origin of replication (ori) from 4957 to 5545;

- gene for ampicillin resistance (AmpR) from 5716 to 6576.

This technical result is also achieved by the fact that the plasmid genetic construct pSVO-Vic provides the synthesis and secretion of the target protein alpaca prochymosin in yeast cells of the species Kluyveromyces lactis, has a nucleotide sequence of SEQ ID No. 3 and contains, in accordance with the physical and genetic map shown in FIG. 4, the following items:

- autoinducible hybrid promoter 341 to 1017, including the core sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the regulatory sequence of the isocitrate lyase (ICL) promoter;

- hybrid signal sequence (Ost2) to ensure the secretion of the protein into the extracellular space from 1018 to 1287;

- the sequence of the gene for alpaca prochymosin (Vic) from 1300 to 2397;

- terminator region of the S. cerevisiae gene (CYC) from 2405 to 2682;

- promoter of the alcohol dehydrogenase gene (ADH1 promoter) from 2691 to 3383;

- gene for resistance to zeocin (BleoR) from 3404 to 3778;

- 5 'sequence of the isocitrate lyase promoter gene (ICL-5') from 3779 to 4738;

- origin of replication (ori) from 5548 to 6136;

- gene for ampicillin resistance (AmpR) from 6307 to 7167.

The claimed plasmids are devoid of the disadvantages of the prototype since as a signal sequence, they used a hybrid signal of the secretion of Ost2, which, in comparison with the signal of α-MF, increases the secretion of heterogeneous proteins up to 20 times (Barrero, JJ, Casler, J.C, Valero, F., Ferrer, P., & Glick , BS An improved secretion signal enhances the secretion of model proteins from Pichia pastoris. Microbial cell factories. 2018; 17 (1), 161).

An autoinducible hybrid promoter, including the core sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAP1) promoter and the regulatory sequence of the isocitrate lyase (ICL1) promoter, on the one hand, ensures efficient synthesis of the target RNA, and, on the other hand, severely suppresses the expression of their genes in the absence of induction. Used in the vector of the gene for resistance to zeocin (BleoR), allows you to adjust the amount introduced into the medium of a selectable marker for more efficient selection of multi-copy clones. The above examples show that the claimed universal plasmid vector pSVO allows you to create target plasmids (for example, pSVO-Gluc and pSVO-Vic), which provide efficient synthesis of target proteins, with tight control until autoinduction.

The invention is illustrated by the following graphic materials. FIG. 1, (A, B, C, D) shows a scheme for constructing a universal recombinant vector pSVO. FIG. 2 shows an electrophoretogram of the amplification products of the pSVO-Vic vector (lane 1) and the pSVO-Gluc vector (lane 2). FIG. 3 shows the pSVO-Gluc recombinant vector for the expression of the luciferata gene from Gaussia princeps in K. lactis cells. FIG. 4 shows data on the luciferase activity of the protein produced by the pSVO-Vic plasmid in K. lactis cells. FIG. 5 shows the pSVO-Vic recombinant vector for expression of the alpaca prochymosin gene in K. lactis cells.

Below are examples 1-3 of a specific implementation of the invention.

Example 1. Construction of the pSVO vector.

The design diagram of the claimed invention is shown in FIG. 1.

Sequence c) of the Ost2 hybrid leader was custom synthesized. The remaining constituents of the engineered vector were amplified using the engineered oligonucleotides (Table 1).

Amlicons were obtained from the template for K. lactis DNA: a) 3'-part of ICL1 promoter, b) 5'-part of GAP promoter and g) 5'-part of ICL1 promoter. An amplicon (i) of the terminator region of the CYC1 gene was obtained from the S. cerevisiae genomic DNA template.

The amplicons of the alcohol dehydrogenase f) gene and the amdS acetamidase gene were obtained from the pKLAC2 vector template (Fig. La). The reaction mixture for PNR in 50 μl contained: 1 mm dNTP solution; 100 pM of each of the primers; 5 μl 10x Pfu DNA polymerase buffer; 0.5 units Pfu DNA polymerase; 20 ngr of template DNA. The reaction mixture was denatured at 95 ° C for 10 minutes, followed by amplification (25 cycles), consisting of denaturation at 94 ° C for 30 seconds, annealing at 59 ° C for 30 seconds and elongation at 72 ° C, the duration was calculated from calculation 1 minute per 1000 bp After the completion of the amplification, the extension of the DNA ends was carried out at 72 ° C for 5 minutes. The reactions were performed on a PCR amplifier (OOO BIS-N, Russia).

Then the obtained parts were combined using annealing into three fragments according to the following scheme 1: a) b) c) 2: i) 3: f) g) (Fig. 1b). The resulting fragments were inserted into the pJet1.2 shuttle vector in order to check the integrity of the nucleotide sequences. After verification, the vectors were treated with the enzymes CciNI, BamHI, XmaI, PspXI (Fig. 1c) for their simultaneous insertion into the pjet1.2 shuttle vector. As a result, on the basis of the commercial vector pJet1.2, the vector pSVO was obtained (Fig.1d), which has the following structural elements:

- autoinducible hybrid promoter 341 to 1017, including the core sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the regulatory sequence of the isocitrate lyase (ICL) promoter

- hybrid signal sequence (Ost2) to ensure the secretion of the protein into the extracellular space from 1018 to 1287;

- region of insertion of the target gene (MCS) from 1297 to 1353;

- terminator region of the S. cerevisiae gene (CYC) from 1360 to 1638;

- promoter of the alcohol dehydrogenase gene (ADH1 promoter) from 1647 to 2339;

- gene for resistance to zeocin (BleoR) from 2360 to 2734;

- 5 'sequence of the isocitrate lyase promoter gene (ICL-5') from 2735 to 3694;

- origin of replication (ori) from 4504 to 5092;

- gene for ampicillin resistance (AmpR) from 5263 to 6122.

The constructed universal vector pSVO was further tested for its ability to express and secrete proteins in the Kluyveromyces lactis system by inserting the genes of target enzymes that are part of the developed plasmid vectors pSVO-Gluc and pSVO-Vic, and their specific activity in the obtained culture fluid was also determined. ...

Example 2. Construction of the pSVO-Gluc vector and detection of expression of the luciferase gene from Gaussia princeps in the culture medium after transformation of Kluyveromyces lactis cells with the constructed plasmid. To construct the pSVO-Gluc vector, the luciferase gene from Gaussia princeps was amplified from the pCDH-EFl-GausLuc-IRES-copGFP plasmid template using the designed primers:

- Gluc-G-F (5'-aaaaaaggatccaaaagaaagcccaccgagaacaacgaag-3 ') and

- Gluc-G-R (5'-atcgacaaaggaaaaggggcctgtcctaggttagtcaccaccggcccccttg-3 '). After the PCR product was ligated into the region of insertion of the target gene of the pSVO vector at the unique sites BamHI / AspA2I, ZAO Sibenzyme, Russia, according to the manufacturer's recommendations. As a result, the vector pSVO-Gluc was obtained (Fig. 3), containing the following structural elements:

- autoinducible hybrid promoter 341 to 1017, including the core sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the regulatory sequence of the isocitrate lyase (ICL) promoter;

- hybrid signal sequence (Ost2) to ensure the secretion of the protein into the extracellular space from 1018 to 1287;

- gene sequence of lucifira from Gaussia princeps (Gluc) from 1300 to 1803;

- the terminator region of the S. cerevisiae gene (CYC) from 1813 to 2091;

- promoter of the alcohol dehydrogenase gene (ADH1 promoter) from 2100 to 2792;

- gene for resistance to zeocin (BleoR) from 2813 to 3187;

- 5 'sequence of the isocitrate lyase promoter gene (ICL-5') from 3188 to 4147;

- origin of replication (ori) from 4957 to 5545;

- gene for ampicillin resistance (AmpR) from 5716 to 6576.

The constructed vector pSVO-Gluc was used to transform the electrocompetent cells of K. lactis strain GG799; for this, PCR was performed using the designed primers:

- J-F (5 '-cgactcactatagggagagcggc-3') and

- J-R (5'-aagaacatcgattttccatggcag-3 ')

Then, the PCR product obtained (Fig. 2, lane 2) in an amount of 1 μg was inserted into K. lactis cells of the GG799 strain by electroporation using a MicroPulser Electroporator (BioRad, USA), according to the manufacturer's recommendations. After electroporation, the cells were plated on YPDS agar medium containing 500, 1000, 2000 μg / ml of zeocin as a selective marker and cultured in a thermostat at 30 ° C for 72 hours. Then, 3 individual colonies from each plate were transferred into liquid YEP medium containing 2%, and grown overnight on an orbital shaker at 30 ° C and 220 rpm. The inoculum, in a ratio of 1: 100, was transferred into an Erlenmeyer flask containing YEP medium containing 2% and cultured for four days at 30 ° C and 220 rpm, taking samples of the culture fluid every day. Then the obtained samples were analyzed for the presence of luciferase activity using native coelenterazine (NanoLight Technologies, USA) as a substrate. The culture medium of HEK293FT cells transfected with plasmid pCDH-EF1-GausLuc-IRES-copGFP served as a positive control. As negative, culture fluid of K. lactis strain GG799. The luciferase activity data are shown in FIG. 4.

Example 3. Construction of pSVO-Vic vector and detection of milk-clotting activity (MA) in culture medium after transformation of Kluyveromyces lactis cells with the constructed plasmid.

To construct the pSVO-Vic vector, the sequence of the alpaca prochymosin gene was custom synthesized as part of the pGH shuttle vector and inserted into the pSVO vector at the unique BamHI / AspA2I sites. As a result, the vector pSVO-Vic was obtained (Fig. 5) containing the following structural elements:

- autoinducible hybrid promoter 341 to 1017, including the core sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the regulatory sequence of the isocitrate lyase (ICL) promoter;

- hybrid signal sequence (Ost2) to ensure the secretion of the protein into the extracellular space from 1018 to 1287;

- the sequence of the gene for alpaca prochymosin (Vic) from 1300 to 2397;

- terminator region of the S. cerevisiae gene (CYC) from 2405 to 2682;

- promoter of the alcohol dehydrogenase gene (ADH1 promoter) from 2691 to 3383;

- gene for resistance to zeocin (BleoR) from 3404 to 3778;

- 5 'sequence of the isocitrate lyase promoter gene (ICL-5') from 3779 to 4738;

- origin of replication (ori) from 5548 to 6136;

- gene for ampicillin resistance (AmpR) from 6307 to 7167.

The constructed vector pSVO-Vic was used for electroporation of K. lactis cells of the GG799 strain. For this, PCR was performed using the designed primers:

- J-F (5'-cgactcactatagggagagcggc-3 ')

- J-R (5'-aagaacatcgattttccatggcag-3 ')

Further, the obtained PCR product (Fig. 2, lane 1) in an amount of 1 μg was inserted into K. lactis cells of strain GG799 by electroporation using a MicroPulser Electroporator (BioRad, USA), according to the manufacturer's recommendations. The cultivation of the obtained individual colonies was carried out as described in example 2. After obtaining samples of the culture medium in them was determined milk-clotting activity (MA) to analyze the presence of the target protein (prochymosin alpaca) in an enzymatically active form. At the beginning, the procedure for activating the obtained samples of the culture medium was carried out. For this, 2.0 M HCl was added to the samples with constant stirring to pH 3.0. The stirring was stopped and the mixture was incubated at pH 3.0 ± 0.1 for 2 hours. After the incubation time, the pH of the sample was adjusted to 5.8 ± 0.1 using 0.5M NaOH. After that, MA was determined in the sample activated in this way.

To determine MA, we used a standardized dry milk substrate (Substrat ST.3) manufactured by VNIIMS (Uglich). To prepare for work 25.0 gr. the substrate was dissolved in 230 ml of 0.01N CaCl2 for 1 hour at room temperature and constant stirring. The pH of the resulting mixture was adjusted to 6.5 ± 0.1 using 1.0M NaOH. The final volume of the solution was brought to 250 ml with 0.01 N CaCl2. The activity of the test samples was determined relative to the industry control sample of rennet (OKO SF), certified by MA, produced by JSC "Moscow rennet plant". To prepare for work, 80 ml of distilled water was added to 1.0 g of OCO SF, stirred for 30 minutes at 20-25 ° C, the volume of the mixture was brought to 100 ml and kept in a water bath at 35 ° C for 15 minutes. Before determining MA, the test samples were kept for 15 minutes at 35 ° C.

MA was determined at 35 ° C. 2.5 ml of a standardized substrate solution was added to a dry glass tube and heated at 35 ° C for 5 minutes. 0.25 ml of the test sample was added to the tube with the substrate, the stopwatch was turned on, the contents of the tube were immediately thoroughly mixed. The duration of coagulation of the substrate was noted by a stopwatch from the moment of sample introduction until the appearance of the first flocs of substrate coagulation. The appearance of flakes was checked by periodically applying a drop of the reaction mixture with a glass rod to the wall of the test tube. Milk-clotting activity (MA) values are shown in Table 2.

The results obtained in the above examples 2-3 show that using the universal recombinant vector pSVO, it was possible to obtain the target plasmid vectors pSVO-Gluc and pSVO-Vic, providing the synthesis of heterogeneous proteins into the extracellular space after transformation of K. lactis cells. It should be noted that the use of the bleomecin resistance gene in the constructed vectors makes it possible to select multi-copy clones of recombinant K. lactis cells with an increase in the antibiotic concentration. The use of a hybrid promoter makes it possible to completely suppress the expression of the target gene until the moment of autoinduction.

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Sequence listing

<110> Federal Budgetary Institution of Science "State Scientific Center

virology and biotechnology "Vector" of the Federal Service for Surveillance in the field

protection of consumer rights and human well-being (FBSI SSC VB "Vector"

Rospotrebnadzor)

<120> Universal recombinant vector pSVO and plasmid genetic

pSVO-Gluc and pSVO-Vic constructs, providing synthesis and secretion of target

proteins in yeast cells of the species Kluyveromyces lactis and obtained with

using the specified generic vector

<160> SEQ ID NO: 3

<210> SEQ ID NO: 1

<211> 6315

<212> PRT

<213> Artificial Sequence

<223> Nucleotide Sequence of the pSVO Vector

<400> 1

1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc

61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc

121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact

181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt

241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc

301 ggcgtaatac gactcactat agggagagcg gccgcaattc atctagataa tcgggtatga

361 ttaacagcga catagctatg tgattatctg ccccattact taaagtaagg tttcactttc

421 ggactaagga cgaatgacac aagacacaga acgctcacac ggttgaaaca ttttttttcc

481 tgatcctttt gaccctgttt gttgtccctc ttttacgatg ggagaattga atcatccgcc

541 attaaaccga atgtgatagt ttttccagtt tttcttcccc tccctacatc catttctaaa

601 gaattcacgg acattttcgc atgaatgggg aaagttgact cagttgactc tatttggttc

661 ctcagcctcg actgctttgc ttcatcttct ctatgggcca atgagctaat gagcacaatg

721 tgctgcgaaa taaagggata tctaatttat attattacat tataatatgt actagtgtgg

781 ttattggtaa ttgtacttaa ttttgatata taaagggtgg atctttttca ttttgaatca

841 gaattggaat tgcaacttgt ctcttgtcac tattacttaa tagtaattat atttcttatt

901 aacctttttt ttaagtcaaa acaccaagga caagaactac tcttcaaagg tatttcaagt

961 tatcatacgt ctcacacacg cttcacagtt tcaagtaaaa aaaaagaata ttacacaatg

1021 agacaagttt ggttctcttg gatcgttggt ttgttcttgt gtttcttcaa cgtttcttct

1081 gctgctccag ttaacactac tactgaagat gagactgctc aaattccagc tgaagctgtt

1141 attggttact ctgatttgga aggtgatttc gatgttgctg ttttgccatt ctctaactca

1201 actaacaacg gtttgttgtt catcaacacc actattgctt ctatcgctgc taaagaagag

1261 ggagtttctt tggaaaaaag agaggctgga tccaaaagag aggctgaagc tagaagagct

1321 catatgtcca tgggcggccg cgatatcgtc gaccctagga caggcccctt ttcctttgtc

1381 gatatcatgt aattagttat gtcacgctta cattcacgcc ctcctcccac atccgctcta

1441 accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt tttttaatag

1501 ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacaaac

1561 gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga

1621 aggctttaat ttgcaagcag cccgggtgtt tccgggtgta caatatggac ttcctctttt

1681 ctggcaacca aacccataca tcgggattcc tataatacct tcgttggtct ccctaacatg

1741 taggtggcgg aggggagata tacaatagaa cagataccag acaagacata atgggctaaa

1801 caagactaca ccaattacac tgcctcattg atggtggtac ataacgaact aatactgtag

1861 ccctagactt gatagccatc atcatatcga agtttcacta ccctttttcc atttgccatc

1921 tattgaagta ataataggcg catgcaactt cttttctttt tttttctttt ctctctcccc

1981 cgttgttgtc tcaccatatc cgcaatgaca aaaaaatgat ggaagacact aaaggaaaaa

2041 attaacgaca aagacagcac caacagatgt cgttgttcca gagctgatga ggggtatctc

2101 gaagcacacg aaactttttc cttccttcat tcacgcacac tactctctaa tgagcaacgg

2161 tatacggcct tccttccagt tacttgaatt tgaaataaaa aaaagtttgc tgtcttgcta

2221 tcaagtataa atagacctgc aattattaat cttttgtttc ctcgtcattg ttctcgttcc

2281 ctttcttcct tgtttctttt tctgcacaat atttcaagct ataccaagca tacaatcaag

2341 caattccaga tctgccacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg

2401 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt

2461 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca

2521 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct

2581 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat

2641 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa

2701 ctgcgtgcac ttcgtggccg aggagcagga ctgactttta cctttgttgt cttatgtgtt

2761 tttactccaa cacccctaac cctaattcca catagatcca accctaattc cgcagaggta

2821 caaataaaca caaccagcta gagatagcac accaccatga ggctaggttg cacataacac

2881 accaccgaat tttttgctac ccacttttct tcctctttgt ctctatacca acccatttag

2941 tacccacaaa taaactctgg ggtgtacggt accaagcttc gccaacgaga atgcaacaaa

3001 tgatgaatat actgtgatgt agtgattgct cattgaataa gtagcgttat tcgaagtaaa

3061 gaatagaaaa tcacaagact ggggttaaag ctgcatcaaa taggcaaatg tgcggcgtat

3121 cgtggtttgc gcgcacggat tatctaacaa gtttttccac cacttaaaaa ttgagggagc

3181 gttccgcata attataattg ctttttttgc tgacatcggc atcctgactt cttcttttgt

3241 agtgctctat ttgtgagtgg tgttaaaaaa acattcaaag tggcatgcta ttctaaggca

3301 atatggaaac atgagagttc ctctttgaat atctggagct ccagcctatt aggcaagttt

3361 tgtcagactg cttgtatgga agaggttttc gggaaaccat attcgactcg gtagagtaag

3421 agatatgtct gatttatcct catatgaaag gtttttaaga atataagaat caagtatcga

3481 aatatttgca aaattttggc actatggccg agtggttaag gcgacagact tgaaatctgt

3541 tgggctctgc ccgcgctggt tcaaatcctg ctggtgtcgt tattttttgg aaataacttt

3601 ttttaactag acaataagcg tttagccggg taatatttat ttatatatat cggctccttc

3661 catggagctg ataccgtaag ctcacctaat ctgcgaattg ctcgagtttt ttatctttct

3721 agaagatctc ctacaatatt ctcagctgcc atggaaaatc gatgttcttc ttttattctc

3781 tcaagatttt caggctgtat attaaaactt atattaagaa ctatgctaac cacctcatca

3841 ggaaccgttg taggtggcgt gggttttctt ggcaatcgac tctcatgaaa actacgagct

3901 aaatattcaa tatgttcctc ttgaccaact ttattctgca ttttttttga acgaggttta

3961 gagcaagctt caggaaactg agacaggaat tttattaaaa atttaaattt tgaagaaagt

4021 tcagggttaa tagcatccat tttttgcttt gcaagttcct cagcattctt aacaaaagac

4081 gtctcttttg acatgtttaa agtttaaacc tcctgtgtga aattgttatc cgctcacaat

4141 tccacacatt atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag

4201 ctaactcaca ttaattgcgt tgcgctcact gccaattgct ttccagtcgg gaaacctgtc

4261 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg

4321 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt

4381 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa

4441 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc

4501 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag

4561 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt

4621 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg

4681 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg

4741 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg

4801 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac

4861 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg

4921 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt

4981 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg

5041 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc

5101 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt

5161 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt

5221 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag

5281 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt

5341 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc

5401 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc

5461 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg

5521 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac

5581 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg

5641 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc

5701 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact

5761 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc

5821 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat

5881 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc

5941 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac

6001 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa

6061 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact

6121 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg

6181 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg

6241 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag

6301 gcgtatcacg aggcc

<210> SEQ ID NO: 2

<211> 6768

<212> PRT

<213> Artificial Sequence

<223> Nucleotide Sequence of the pSVO-Gluc Vector

<400> 2

1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc

61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc

121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact

181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt

241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc

301 ggcgtaatac gactcactat agggagagcg gccgcaattc atctagataa tcgggtatga

361 ttaacagcga catagctatg tgattatctg ccccattact taaagtaagg tttcactttc

421 ggactaagga cgaatgacac aagacacaga acgctcacac ggttgaaaca ttttttttcc

481 tgatcctttt gaccctgttt gttgtccctc ttttacgatg ggagaattga atcatccgcc

541 attaaaccga atgtgatagt ttttccagtt tttcttcccc tccctacatc catttctaaa

601 gaattcacgg acattttcgc atgaatgggg aaagttgact cagttgactc tatttggttc

661 ctcagcctcg actgctttgc ttcatcttct ctatgggcca atgagctaat gagcacaatg

721 tgctgcgaaa taaagggata tctaatttat attattacat tataatatgt actagtgtgg

781 ttattggtaa ttgtacttaa ttttgatata taaagggtgg atctttttca ttttgaatca

841 gaattggaat tgcaacttgt ctcttgtcac tattacttaa tagtaattat atttcttatt

901 aacctttttt ttaagtcaaa acaccaagga caagaactac tcttcaaagg tatttcaagt

961 tatcatacgt ctcacacacg cttcacagtt tcaagtaaaa aaaaagaata ttacacaatg

1021 agacaagttt ggttctcttg gatcgttggt ttgttcttgt gtttcttcaa cgtttcttct

1081 gctgctccag ttaacactac tactgaagat gagactgctc aaattccagc tgaagctgtt

1141 attggttact ctgatttgga aggtgatttc gatgttgctg ttttgccatt ctctaactca

1201 actaacaacg gtttgttgtt catcaacacc actattgctt ctatcgctgc taaagaagag

1261 ggagtttctt tggaaaaaag agaggctgga tccaaaagaa agcccaccga gaacaacgaa

1321 gacttcaaca tcgtggccgt ggccagcaac ttcgcgacca cggatctcga tgctgaccgc

1381 gggaagttgc ccggcaagaa gctgccgctg gaggtgctca aagagatgga agccaatgcc

1441 cggaaagctg gctgcaccag gggctgtctg atctgcctgt cccacatcaa gtgcacgccc

1501 aagatgaaga agttcatccc aggacgctgc cacacctacg aaggcgacaa agagtccgca

1561 cagggcggca taggcgaggc gatcgtcgac attcctgaga ttcctgggtt caaggacttg

1621 gagcccatgg agcagttcat cgcacaggtc gatctgtgtg tggactgcac aactggctgc

1681 ctcaaagggc ttgccaacgt gcagtgttct gacctgctca agaagtggct gccgcaacgc

1741 tgtgcgacct ttgccagcaa gatccagggc caggtggaca agatcaaggg ggccggtggt

1801 gactaaccta ggacaggccc cttttccttt gtcgatatca tgtaattagt tatgtcacgc

1861 ttacattcac gccctcctcc cacatccgct ctaaccgaaa aggaaggagt tagacaacct

1921 gaagtctagg tccctattta ttttttttaa tagttatgtt agtattaaga acgttattta

1981 tatttcaaat ttttcttttt tttctgtaca aacgcgtgta cgcatgtaac attatactga

2041 aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag cagcccgggt

2101 gtttccgggt gtacaatatg gacttcctct tttctggcaa ccaaacccat acatcgggat

2161 tcctataata ccttcgttgg tctccctaac atgtaggtgg cggaggggag atatacaata

2221 gaacagatac cagacaagac ataatgggct aaacaagact acaccaatta cactgcctca

2281 ttgatggtgg tacataacga actaatactg tagccctaga cttgatagcc atcatcatat

2341 cgaagtttca ctaccctttt tccatttgcc atctattgaa gtaataatag gcgcatgcaa

2401 cttcttttct ttttttttct tttctctctc ccccgttgtt gtctcaccat atccgcaatg

2461 acaaaaaaat gatggaagac actaaaggaa aaaattaacg acaaagacag caccaacaga

2521 tgtcgttgtt ccagagctga tgaggggtat ctcgaagcac acgaaacttt ttccttcctt

2581 cattcacgca cactactctc taatgagcaa cggtatacgg ccttccttcc agttacttga

2641 atttgaaata aaaaaaagtt tgctgtcttg ctatcaagta taaatagacc tgcaattatt

2701 aatcttttgt ttcctcgtca ttgttctcgt tccctttctt ccttgtttct ttttctgcac

2761 aatatttcaa gctataccaa gcatacaatc aagcaattcc agatctgcca ccatggccaa

2821 gttgaccagt gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg

2881 gaccgaccgg ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg

2941 ggacgacgtg accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct

3001 ggcctgggtg tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc

3061 cacgaacttc cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg

3121 gcgggagttc gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca

3181 ggactgactt ttacctttgt tgtcttatgt gtttttactc caacacccct aaccctaatt

3241 ccacatagat ccaaccctaa ttccgcagag gtacaaataa acacaaccag ctagagatag

3301 cacaccacca tgaggctagg ttgcacataa cacaccaccg aattttttgc tacccacttt

3361 tcttcctctt tgtctctata ccaacccatt tagtacccac aaataaactc tggggtgtac

3421 ggtaccaagc ttcgccaacg agaatgcaac aaatgatgaa tatactgtga tgtagtgatt

3481 gctcattgaa taagtagcgt tattcgaagt aaagaataga aaatcacaag actggggtta

3541 aagctgcatc aaataggcaa atgtgcggcg tatcgtggtt tgcgcgcacg gattatctaa

3601 caagtttttc caccacttaa aaattgaggg agcgttccgc ataattataa ttgctttttt

3661 tgctgacatc ggcatcctga cttcttcttt tgtagtgctc tatttgtgag tggtgttaaa

3721 aaaacattca aagtggcatg ctattctaag gcaatatgga aacatgagag ttcctctttg

3781 aatatctgga gctccagcct attaggcaag ttttgtcaga ctgcttgtat ggaagaggtt

3841 ttcgggaaac catattcgac tcggtagagt aagagatatg tctgatttat cctcatatga

3901 aaggttttta agaatataag aatcaagtat cgaaatattt gcaaaatttt ggcactatgg

3961 ccgagtggtt aaggcgacag acttgaaatc tgttgggctc tgcccgcgct ggttcaaatc

4021 ctgctggtgt cgttattttt tggaaataac tttttttaac tagacaataa gcgtttagcc

4081 gggtaatatt tatttatata tatcggctcc ttccatggag ctgataccgt aagctcacct

4141 aatctgcgaa ttgctcgagt tttttatctt tctagaagat ctcctacaat attctcagct

4201 gccatggaaa atcgatgttc ttcttttatt ctctcaagat tttcaggctg tatattaaaa

4261 cttatattaa gaactatgct aaccacctca tcaggaaccg ttgtaggtgg cgtgggtttt

4321 cttggcaatc gactctcatg aaaactacga gctaaatatt caatatgttc ctcttgacca

4381 actttattct gcattttttt tgaacgaggt ttagagcaag cttcaggaaa ctgagacagg

4441 aattttatta aaaatttaaa ttttgaagaa agttcagggt taatagcatc cattttttgc

4501 tttgcaagtt cctcagcatt cttaacaaaa gacgtctctt ttgacatgtt taaagtttaa

4561 acctcctgtg tgaaattgtt atccgctcac aattccacac attatacgag ccggaagcat

4621 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc

4681 actgccaatt gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca

4741 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc

4801 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg

4861 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa

4921 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga

4981 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag

5041 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct

5101 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg

5161 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc

5221 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt

5281 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta

5341 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac

5401 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc

5461 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat

5521 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc

5581 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt

5641 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta

5701 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct

5761 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg

5821 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga

5881 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt

5941 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt

6001 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt

6061 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat

6121 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc

6181 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc

6241 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat

6301 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag

6361 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt

6421 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc

6481 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa

6541 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg

6601 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa

6661 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac

6721 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggcc

<210> SEQ ID NO: 3

<211> 7359

<212> PRT

<213> Artificial Sequence

<223> Nucleotide Sequence of the pSVO-Vic Vector

<400> 3

1 gcccctgcag ccgaattata ttatttttgc caaataattt ttaacaaaag ctctgaagtc

61 ttcttcattt aaattcttag atgatacttc atctggaaaa ttgtcccaat tagtagcatc

121 acgctgtgag taagttctaa accatttttt tattgttgta ttatctctaa tcttactact

181 cgatgagttt tcggtattat ctctattttt aacttggagc aggttccatt cattgttttt

241 ttcatcatag tgaataaaat caactgcttt aacacttgtg cctgaacacc atatccatcc

301 ggcgtaatac gactcactat agggagagcg gccgcaattc atctagataa tcgggtatga

361 ttaacagcga catagctatg tgattatctg ccccattact taaagtaagg tttcactttc

421 ggactaagga cgaatgacac aagacacaga acgctcacac ggttgaaaca ttttttttcc

481 tgatcctttt gaccctgttt gttgtccctc ttttacgatg ggagaattga atcatccgcc

541 attaaaccga atgtgatagt ttttccagtt tttcttcccc tccctacatc catttctaaa

601 gaattcacgg acattttcgc atgaatgggg aaagttgact cagttgactc tatttggttc

661 ctcagcctcg actgctttgc ttcatcttct ctatgggcca atgagctaat gagcacaatg

721 tgctgcgaaa taaagggata tctaatttat attattacat tataatatgt actagtgtgg

781 ttattggtaa ttgtacttaa ttttgatata taaagggtgg atctttttca ttttgaatca

841 gaattggaat tgcaacttgt ctcttgtcac tattacttaa tagtaattat atttcttatt

901 aacctttttt ttaagtcaaa acaccaagga caagaactac tcttcaaagg tatttcaagt

961 tatcatacgt ctcacacacg cttcacagtt tcaagtaaaa aaaaagaata ttacacaatg

1021 agacaagttt ggttctcttg gatcgttggt ttgttcttgt gtttcttcaa cgtttcttct

1081 gctgctccag ttaacactac tactgaagat gagactgctc aaattccagc tgaagctgtt

1141 attggttact ctgatttgga aggtgatttc gatgttgctg ttttgccatt ctctaactca

1201 actaacaacg gtttgttgtt catcaacacc actattgctt ctatcgctgc taaagaagag

1261 ggagtttctt tggaaaaaag agaggctgga tccaaaagat ctggtatcac tagaatccca

1321 ttgcataagg gtaagacttt gagaaaggcc ttgaaagaac atggtttgtt ggaagatttc

1381 ttgcagagac aacaatacgc cgtttcttct aagtattcct cattgggtaa agtggctaga

1441 gaaccattga cttcatactt ggattctcag tacttcggta agatctacat tggtactcca

1501 ccacaagaat tcaccgttgt tttcgatact ggttcttctg atttgtgggt tccatctatc

1561 tactgtagat ctaacgtgtg caagaaccat catagattcg atccaagaaa gtcctctacc

1621 ttcagaaatt tgggtaagcc attgtctatc cattacggta ctggttcaat ggaaggtttt

1681 ttgggttacg ataccgttac cgtttctaac atcgttgatc caaatcaaac cgttggtttg

1741 tctactgaac aaccaggtga agttttcacc tactctgaat tcgatggtat cttgggttta

1801 gcttatccat ctttggcttc cgaatattct gttccagtgt tcgataacat gatggacaga

1861 catttggttg ctcaggattt gttctctgtg tacatggaca gaaatggtca aggttctatg

1921 ttgactttgg gtgctattga tccatcttac tacactggtt cattgcattg ggttccagtt

1981 actgttcaac aatactggca attcactgtt gattctgtta ccattaacgg tgttgctgtt

2041 gcttgtgttg gtggttgtca agctattttg gatacaggta cttcagtttt gttcggtcca

2101 tcttctgata tcttgaagat ccaaaaagct atcggtgcta ccgaaaacag atatggtgaa

2161 ttcgacgtta actgcggttc tttgagatct atgccaactg ttgtgttcga gattaacggt

2221 agagattatc cattgtctcc atctgcttac acctctaaag atcaaggttt ctgtacttct

2281 ggtttccagg gtgataacaa ctcagaattg tggattttgg gtgacgtttt catcagagag

2341 tactactcag ttttcgacag agctaacaat agagttggtt tggctaaggc tatctgacct

2401 aggacaggcc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg cttacattca

2461 cgccctcctc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag

2521 gtccctattt atttttttta atagttatgt tagtattaag aacgttattt atatttcaaa

2581 tttttctttt ttttctgtac aaacgcgtgt acgcatgtaa cattatactg aaaaccttgc

2641 ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa gcagcccggg tgtttccggg

2701 tgtacaatat ggacttcctc ttttctggca accaaaccca tacatcggga ttcctataat

2761 accttcgttg gtctccctaa catgtaggtg gcggagggga gatatacaat agaacagata

2821 ccagacaaga cataatgggc taaacaagac tacaccaatt acactgcctc attgatggtg

2881 gtacataacg aactaatact gtagccctag acttgatagc catcatcata tcgaagtttc

2941 actacccttt ttccatttgc catctattga agtaataata ggcgcatgca acttcttttc

3001 tttttttttc ttttctctct cccccgttgt tgtctcacca tatccgcaat gacaaaaaaa

3061 tgatggaaga cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt

3121 tccagagctg atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc

3181 acactactct ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat

3241 aaaaaaaagt ttgctgtctt gctatcaagt ataaatagac ctgcaattat taatcttttg

3301 tttcctcgtc attgttctcg ttccctttct tccttgtttc tttttctgca caatatttca

3361 agctatacca agcatacaat caagcaattc cagatctgcc accatggcca agttgaccag

3421 tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct ggaccgaccg

3481 gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc gggacgacgt

3541 gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc tggcctgggt

3601 gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt ccacgaactt

3661 ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg ggcgggagtt

3721 cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc aggactgact

3781 tttacctttg ttgtcttatg tgtttttact ccaacacccc taaccctaat tccacataga

3841 tccaacccta attccgcaga ggtacaaata aacacaacca gctagagata gcacaccacc

3901 atgaggctag gttgcacata acacaccacc gaattttttg ctacccactt ttcttcctct

3961 ttgtctctat accaacccat ttagtaccca caaataaact ctggggtgta cggtaccaag

4021 cttcgccaac gagaatgcaa caaatgatga atatactgtg atgtagtgat tgctcattga

4081 ataagtagcg ttattcgaag taaagaatag aaaatcacaa gactggggtt aaagctgcat

4141 caaataggca aatgtgcggc gtatcgtggt ttgcgcgcac ggattatcta acaagttttt

4201 ccaccactta aaaattgagg gagcgttccg cataattata attgcttttt ttgctgacat

4261 cggcatcctg acttcttctt ttgtagtgct ctatttgtga gtggtgttaa aaaaacattc

4321 aaagtggcat gctattctaa ggcaatatgg aaacatgaga gttcctcttt gaatatctgg

4381 agctccagcc tattaggcaa gttttgtcag actgcttgta tggaagaggt tttcgggaaa

4441 ccatattcga ctcggtagag taagagatat gtctgattta tcctcatatg aaaggttttt

4501 aagaatataa gaatcaagta tcgaaatatt tgcaaaattt tggcactatg gccgagtggt

4561 taaggcgaca gacttgaaat ctgttgggct ctgcccgcgc tggttcaaat cctgctggtg

4621 tcgttatttt ttggaaataa ctttttttaa ctagacaata agcgtttagc cgggtaatat

4681 ttatttatat atatcggctc cttccatgga gctgataccg taagctcacc taatctgcga

4741 attgctcgag ttttttatct ttctagaaga tctcctacaa tattctcagc tgccatggaa

4801 aatcgatgtt cttcttttat tctctcaaga ttttcaggct gtatattaaa acttatatta

4861 agaactatgc taaccacctc atcaggaacc gttgtaggtg gcgtgggttt tcttggcaat

4921 cgactctcat gaaaactacg agctaaatat tcaatatgtt cctcttgacc aactttattc

4981 tgcatttttt ttgaacgagg tttagagcaa gcttcaggaa actgagacag gaattttatt

5041 aaaaatttaa attttgaaga aagttcaggg ttaatagcat ccattttttg ctttgcaagt

5101 tcctcagcat tcttaacaaa agacgtctct tttgacatgt ttaaagttta aacctcctgt

5161 gtgaaattgt tatccgctca caattccaca cattatacga gccggaagca taaagtgtaa

5221 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgccaat

5281 tgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg

5341 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc

5401 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac

5461 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa

5521 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca

5581 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc

5641 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata

5701 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta

5761 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca

5821 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga

5881 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg

5941 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg

6001 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg

6061 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag

6121 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa

6181 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat

6241 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc

6301 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc

6361 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc

6421 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc

6481 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc

6541 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt

6601 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc

6661 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa

6721 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt

6781 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg

6841 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc

6901 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa

6961 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt

7021 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt

7081 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag

7141 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta

7201 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat

7261 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat

7321 catgacatta acctataaaa ataggcgtat cacgaggcc

<---

Claims (30)

1. Universal recombinant vector pSVO for creating plasmid genetic constructs that provide synthesis and secretion of target proteins in yeast cells of the species Kluyveromyces lactis, having the nucleotide sequence SEQ ID No. 1 and containing, in accordance with the physical and genetic map shown in FIG. 1d, the following elements: - autoinducible hybrid promoter 341 to 1017, including the core sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the regulatory sequence of the isocitrate lyase (ICL) promoter; - hybrid signal sequence (Ost2) to ensure the secretion of the protein into the extracellular space from 1018 to 1287; - region of insertion of the target gene (MCS) from 1297 to 1353; - terminator region of the S. cerevisiae gene (CYC) from 1360 to 1638; - promoter of the alcohol dehydrogenase gene (ADH1 promoter) from 1647 to 2339; - gene for resistance to zeocin (BleoR) from 2360 to 2734; - 5 'sequence of the isocitrate lyase promoter gene (ICL-5') from 2735 to 3694; - origin of replication (ori) from 4504 to 5092; - gene for ampicillin resistance (AmpR) from 5263 to 6122. 2. Plasmid genetic construct pSVO-Gluc, providing the synthesis and secretion of the target luciferase protein from Gaussia princeps in yeast cells of the species Kluyveromyces lactis, having the nucleotide sequence SEQ ID No. 2 and containing, in accordance with the physical and genetic map shown in FIG. 3, the following items: - autoinducible hybrid promoter 341 to 1017, including the core sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the regulatory sequence of the isocitrate lyase (ICL) promoter; - hybrid signal sequence (Ost2) to ensure the secretion of the protein into the extracellular space from 1018 to 1287; - the sequence of the luciferase gene from Gaussia princeps (Gluc) from 1300 to 1803; - the terminator region of the S. cerevisiae gene (CYC) from 1813 to 2091; - promoter of the alcohol dehydrogenase gene (ADH1 promoter) from 2100 to 2792; - gene for resistance to zeocin (BleoR) from 2813 to 3187; - 5 'sequence of the isocitrate lyase promoter gene (ICL-5') from 3188 to 4147; - origin of replication (ori) from 4957 to 5545; - gene for ampicillin resistance (AmpR) from 5716 to 6576. 3. Plasmid genetic construct pSVO-Vic, providing synthesis and secretion of the target protein alpaca prochymosin in yeast cells of the species Kluyveromyces lactis, having the nucleotide sequence SEQ ID No. 3 and containing, in accordance with the physical and genetic map shown in FIG. 5, the following items: - autoinducible hybrid promoter 341 to 1017, including the core sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter and the regulatory sequence of the isocitrate lyase (ICL) promoter; - hybrid signal sequence (Ost2) to ensure the secretion of the protein into the extracellular space from 1018 to 1287; - the sequence of the gene for alpaca prochymosin (Vic) from 1300 to 2397; - terminator region of the S. cerevisiae gene (CYC) from 2405 to 2682; - promoter of the alcohol dehydrogenase gene (ADH1 promoter) from 2691 to 3383; - gene for resistance to zeocin (BleoR) from 3404 to 3778; - 5 'sequence of the isocitrate lyase promoter gene (ICL-5') from 3779 to 4738; - origin of replication (ori) from 5548 to 6136; - gene for ampicillin resistance (AmpR) from 6307 to 7167.

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