A DNA fragment and its vector as well as a breeding method and application of a new breed of lean pigs

Abstract

The present invention discloses a novel DNA fragment and a vector containing the DNA fragment, as well as a method for preparing a lean meat gene-edited pig by using the DNA fragment or the vector, a method for breeding a new breed of lean meat pigs and the application of the DNA fragment or the vector in the field of animal husbandry, so as to obtain a new breed of lean meat high-quality pigs with higher intramuscular fat, faster growth rate and higher fertility.

Description

Technical Field

[0001] The present invention relates to the field of animal breeding, and in particular to a DNA fragment and its carrier, and a breeding method and application of a new breed of lean meat pigs. Background Art

[0002] In recent years, people's demand for pork consumption has shifted from quantity to quality. High-quality and delicious pork is very popular among consumers. Coupled with my country's traditional cooking culture, there is a strong demand for high-end pork ingredients. At present, more than 90% of the pork supplied on the market are crossbred from lean-type pigs of three or four lines. They usually have high production efficiency, high lean meat rate, low fat content, and poor flavor and taste. Studies have shown that on average, every 1% increase in lean meat rate will reduce the intramuscular fat (IMF) content by 0.07%, which leads to a continuous decline in pork quality (de Vries et al., 1994). Some scholars believe that the best taste experience can be achieved when the IMF content of the longissimus dorsi muscle between the second and third intercostal spaces of pigs is 2% to 3.5% (Blanchard et al., 1999, Fernandez et al., 1999, Kipfmüller et al., 2000), while the IMF content of the longissimus dorsi muscle of lean pigs is generally 1-1.5%, and only a few reach the 2% threshold level ( Link, Werner, & Wicke, 2007). Due to their slow growth rate, low feed conversion rate, high fat content, low production efficiency, and low meat yield, high-quality local pig breeds in my country are not popular among farmers and vendors, and their market share is less than 2%. Cultivating lean meat pig breeds with high production efficiency, low cost, and high IMF is the future development trend of pig breeding in my country.

[0003] Glucose is the main fuel for mammals to maintain normal body functions. Gluconeogenesis is the process of converting lactate, amino acids, glycerol and other non-carbohydrate substrates into glucose, and plays a vital role in glucose homeostasis. Phosphoenolpyruvate carboxykinase (PCK) is considered to be a key enzyme for gluconeogenesis in the liver and kidney gluconeogenic organs. It is highly conserved in different species. All vertebrates have two PCK isoforms, namely mitochondrial form (PCK2) and cytoplasmic form (PCK1). In pigs, PCK1 and PCK2 have an amino acid similarity of 71% (NCBI analysis). PCK2 is widely expressed in different tissues and organs, while PCK1 is highly enriched in gluconeogenic organs such as kidney, liver and intestine. Most of the current knowledge about PCK comes from studies on mice and rats, among which PCK1 is the main component of phosphoenolpyruvate carboxykinase in the liver and is the first rate-limiting step in regulating hepatic gluconeogenesis, responsible for catalyzing the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) (Yang et al., 2009). Studies have shown that overexpression of PCK1 in mouse liver (7-fold) leads to excessive glucose production (Beale et al., 2017); mice with systemic PCK1 knockout die within 2-3 days after birth, and their tricarboxylic acid cycle rate is only about 10% of the normal value (She et al., 2000). PCK1 expression is regulated by cAMP, protein kinase and glucocorticoids. Under starvation conditions, cAMP or protein kinase A (PKA) is activated, upregulating the PCK1 gene by 5 and 30 times, respectively (Liu et al., 1991, glucocorticoids can upregulate PCK1 gene expression similar to cAMP, and insulin inhibits PCK1 gene expression. The half-life of the 3'UTRPCK1 mRNA containing the pig's own gene is only 1.2 hours (Hajarnis et al., 2005).

[0004] Triglyceride (TG) is the main lipid storage and energy source. Glycerol 3-phosphate is an essential precursor of TG, which can be synthesized from pyruvate, called glycerogenesis. Glycerogenesis is essential for maintaining the triglyceride / fatty acid cycle. Glycerogenesis is a truncated gluconeogenesis that shares many gluconeogenesis enzymes. PCK1 is also the rate-limiting enzyme in glycerogenesis, catalyzing the conversion of oxaloacetate OAA to PEP, and then to 3-phosphoglycerate in adipose tissue and liver. Studies have shown that adipose-specific knockout of PCK1 leads to lipodystrophy in a small number of mice due to loss of glycerogenesis function, and reduced fatty acid re-esterification; overexpression of PCK1 in adipose tissue increases fatty acid re-esterification, leading to obesity (Beale et al., 2017). The phenotype regulated by PCK1 depends largely on the differential expression of PCK1 and key enzymes of metabolic pathways in different organs, such as glucose-6-phosphatase (G6PC), which is the last step in regulating liver gluconeogenesis and is highly expressed in the liver, but almost not expressed in adipose tissue. Therefore, PCK1 in adipose tissue mainly flows to the pathway of glycerogenesis. PCK is also the link between upstream gluconeogenesis, glycolysis and downstream tricarboxylic acid cycle metabolites, playing the role of glucose biosynthesis (gluconeogenesis), glycerol biosynthesis (glycerologenesis), serine biosynthesis, and amino acid metabolism in different tissues and organs (Yu et al., 2021).

[0005] Compared with lean pig breeds, local pig breeds deposit more fat, have higher subcutaneous and intramuscular fat production and desaturation capacity, and lower lipolysis capacity. Transcriptomic and proteomic studies have shown that the expression of genes and proteins related to adipocyte differentiation, adipogenesis, desaturation, lipolysis, fatty acid transport and immune response in intramuscular fat of local pigs is upregulated compared with lean pigs, such as fatty acid synthase (FAS), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PDH), acetyl-CoA carboxylase (ACACA) and stearoyl-CoA-desaturase (SCD) related to adipogenesis. Hakimi et al. (2007) used 2kb mouse skeletal muscle a-actin promoter and mouse 1976bp PEPCK-C cDNA and bovine bGH downstream regulatory region, and found that specific overexpression of PEPCK-C gene in mouse skeletal muscle greatly improved the mouse's exercise capacity and lactate clearance ability (8.12Vs.3.7mM). The content of muscle triglyceride in transgenic mice increased with the increase of PEPCK-C expression. Unfortunately, the transgenic mice increased their food intake by 60%, but their body weight was only half of that of the control mice, and the excessive feed consumed could not be converted into valuable muscle (Hakimi et al., 2007, Hanson and Hakimi, 2008). Then Chinese scientists applied the skeletal muscle-specific expression of PEPCK-C technology to Tibetan miniature pigs, and constructed a promoter containing 2kb of pig skeletal muscle α-actin, driven by mouse skeletal muscle-specific MCK enhancer, pig PEPCK-C cDNA and the 3' untranslated region of the bovine growth hormone gene (bGH). They used random integration to prepare PEPCK-C gene-transgenic Tibetan pigs, and found that the PEPCK-C protein content in the psoas, gluteal, leg and hind leg muscles was significantly higher than that in these parts of the muscles of transgenic negative pigs. However, there was no significant difference in the PEPCK-C protein content in the muscle tissue of the four parts of the transgenic positive pigs. The authors found that the transgenic pork had increased marbling, increased oil red staining area of ​​the hind leg muscle and psoas major muscle, and significantly increased total fat content (p < 0.05) (Ren et al., 2017); unlike mice, the weight gain of piglets with the PEPCK-C gene at 2 and 3 months of age was 14.53% and 9.28% higher than that of ordinary pigs (only 2 pigs were tested), but the growth rate gradually decreased during the subsequent growth. After adulthood, the transgenic cloned pigs all had reproductive problems such as prolonged estrus and repeated failure of mating after estrus, so the breeding value was not great (Gao Hui, 2013). Local pigs have high intramuscular fat, and further increasing intramuscular fat on this basis is not meaningful, but will make consumers worry about health problems caused by excessive fat intake.

[0006] Therefore, it is necessary to study and prepare a high-quality fast-growing lean meat pig breed with higher intramuscular fat, faster growth rate, and higher reproductive capacity, successfully overcome the shortcomings of the existing technology, and make it have greater breeding value and transformation potential. Summary of the invention

[0007] The purpose of the present invention is to provide a new DNA fragment and a vector containing the DNA fragment, as well as a method for preparing lean gene-edited pigs by using the DNA fragment or vector, a method for breeding new lean pig varieties and the application of the DNA fragment or vector in the field of animal husbandry, so as to obtain a lean high-quality pig variety or method with higher intramuscular fat, faster growth rate and higher fertility.

[0008] According to the first aspect of the present invention, a DNA fragment is provided, which can be used to prepare gene-edited pigs and ectopically express the PCK1 gene in the pigs, so that the gene-edited pigs prepared by using the DNA fragment can obtain higher intramuscular fat.

[0009] In certain embodiments, the DNA fragment comprises a sequence as shown in SEQ ID NO:4. The DNA fragment is a brand-new DNA fragment after optimization. The gene-edited pig prepared by it can achieve extensive ectopic expression of the PCK1 gene in its tissues, and the gene-edited pig has higher intramuscular fat, faster growth rate, higher fertility and stable inheritance, and has greater breeding value and transformation potential. Compared with the DNA fragments used in the prior art construction, the -2kb region upstream of the transcription start site of the human or pig ASMA gene is directly used as the promoter, while the -2.4 to -2.0kb enhancer region, the first exon, the first intron and the 3' end downstream regulatory region are missing, resulting in a decrease in the expression efficiency of the target gene; the DNA fragment in this application adopts a combined promoter strategy to minimize the introduction of exogenous genes, while creating a better genomic environment, improving biosafety, making up for the weak defects of the pig endogenous ASMA promoter, and increasing the expression level and tissue extensiveness of PCK1. Moreover, the original 3'UTR of PCK1 gene was replaced in this DNA fragment, thus overcoming the defect that the half-life of PCK1 mRNA containing the complete 3'UTR of its own gene is only 1.2 hours (Hajarnis et al., 2005), and enabling the long-term stable expression of PCK1 mRNA.

[0010] In certain embodiments, the DNA fragment is site-specifically integrated into chromosome 13 between 65754555-65758570bp of the Sscrofa11.1 version of the genome of the gene-edited pig.

[0011] According to the second aspect of the present invention, there is provided an application of the DNA fragment in preparing a gene-edited pig that can widely ectopically express the PCK1 gene. Widespread ectopic expression refers to ectopic expression in any one or more tissues of the testicles, kidneys, lungs, spleen, brain, intestines, stomach, heart, and skeletal muscle of the gene-edited pig, but is not limited to the above-mentioned tissues. Therefore, compared with the prior art which can only translocate PEPCK-C or PCK1 genes to skeletal muscle for specific expression and only increase glycerol noeogenesis in skeletal muscle, the present application not only increases skeletal muscle glycerol noeogenesis, but also translocates the endogenous PCK1 gene of pigs to tissues and organs such as the lungs, testicles, stomach, spleen, heart, glands, uterus, brain, and skin, increases the expression of endogenous PCK1 in digestive organs such as the liver, stomach, jejunum, ileum, and cecum, reduces the expression of PCK1 in the kidneys, reshapes the pathways of glucose and lipid metabolism in pigs, increases the energy synthesis and reserve efficiency of corresponding tissues and organs of pigs, and gives full play to the PCK1's ability to regulate genes in the body more widely, ultimately increasing the intramuscular fat content of gene-edited pigs, improving semen quality, improving later growth rate, and improving embryo quality and reproductive performance.

[0012] According to the third aspect of the present invention, a vector containing the DNA fragment is provided, and the nucleotide sequence of the vector is shown in SEQ ID NO: 3. Thus, by transfecting cells with the vector and screening, a positive cell line in which the DNA fragment contained in the vector is site-specifically integrated between 65754555-65758570bp of chromosome 13 can be obtained, and then the positive cell line is used as a nuclear donor cell for nuclear transplantation to prepare the corresponding gene-edited pig, and the obtained gene-edited pig can achieve extensive ectopic expression of the PCK1 gene in various tissues, thereby changing the in vivo regulation of the gene-edited pig, especially the regulation of gluconeogenesis and glycerol neogenesis in various tissues, regulating the metabolic situation, and ultimately improving the intramuscular fat content of the gene-edited pig, improving the semen quality, improving the late growth rate, improving the embryo quality and reproductive performance, etc.

[0013] According to the fourth aspect of the present invention, a vector containing the DNA fragment is provided, and the nucleotide sequence of the vector is shown in SEQ ID NO: 5 or SEQ ID NO: 6. By transfecting cells with the vector and screening, a positive cell line with site-specific integration between 65754555-65758570bp of chromosome 13 can be obtained, and then the positive cell line can be used as a nuclear donor cell for nuclear transplantation to prepare the corresponding gene-edited pig, and the obtained gene-edited pig can ectopically express the PCK1 gene in its tissue.

[0014] According to the fifth aspect of the present invention, a method for preparing a lean meat type gene-edited pig is provided, wherein the method obtains a lean meat type high-quality gene-edited pig that can widely ectopically express the PCK1 gene in tissues by integrating a vector with a nucleotide sequence such as SEQ ID NO:3 into between 65754555-65758570bp of chromosome 13 of pig, and the tissues include any one or more tissues of testis, kidney, lung, spleen, brain, intestine, stomach, heart, and skeletal muscle, but not limited to the above tissues. Or by integrating a vector with a nucleotide sequence such as SEQ ID NO:5 or SEQ ID NO:6 into between 65754555-65758570bp of chromosome 13 of pig, a high-quality lean meat type gene-edited pig that can ectopically express the PCK1 gene in tissues is obtained. As a result, the lean meat type gene-edited pig has higher intramuscular fat, faster growth rate, higher fertility and stable inheritance, and has greater breeding value and transformation potential.

[0015] According to the sixth aspect of the present invention, a method for breeding a new breed of lean meat pigs is provided, and the method comprises the following steps: S1: first synthesizing the DNA fragment, and then adding homologous arms to both ends of the obtained DNA fragment to construct a homologous recombination vector, wherein the homologous arms can undergo homologous recombination with the sequence between 65754555-65758570bp of chromosome 13 of the pig genome Sscrofa11.1 version; or directly constructing and synthesizing the vector; S2: transfecting the obtained vector into pig cells to obtain a site-specific integrated pig positive cell line; S3: using the obtained pig positive cell line as a donor cell for nuclear transplantation to obtain a primary gene-edited pig; S4: breeding the obtained primary gene-edited pig to obtain a new breed of lean meat quality pigs. By using the vector for site-specific integration to prepare the primary gene-edited pig, it can be stably inherited to the next generation, thereby achieving pig breeding and obtaining a new breed of lean meat quality pigs. The existing technology uses random integration technology for exogenous fragments, and the integration site and copy number are uncontrollable, which affects the expression of the target gene and the safety of the endogenous gene. Compared with the existing technology, the present application uses site-specific integration technology mediated by homologous recombination, which can be inserted into the target site through natural recombination or with the help of various gene editing tools to assist homologous recombination, which is conducive to obtaining pigs with completely identical genotypes, the same genomic environment, and good consistency in PCK1 gene expression, which is conducive to the preparation of a new breed of high-quality pigs with completely consistent genotypes and phenotypes. Breeding population.

[0016] According to a seventh aspect of the present invention, there is provided a use of the DNA fragment or vector for increasing the intramuscular fat content of lean pigs.

[0017] According to the eighth aspect of the present invention, there is provided an application of the DNA fragment or vector in increasing the ectopic expression of PCK1 gene in various tissues of lean pigs, wherein the tissues include any one or more of testicles, kidneys, lungs, spleen, brain, intestines, stomach, heart, and skeletal muscle, but are not limited to the above tissues.

[0018] According to the ninth aspect of the present invention, there is provided a use of the DNA fragment or vector in breeding a new breed of lean meat pigs.

[0019] According to the tenth aspect of the present invention, there is provided a use of the DNA fragment or vector for improving the intramuscular fat, later growth rate and reproductive performance of lean pigs.

[0020] Beneficial effects of this application:

[0021] 1. The expression level of the CE combined regulatory element DNA fragment (sequence shown in SEQ ID NO: 4) obtained in the present application in skeletal muscle is significantly higher than that in the prior art;

[0022] 2. The intramuscular fat content of the longissimus dorsi muscle of the gene-edited high-quality pigs prepared in this application is 50% higher than that of the control pigs, reaching 3.11% (wild-type pig IMF 2.0%), which is significantly higher than the existing technology and reaches the standard of lean high-quality pigs (2.5%). <IMF<3.5%)((Blanchard et al.,1999,Fernandez et al.,1999,Kipfmül ler etal.,2000);

[0023] 3. The high-quality lean meat gene-edited pigs obtained by this application have a growth rate of 25.75% higher than that of the control pigs in the later stage of growth, reaching 1210.44g / day, which is significantly higher than the 962.16g / day of the control pigs, and the reproductive performance is also improved;

[0024] 4. The HC gene-edited pigs obtained in this application have the potential to improve the reproductive capacity of breeding pigs, and their semen quality and reproductive capacity of sows after mating are significantly improved;

[0025] 5. The prior art increases the fat content in the skeletal muscle of local pigs (Tibetan pigs), which is of little significance to local pigs with rich intramuscular fat, but increases consumers' concerns about health problems caused by excessive fat intake; the present application migrates the glycerol angiotensin metabolism pathway to the skeletal muscle of lean pigs, making up for the disadvantage of poor fat deposition in lean skeletal muscle, improving the quality, taste and juiciness of lean pork, and making it more valuable as a commodity;

[0026] 6. The prepared HM-type gene-edited pigs can highly express the PCK1 gene in skeletal muscle, and can significantly increase the intramuscular fat content compared with the control group; the PCK1 gene can also be ectopically expressed in other tissues of the pig, but the expression level is not as high as that of the HC-type gene-edited pigs;

[0027] 7. HO-type cell lines can be used to prepare HO-type gene-edited pigs, which ectopically express the PCK1 gene only in skeletal muscle. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of the comparison of skeletal muscle promoter activity are shown in Figure a. The schematic diagram of the skeletal muscle promoter structure from three sources is shown in Figure b. The different letters in Figure b indicate significant differences (p<0.05).

[0029] Figure 2 The results of the prediction of cis-acting elements of the porcine skeletal muscle ASMA promoter: Figure a shows the online prediction of the Ssapi promoter characteristics by Cister software, and Figure b shows the functional element characteristics with a threshold of 0.3 or above;

[0030] Figure 3 The results of the effect of intron Intron1 on the activity of porcine skeletal muscle ASMA promoter are shown in Figure a. The construction map of porcine skeletal muscle promoter is shown in Figure b. The different letters in Figure b indicate significant differences (p<0.05).

[0031] Figure 4 The results of the effect of exogenous enhancers on the activity of pig skeletal muscle promoters are shown in Figure a. The results of the construction of skeletal muscle promoters are shown in Figure b. Different letters in Figure b indicate significant differences (p<0.05).

[0032] Figure 5 The results of the activity analysis of skeletal muscle promoters on different cells are shown in Figure a. Figure a shows the transfection effect of C2C12 myotubes cells, and Figure b shows the comparison of promoter activity in C2C12 myotubes cells. In Figure b, * indicates significant difference (p<0.05), and ** indicates extremely significant difference (p<0.01).

[0033] Figure 6 The results of the conservation and expression profile analysis of the porcine PCK1 gene are shown in Figure a. The structure of the porcine PCK1 genome is shown in Figure b. The evolutionary tree of the PCK1 gene is shown in Figure c. The homology comparison results of the PCK1 proteins from porcine, human, mouse, chicken and cattle sources are shown in Figure d. The transcriptome expression analysis results of the PCK1 gene in Duroc pigs are shown in Figure d.

[0034] Figure 7The results of PCK1 gene expression driven by porcine ASMA promoter: Figure a shows the construction process of HR-Cssap-PCK1 plasmid, Figure b shows the structure of three HR sites (chr.13:65754555-65758570(-)), and Figure c shows the results of three plasmids overexpressing PCK1 gene in C2C12myotubes: Different letters in Figure c indicate significant differences (p<0.05);

[0035] Figure 8 The results of screening cell lines specifically expressing PCK1 gene in skeletal muscle are shown in Figure a: the structure of site-specific integration of HR locus, Figure b: PCR identification of HR-Cssap-PCK1 monoclonal cells, Figure c: PCR identification of HR-Mssap-PCK1 monoclonal cells, Figure d: PCR identification of HR-Ssap-PCK1 monoclonal cells, Figure e: Sanger sequencing, Figure f: statistics of gene KI efficiency of three cell lines, and Figure g: Cre-Loxp marked deletion cell diagram;

[0036] Fig. 9 Preparation and detection results of gene-edited pigs expressing ectopic PCK1: Figure A is a schematic diagram of the location of the site-specific integration site, Figure B is a picture of a gene-edited positive pig, and Figure C is a detection result diagram. In the figure, the individual numbers of HM type (MCK enhancer) are: 22-80300, 22-80301, 22-80400, 22-800402, 22-800403, D15, D21, D14, and the amplification size is 1293bp; the individual numbers of HC type (CE) enhancers are: 22-800401, 22-800500, 22-800502, D6, D5, D7, D10, D11, D12, D1, D18, and the amplification size is 1387bp;

[0037] Fig.10 This is a graph comparing the expression levels of the target gene PCK1 in gene-edited adult boars of HC and HM types;

[0038] Fig.11 These are the test results of the high-quality performance and production performance of the F1 generation of gene-edited pigs that are gene-edited positive pigs: Figures A and B are the comparison of the expression levels of the PCK1 gene in skeletal muscle: Figure A is the longissimus dorsi muscle, and Figure B is the biceps femoris muscle. In the figures, ** indicates extremely significant difference p<0.01, *** indicates extremely significant difference p<0.001, and **** indicates very, very significant difference p<0.0001. Figure C is the comparison of intramuscular fat content. Figures D and E are the comparison of growth rates at different growth stages (0-169dVS.169-196d), and * indicates significant difference p<0.05. Figure F is the comparison of body shape and appearance, where HC is HC type F1 generation high-expression gene-edited pig, HM is HM type F1 generation gene-edited pig, and WT is the control pig. DETAILED DESCRIPTION

[0039] The invention is further described in detail below with reference to the accompanying drawings and examples. Unless otherwise specified, the reagents and carrier tools used in this application are all commercially available.

[0040] Example 1 Skeletal muscle ASMA promoter optimization

[0041] 1.1 Analysis of ASMA promoter in skeletal muscle from different sources

[0042] Download the 5'UTR sequence of the ASMA gene coding region of pig, human and mouse from the UCSC official website (https: / / genome.ucsc.edu / ), which is about 3.0-3.5 kb. Use PCR technology to amplify the target fragment of the ASMA regulatory region (such as Figure 1 As shown in a, Figure 1 a, Ssapi represents porcine skeletal muscle ASMA promoter, Msapi represents mouse skeletal muscle ASMA promoter, and Hsapi represents human skeletal muscle ASMA promoter). PCR amplification was performed using the primer pairs in Table 1, and the PCR products were then cloned into the saII site in the dual luciferase pEZX vector MSC to construct vectors pEZX-Ssapi, pEZX-Hsapi, and pEZX-Msapi. The constructed vector plasmid was transfected into C2C12 cells using LTX transfection reagent at a plasmid:liposome ratio of 1:3. It was found that the mouse skeletal muscle ASMA promoter Msapi had the highest activity, followed by the human skeletal muscle ASMA promoter Hsapi, while the porcine skeletal muscle ASMA promoter Ssapi had the lowest activity (results as shown in Figure 1 As shown in b, Figure 1 In b, Ssapi represents the pEZX-Ssapi vector transfection group, Hsapi represents the pEZX-Hsapi vector transfection group, Msapi represents the pEZX-Msapi vector transfection group, and Control represents the pEZX blank plasmid transfection control group).

[0043] Table 1 PCR amplification primer pairs

[0044]

[0045] 1.2 Effect of the first intron on the activity of porcine promoter

[0046] In order to better understand the characteristics of the porcine skeletal muscle ASMA promoter Ssapi and obtain higher promoter activity, this study used Cister software (https: / / zlab.bu.edu / -mfrith / cister.shtml) to predict the common regulatory sites of the promoter online. The results showed that these common cis-acting elements are mainly concentrated between 2.0kb and 3.0kb, including TATA box, Sp1, CRE, ERE, NF-1, E2F, Mef-2, Myf, CCAAT box and AP-1 (such as Figure 2 a), where Myf is myogenin and Mef-2 is myocyte promoting factor, which regulate myocyte generation and differentiation respectively. Generally, cis-acting elements are distributed in any position and any direction in the promoter. These functional elements can be recognized by transcription factors, thereby regulating the activity of the promoter. Among these predicted cis-acting elements, there are three serum response factor (SRF) elements, which are very important for the growth and development of skeletal muscle; there are two transcription enhancer factors (Tef), which are conducive to the expression of subsequent genes ( Figure 2 b).

[0047] Introns are non-coding RNAs in eukaryotic cells and are of great significance for the regulation of gene expression. In order to study the effect of the first intron (Intron1) on the promoter activity of porcine skeletal muscle ASMA, this experiment constructed a promoter sequence without introns into the saII site of MSC in the pEZX expression vector (such as Figure 3 As shown in Figure 1, Ssapi indicates that the first intron is present, and Ssap indicates that the first intron is not present), the vector pEZX-Ssap was constructed. Then the pEZX-Ssapi vector containing the first intron, the pEZX-Ssap vector without the first intron, and the blank pEZX vector were transfected into cells and tested. The results are shown in Figure 1. Figure 3 As shown in b, the results showed that the first intron Intron1 could significantly reduce the activity of porcine skeletal muscle ASMA promoter by about 44.2% (the results are shown in Figure 3 As shown in b, Ssap represents the pEZX-Ssap vector transfection group, Ssapi represents the pEZX-Ssapi vector transfection group, and Control represents the pEZX blank plasmid transfection control group), which reveals the negative regulatory effect of Intron1 on the ASMA promoter of porcine skeletal muscle.

[0048] 1.3 Effects of exogenous enhancers on porcine promoter activity

[0049] Enhancers regulate the transcription and expression of related genes through spatial folding or interaction of chromosomes. In order to make up for the weak regulatory ability of the porcine ASMA promoter, this experiment placed the ubiquitously expressed CMV enhancer (CE) and the skeletal muscle-specific expression MCK enhancer at the 5' end of the porcine skeletal muscle ASMA promoter Ssap (such as Figure 4 As shown in a, Cssap in the figure indicates a CMV enhancer structure, and Mssap indicates a MCK enhancer structure) speI site, C2C12 cells were used to verify the effect of exogenous enhancers on the activity of the porcine skeletal muscle ASMA promoter. The results are shown in Figure 4 As shown in b, the results showed that the CMV enhancer increased the activity of porcine ASMA by 3.31 times, while the MCK enhancer had no significant difference in the effect on ASMA compared with the control group ( Figure 4 b, in the figure, Cssap indicates the detection result of containing CMV enhancer, Mssap indicates the detection result of containing MCK enhancer, Ssap indicates the detection result of not containing enhancer, and Control indicates the pEZX blank plasmid control group).

[0050] In C2C12 myotubes cells, the activities of promoters from other sources were compared. The results are as follows Figure 5 As shown: It was found that the CE enhancer combination of porcine ASMA promoter (abbreviated as Cssap) had the highest activity. After adding the CMV enhancer, the activity of the porcine skeletal muscle ASMA promoter increased 2.84 times compared with the original Ssap. Moreover, the promoter activities of human and mouse promoters were significantly increased in the absence of Intron1, among which the human promoter had the strongest activity; while the MCKenhancer enhancer had almost no effect on the activity of the ASMA promoter (abbreviated as Mssap).

[0051] In summary, the best ASMA promoter activity can be obtained by using the CE combined regulatory element: that is, a porcine skeletal muscle ASMA promoter without the first intron is selected, and a CMV enhancer is added to the 5' speI site of the promoter, and finally an optimized porcine skeletal muscle ASMA promoter combined regulatory element (represented by "Cssap") is obtained. The nucleotide sequence of the ASMA promoter (Cssap) containing the CE combined regulatory element is shown in SEQ ID NO: 1.

[0052] Example 2 Cloning of PCK1 gene and screening of positive cell lines expressing PCK1 gene 2.1 Conservation and expression profile analysis of porcine PCK1 gene

[0053] The porcine PCK1 gene encodes PCK1 protein. The gene is 6016 bp long and is located at positions 57,930,507-57,936,523 on porcine chromosome 17. It contains 10 exons and 9 introns ( Figure 6 a), where the coding sequence is 1869 bp long and can be translated into 622 amino acids. The evolutionary history of the PCK1 gene in the Ensembl database shows that there are 457 genes homologous to the pig PCK1 gene, totaling 425 species, among which domestic pigs and wild boars have the highest gene homology, followed by whales and even-toed ungulates ( Figure 6 b). By comparing the amino acid sequences of PCK1 from pig, human, mouse, chicken and cattle, it was found that the protein is conserved among the above species ( Figure 6 c). The similarity of porcine PCK1 protein with human, mouse, chicken and bovine proteins was 86.5%, 86.8%, 80.4% and 89.9%, respectively. In addition, in the transcriptome sequencing of Duroc pigs, among all non-liver and adipose tissues, the expression of PCK1 in the renal cortex was the highest, followed by the intestinal tissue; a small amount of PCK1 was expressed in the omentum of boars, but not or at very low levels in sows; no expression of the PCK1 gene was detected in other tissues ( Figure 6 d, data from The Roslin Institute).

[0054] 2.2 Porcine ASMA promoter drives PCK1 gene expression

[0055] In order to prove that the porcine skeletal muscle ASMA promoter can promote the expression of PCK1 gene, this experiment firstly uses the screened ASMA promoter (Cssap) sequence containing the combined regulatory element and the PCK1 gene sequence to form a new DNA fragment Cssap-PCK1. The sequence of the DNA fragment Cssap-PCK1 is shown in SEQ ID NO:4. The left homology arm LA is added to the DNA fragment and artificially synthesized into LA-Cssap-PCK1 vector. The nucleotide sequence of the vector is shown in SEQ ID NO:2. Then, the LA-Cssap-PCK1 vector is used as the backbone, and pPB-MusPSP-neo-EGFP-BgEgXyAp (see CN201310343271) is used as the template to PCR amplify the right homology arm (RA) of the neoGFP-HR site, and insert it into the EcoRV site in the LA-Cssap-PCK1 plasmid to construct the HR-Cssap-PCK1 vector plasmid. The size of the plasmid is about 15kb ( Figure 7 a), the nucleotide sequence of which is shown in SEQ ID NO:3.

[0056] Subsequently, based on the HR-Cssap-PCK1 vector plasmid, the same method was used to construct HR-Mssap-PCK1 (the CMV enhancer was replaced by MCK enhancer on the basis of the HR-Cssap-PCK1 plasmid, and its nucleotide sequence is shown in SEQ ID NO:5) and HR-Ssap-PCK1 plasmid (the CMV enhancer was removed on the basis of the HR-Cssap-PCK1 plasmid, and its nucleotide sequence is shown in SEQ ID NO:6) ( Figure 7 b). C2C12 myotube cells were transfected by electroporation and lipofectamine LTX transfection, respectively. The results showed that the expression of PCK1 gene in C2C12 myotubes was affected by the transfection method ( Figure 7 c), among which, electrofection was better than liposome transfection, and the PCK1 genes of the three plasmids were all successfully expressed; under liposome transfection conditions, HR-Cssap-PCK1 and HR-Ssap-PCK1 could both significantly express the PCK1 gene, while the amount of PCK1 expressed by HR-Mssap-PCK1 was extremely low, with no significant difference compared with the control group (P<0.05).

[0057] Electrotransfection method: C2C12 cells were electrotransfected using Cell Line NucleofectorKit V. The specific transfection steps were as follows: C2C12 cells with a density of 80% were digested and counted. Taking a 6-well plate as an example, 1×10 6 The cells were centrifuged (90×g, 5min), resuspended in DPBS, centrifuged again, and 82μL Cell Line Nucleofector Solution V and 18μL Supplement were mixed and resuspended. 5μg of the plasmid to be transfected was added and mixed, and then the mixture was transferred to a 2mm electroporation cup. The electroporation program was B-032. Immediately after electroporation, 500μL of preheated differentiation medium was added to the electroporation cup, and then all the mixtures were transferred to a 6-well plate containing 1.5mL differentiation medium for culture, with 3 replicates per group. C2C12 myotubes were collected 48h-60h after electroporation for subsequent quantitative detection of gene expression.

[0058] 2.3 Screening of edited cell lines with ectopic expression of PCK1 gene

[0059] In order to obtain an edited cell line that ectopically expresses the PCK1 gene, this experiment used the principle of homologous recombination to site-specifically integrate the HR-Cssap-PCK1, HR-Mssap-PCK1, and HR-Ssap-PCK1 donor plasmids into the position between 65754555-65758570bp of chromosome 13 of the pig genome Sscrofa11.1 version. The specific process is as follows:

[0060] Screening method for site-specific integration gene editing of Duroc pig PEF cells: Resuscitate F0 generation Duroc pig PFFs cells to 10cm cell culture dishes, and perform electrotransfection according to the Basic NucleofectorTM Kit for Primary Mammalian Fibroblasts instructions when the cell density is 80%. Select A-033 for the electrotransfection program, and the plasmid is 10μg of the target plasmid (specifically the following three target donor plasmids: HR-Cssap-PCK1, HR-Mssap-PCK1, HR-Ssap-PCK1). Using the limiting dilution method, pick monoclonal cells after 10-15 days of culture. Use trypsin green fluorescent cells to transfer to a 48-well plate for continued culture. When the cells are gradually passaged to a 12-well plate, take 1 / 3 of the cells for DNA extraction for PCR and sequencing to identify positive cells; the remaining cells are frozen to ensure that they are consistent with the cell number identified by PCR.

[0061] The site of integration in the positive cell line is located between 65754555-65758570 bp of chromosome 13 of the pig genome Sscrofa11.1 version ( Figure 8 a), the results of PCR identification of monoclonal cells are as follows Figure 8 bd, some Sanger sequencing results are shown in Figure 8 e. The positive monoclonal cells were counted and the gene KI efficiencies of HR-Cssap-PCK1, HR-Mssap-PCK1 and HR-Ssap-PCK1 were 20.8%, 23.2% and 20.0%, respectively ( Figure 8 f). The positive monoclonal cells with correct sequencing were treated with Cre enzyme, and the results showed that the green fluorescence was completely deleted ( Figure 8 g), three editing-positive cell lines of site-directed integration of PCK1 gene without marker gene were obtained, which were named HC cell line (donor plasmid HR-Cssap-PCK1), HM cell line (donor plasmid HR-Mssap-PCK1), and HO cell line (donor plasmid HR-Ssap-PCK1).

[0062] In the HC type positive cell line, a DNA fragment Cssap-PCK1 was site-specifically integrated at the position between 65754555-65758570bp of chromosome 13 of the pig genome Sscrofa11.1 version. The sequence of the DNA fragment Cssap-PCK1 is shown in SEQ ID NO:4.

[0063] Example 3 Construction and detection of site-specific integration PCK1 gene-edited pigs (Duroc) 3.1 Construction and identification of site-specific integration PCK1 gene-edited pigs

[0064] The three site-specific integration positive cell lines expressing the PCK1 gene obtained in Example 2 were used as nuclear donor cells, and the site-specific integration gene-edited pigs were prepared by somatic cell nuclear transplantation. The specific method is as follows:

[0065] First, mature oocytes are obtained through collection and in vitro maturation culture. Then, after the oocytes mature, the oocytes are enucleated and the nuclear donor cells are injected into the perivitelline space to complete the embryo reconstruction process. The reconstructed embryos are then placed in embryo culture medium for recovery and culture for 1 hour, and the reconstructed eggs are transferred to the fusion medium in batches for activation, followed by reconstructed embryo culture. Finally, when the reconstructed embryos are at the 2-cell or 4-cell stage, they are transplanted into the uterus of a healthy surrogate sow, and the piglets are tested after they are born.

[0066] Somatic cell cloned embryos were prepared using the monoclonal positive cell line after the marker was deleted as the nuclear donor cell, and then transplanted into the uterine oviducts of 6 surrogate sows. Among them, 3 sows became pregnant, with a farrowing rate of 50%, an average of 7 litters, and an average of 4.33 live piglets. All cloned pigs were tested positive, and their left and right homologous arms, promoters, and target genes were fully integrated between 65754555-65758570bp of chromosome 13 ( Fig. 9 AC), the gene-edited pigs developed well. HC and HM gene-edited positive pigs were obtained: HC type refers to gene-edited pigs prepared using HC positive cell lines as nuclear donor cells, and the donor plasmid used is HR-Cssap-PCK1; HM type refers to gene-edited pigs prepared using HM positive cell lines as nuclear donor cells, and the donor plasmid used is HR-Mssap-PCK1. After testing, the DNA fragment Cssap-PCK1 was site-integrated between 65754555-65758570bp on chromosome 13 in HC gene-edited positive pigs. The donor plasmid site-integration site in HM gene-edited positive pigs is also between 65754555-65758570bp on chromosome 13.

[0067] 3.2 Detection of ectopic expression of target genes

[0068] The expression of PCK1 gene mRNA in various tissues of adult Duroc boars in HC and HM gene-edited positive pigs was compared with that in WT non-gene-edited control pigs. The results showed that ( Fig.10): The expression of PCK1 mRNA in the liver of HM-type gene-edited positive pigs relative to WT pigs. The expression of PCK1 mRNA in the liver of HM-type gene-edited pigs was 3.5 times higher than that of WT control pigs, and even 1.7 times higher than that of HC type, indicating that its gluconeogenesis ability is strong. In addition, compared with WT control pigs, it was increased in testis, cecum, and lard, slightly increased in skeletal muscle (longissimus dorsi, biceps femoris, trapezius muscle), and the expression of PCK1 mRNA in the kidney decreased significantly, only 8.39% of WT control pigs, and there was no significant change in other tissues. Compared with WT control pigs, HC-type gene-edited positive pigs, except for a slight decrease in kidney PCK1 mRNA expression (about 1 / 3), all other tissues increased to varying degrees, among which the expression in muscle, stomach, heart, testis, spleen, and lung increased significantly. The expression of PCK1 in the liver was about 2 times higher than that in WT control pigs, which is conducive to improving gluconeogenesis; the expression of PCK1 in the brain was 28 times higher than that in WT control pigs;

[0069] Compared with HM-type gene-edited positive pigs, the mRNA expression of PCK1 gene in HC-type gene-edited positive pigs increased by 5-20 times in skeletal muscle, heart, stomach, lung, and testis ( Fig.10 In the liver, the main organ of gluconeogenesis, HM increased by 1.7 times compared with HC, which seems to indicate that HM has a more significant gluconeogenesis ability; in non-gluconeogenic organs, the PCK1 mRNA expression level of HC pigs is higher than that of HM, which will significantly enhance their glycerol neogenesis ability and create conditions for intramuscular fat deposition (see Fig.10 3.3 Testing of the quality and production performance of gene-edited F1-generation pigs

[0070] The obtained HC and HM type primary gene-edited pigs were bred to obtain the corresponding F1 generation gene-edited pigs, and the quality performance and production performance of the F1 generation were tested. The results are as follows Fig.11 As shown in the results, the new breeding material constructed for ectopic expression of endogenous porcine phosphoenolpyruvate carboxykinase (PCK1) successfully expressed the PCK1 gene ectopically in other tissues and could be stably inherited to the next generation, especially ectopic expression in pig skeletal muscle, significantly increasing the expression of endogenous PCK1 in skeletal muscles such as the longissimus dorsi and biceps femoris ( Fig.11 A, B), remodeling the pathway of glucose and lipid metabolism in muscles. Compared with WT ordinary Duroc control pigs, the intramuscular fat content of the longissimus dorsi muscle of HC gene-edited Duroc pigs increased significantly by 50% (p = 0.011), reaching 3.11% (wild-type pig IMF 2.0%), while the HM type did not reach a significant difference ( Fig.11C); The growth rate of HC gene-edited pigs in the late growth period (170-196 days) increased by 25.75%, reaching 1210.44 g / day, which was significantly higher than the control pigs 962.16 g / day, while the growth rate of HM was lower than that of the control pigs, but it did not reach a statistical difference (the results are shown in Table 2 and Fig.11 D); however, there was no significant difference in growth rate between HC, HM and control group from 0 to 169 days of age ( Fig.11 E); Compared with wild-type pigs, the HC gene-edited pigs have obvious changes in appearance and shape, with ruddy skin, short hair, and fuller muscles (see Fig.11 F).

[0071] Table 2 Growth rate of F1 generation of gene-edited positive pigs in the later stage

[0072]

[0073] Note: ADG stands for Average Day Gain, average daily weight gain g / day.

[0074] 3.4 Results of reproductive ability test of F1 gene-edited pigs

[0075] When the F1 generation of gene-edited positive pigs were grown up, the semen of the boars was collected. It was found that the three HM-type boars had difficulty in collecting semen and could not collect semen manually. The HC-type high-quality boars could collect semen normally. The Spanish semen analysis system SCA (SCA-V-P03) was used to analyze the sperm motility. Compared with other different breeds and individuals, the sperm motility, density, forward PR%, curve speed (μm / s), swing index, whipping frequency (Hz) and other quality indicators of HC gene-edited pigs were significantly better than those of other pigs, suggesting that they have strong reproductive potential. The results are shown in Table 3:

[0076] Table 3 Comparative analysis of semen quality of HC gene-edited pigs

[0077]

[0078] The F0 generation HC and HM gene-edited positive boars were used to mate with wild-type WT and environmentally friendly HB sows (see patent CN202210654651.7) to expand the F1 generation. The F1 generation was statistically analyzed, and the results are shown in Table 4: The total piglets, healthy piglets and live piglets of the offspring of HC boars were significantly better than those of HM and the large group production results of the same period, indicating that the reproductive capacity of HC pigs has an improving trend.

[0079] Table 4 Comparison of F1 generation reproductive performance

[0080]

[0081] Note: The male and female pig breeds in the table are Duroc, HB: grain-saving and environmentally friendly gene-edited pig, HC: HC gene-edited pig, HM: HM gene-edited pig, WT wild-type pig.

[0082] In summary, when the new DNA fragment Cssap-PCK1 (nucleotide sequence as shown in SEQ ID NO: 4) is site-integrated at the 65754555-65758570bp position of chromosome 13 of the pig, the pig can ectopically express the PCK1 gene in skeletal muscle, and the intramuscular fat content of the longissimus dorsi muscle of Duroc pigs can be increased by 50% compared with the control pigs, reaching 3.11% (2.0% for wild-type Duroc pigs), which is much higher than the existing technology. Moreover, the pig also widely ectopically expresses the PCK1 gene in other tissues, and the growth rate of the pig in the late growth period (170-196 days) is 25.75% higher than that of the control pigs, reaching 1210.44g / day, which is significantly higher than 962.16g / day of the control pigs, and has greater economic value than early growth, saving more feed. At the same time, the reproductive performance of the pig is also significantly improved.

[0083] Sequences involved in this application:

[0084]

[0085] Nucleotide sequence of the LA-Cssap-PCK1 vector (SEQ ID NO: 2):

[0086] TTGAGCAGGTGTACGAGGACGAGCCCAATTTCTCTATATTCCCACAGTCTTGAGTTTGTGTCACAAAATAATTATAGTGG

[0087] GGTGGAGATGGGAAATGAGTCCAGGCAACACCTAAGCCTGATTTTATGCATTGAGACTGCGTGTTATTACTAAAGATCTT

[0088] TGTGTCGCAATTTCCTGATGAAGGGAGATAGGTTAAAAAGCACGGATCTACTGAGTTTTACAGTCATCCCATTTGTAGAC

[0089] TTTTGCTACACCACCAAAGTATAGCATCTGAGATTAAATATTAATCTCCAAACCTTAGGCCCCCTCACTTGCATCCTTAC

[0090] CCTAGGTTAATTAAGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATA

[0091] TGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATA

[0092] ATGAC GTATGTTC C CATAGTAAC G C CAATAGG GACTTT CCATTGAC GTCAATG G GTGGAGTATTTAC G GTAAACTG CC CA

[0093] CTTG G CAGTACATCAAGTGTATCATATG C CAAGTAC G C CC C CTATTGAC GTCAATGACGGTAAATG G CC C G C CTG GCATT

[0094] ATG C C CA GTACAT GAC C TTAT G G GACTTTC CTAC TT G G C AGTACAT CTACGTATTAG TC AT C G C TATTAC CATG TTAATT

[0095] AAC GTACGAGTAACC CG CTTACTCAG GCGAGTGCAAGCTGCTG CTGTGTCCCCAAAGTGTACATGTATGGAG GCG GCCTT

[0096] CCAC CTGCC CCTCAGGTCTGTC CCTAAGC CAATGAGAATTCAG CAGCACCAGAATGGCCTGGAG CTGCC CCATGGTGACA

[0097] GTGGGGGAGTATTTAAAGTGGCC CAAC CAGGGCTGAGAGCAGACATCTC CACTGGGCATCTCAGGGCC CATCTC CC CAAG

[0098] GTGTG CTG GACTAGCTAGATCTCTG CTAG C CT GTGAGTGTTGTGTTCATAGCACTTCATGTGTGCTTGAAG C CAATTAGA

[0099] AAAGCTTGCTATAGGAGAGAAAAGAGCTG CACTGAGCACC CTCCTTC CCCTTTAAATGTCAACAGATTAGGAGTCAGTGA

[0100] ATGACAG CACAC CTCTTGCTAC CTTAGAGAC CAAAATTTAAG CTACTC CCCTTAAGCTATAGCTAGAGTG CAC CTGC CAG

[0101] TGTCTTTAGTC C C CACTGATGGAACAG GAC C CAAGGTATTGAAGATGGAACATAGTTATTCATT CATC CT CTAATTTAAA

[0102] AAGCTGGATATG CTGTACAGCAGAAATTGAC GGAACAATGTAAATCAACTATAACAGAAGAAATAAAAACCTGG GGGGAA

[0103] AGAAGCTGACTATGAAAC C C CAGGAG CTTTCTACATGG GC CTGGACTCACCAAACTCTTTATTTTGTAATGGACTTCTGA

[0104] CATTTTTAGGAAG GG CTGTC CTGATGTG GGCTATAGAAGAGG GTTTCACATGCTTCTTCAAGAGGAC CCACACTGTC C CA

[0105] GTTGCTGAGTCC CACCAC CAGATGCTAGTGGCAGCTATTTGGG GAACACTTAGGCACTACAAAAAAATGAGTGATTC CAT

[0106] TCTG G CTCACAC CATATC C CTGATGTAC C C CTTAAAG CATGTCACTGAGTTCATCACAGAAAATT GTTTC C C CTGTG C CT

[0107] TCCACAACAAGGTTAGAGCTGTCCTTGGGGCCAGGGGAAGGGGGCAGGGAGTGAGAAGCACCAACTGGATAACCTCCTCT

[0108] GAC C C C CACTC CAC CTTAC CATAAGTAGATC CAAATC CTT CTAGAAAATTAG GAAG GCATATC C C CATATAT CAG C GATA

[0109] TAAATAGAACTGCTTCAGC GCTCTGGTAGACG GTGACTCTCCAAGGTGGACTGG GAGGCAGCCTGGCCTTGGCTGGGCAT

[0110] CGTCCTCTAAATAGAAAGATGAACTTGTTCAGC CTTTCCAGAAGGAAAACTGCTGC CCAGCCTACAGTGCAAC GTCCTTG

[0111] TCTTC CATCTG GAG GAAGCACG GGTGACATATCATCTAGTAAGG GCAC CTCTCTGTTTCCACCTCCAG GTC GAGGG GTGT

[0112] GACCCTTACTTCTCAGCCTCAAGGGAGGGACACTCAACC CCCCAAAAAGACATGAGGGCGCTCAGCTCGGCCCAC CGCAC

[0113] CC CGGACCGGAGCC GTCACC CC CCGAAATTCACTC CCTTCACAAGC CCC CAAG CGCGTTCTCTGGTGCG GACTGCTC CGG

[0114] GGC CCTG GCTTTGTGC CCAGCGTTGTCAGAGCCACC GC CCTGAGCCTGTC CCC GGGAGCCCC GCGC CTCCTCCCAC CGCT

[0115] CC GCTCTCG CGCC CC GCGGC CAGTTGTCTGCC CCGAGACAG CTGCGC GCC CTCCCGCTGC CGGTGGC CCTCTC CGGTGGG

[0116] GGTGGGGACCGACAGGGTCAGCCCTCCGGATCCGGGGCGCTCCGGGTAGCGGGGAGAAGTGATCGCTGGGGAGCTGGGGG

[0117] AG GGGTCG CCTTCCTGC CCTACC CAGGACTC C GGGTG CGAC CG CTC CTCTATCTCTCCAGC CCAC CACCACTC CAC CACT

[0118] TGGACACGTCTCC CTCCTCC CTGGAGTC GCTCTAGAGGGTTTGGGGGTCTGAGTAAAGAACCCGAAGTAGGGATACAGTG

[0119] TGGCGGCACCTTCCAGAGGCCCCGGGCGCAGGGTAGACCGGGGCGGGGCGGCCCGCGGACAGGTGCAGCCCCAGGCGCAG

[0120] GCGCACTCGCGCCTC CCGGCGCAGGCGGTGAAC CTCGCCCCACCCCAGCCCCTCCGGGGGGCAGCTGGGCCGGGTCGGGA

[0121] GGG GCC CACCAGC CC GGGAGACACTC CATATACG GCCAGG CC CGCTTTAC CTGGG CTCCGGC CAGGC CGCTCCTTCTTTG

[0122] GTCAGCACAGGGGACCCGGGCGGGGCCCAGGCCGCTAACCCGCCGGGGGAGGGGGCTCCAGTGCCCAACAC CCAAATAT

[0123] GGCTCGAGAAGGGGAGCGACATTCCTGTGAGGCGGCTCGGGGGGAGAACCCGCGGGGCTATATAAAACCTGAGC GTGGGGA

[0124] CCAGCGGCCACCGCAGCGGACAGCGCCGAGAGAAGCCTCGCTTCCCTCCCGCGGCGACCAGGGCCCCAGCCGGAGAGCAG

[0125] CAGCAATTGAAACC CGACGC CGGC GCGCCACCATGCCTCCTCAGCTCTCAAACGGC CTCAACCACTCGGC CAAAGTCGTC

[0126] CGGGGCACCCTGGACAGCCTGCC CCAGGCCGTGC GGGATTTCGTGGAGAGCAGCGCCAAGCTGTGCCAGCCTGACCAAAT

[0127] CCACATCTGCGATGGCTCCCGAGGAGGAGAACCAGCAGCTGCTGAGCCACATGGAGGAAGAGGGTGTCATCAAGAGGCTGA

[0128] AGAAGTATGACAACTGCTGGTTGG CTCTCACAGACC CCAGG GACGTGGCCAGAATTGAAAGCAAGAC GGTCATCATTACT

[0129] CAGGAGCAAAGGGATGCGGTGCCCATCCC CAGAAGCGGCCTCAGCCAGCTGGGTCGCTGGATGTCCCCGGAGGACTTCGA

[0130] GAAAG CCTTCAATGC CC GGTTTC CG GGGTG CATGAAGG GTCG CACGATGTATGTCATC CCGTTCAGCATG GGG CC CCTGG

[0131] GCTCGC CC CTGTC CAAGATTGGCATC GAG CTGACGGACTC GC CCTAC GTGGTGGCCAGCATGCG CATCATGACGC GGATG

[0132] GGCTCGGC CGTCCTGGACGC GCTGGGGGCGGGC GAGTTCATCAAGGGCCTCCACTCCGTGGGGTGCCC CCTGCCTTTGAA

[0133] AAAGCC CTTGGTTAACAACTGGGC CTGTAACC CAGAGCTGACG CTCATTGCC CACCTGC CCGAC CG CC GAGAAATCATCT

[0134] CCTTCGGGAGCGGGTACGGCGGGAACTCACTCCTTGGGAAGAAGTGCTTTGCCCTGAGGATGGCCAGCCGGCTGGCCAAG

[0135] GAGGAGGGGTGGCTGGCAGAGCACATGCTGATC CTGGGCGTAAC CAACCCCGAGGGCCAGAAGAAATACTTTGCCGCAGC

[0136] TTTC CCCAGC GCCTGC GGGAAGAC CAACCTGGCTATGATGAACCC CACGCTCCCGGGCTGGAAAGTGGAGTGCGTGGGGG

[0137] AC GACATCGC CTG GATGAAATTCGACCAACAAGGTAATTTACG GGC CATCAACCCAGAGAACGG GTTTTTTGGC GTTGCT

[0138] CC CGGAACCTCTGTGAAGACAAACC CCAATGC CATCAAAAC CATCCAAAGCAACACCATCTTCACCAAC GTGGC CGAGAC

[0139] CAGCGACGGGGGCGTTTACTGGGAAGGCATCGACCAGC CGCTGGCCC CGGGCATCAAGCTCACTTCCTGGAAGGGCACAG

[0140] AGTGGGACCC CAAGGATGG GGAAC CTTGC GCTCACC CCAACTCGC GGTTCTGCAC CCCTGCCAGCCAGTGCC CCATCATC

[0141] GAC CCTGC CTGGGAGGC CC CGGAAGGCGTGC CCATTGAGGGCATCATCTTC GGAGGGC GCCGCC CTGCTGGC GTC CCTCT

[0142] GGTGTACGAGGCTCTCAGCTGGCAGCACGGCGTGTTCGTGGGGGC GGCCATGAGGTCCGAGGCCACCGCGGCTGCGGAGC

[0143] ACAAG GGCAAAGTGATTATGCAC GACC CCTTCGC CATG CG GCC GTTCTTCGGCTACAACTTC GGCAAATAC CTGG CC CAC

[0144] TGGCTCAGCATGGCCCAGCACC CGGCGGCCAAGCTGCC CAAGATCTTC CACGTCAACTGGTTCCGGAAGGACAAGGACGG

[0145] CAGGTTCCTCTGGCCGGGCTTCGGCGAGAACTGCCGGGTGCTGGC GTGGATGTTCGACC GCGTCCAGGGGAAGGGCGGCG

[0146] CCAGGCTCACGCC CATCGGCTACGTCCCC GAGGAGGCC GC CCTGGACCTGCGGGGCCTGGAGGCCATCC GCGTGAGCGAG

[0147] CTCTTCCAGGTCTCCAAGGAGTTCTGGGAGGAGGAGGCGGACGAGATCCACAAGTACCTGGAGGAGCAGGTGAACGCAGA

[0148] CCTGCC CTACGAAATCCAGAGCGAGGTC CTGGCCCTGAAGCAGAGAATCAGC CAGATGTGAGGCGCGCCAGGCCTACC CC

[0149] ATTCCAACAGCTGCGACTTCTC CTCAGGACGACGAATCTGCTCAAGTGCAGTGGCTGACCTCGAGCCAC CAAGCG GCAAC

[0150] TG CT CTC C CAC C G C CT GC GTTG CTG CTG C C CAAAC CGACACAATGTTTATAAC GT GTACATACATTAACTTATTTAC CT C

[0151] ATTTTGTTATTTTTAAAACGAAGCCCTGTGGAAGGAAATGGAAAACTTGAAGCATTAAACTCAGCCATTCTGTTATGCTG

[0152] CGTAAAATTGTCTGCTGTGTTTATTTGCGGGGGTGAGGAGCGGCGGGGGTGGGGAGCGGCGGGGGTCGGGAGGAAGAGGG

[0153] ACCCCCAGCTTTCTACCTCAGCGTCACTTTTCACAATACTCTGAACGAATGCACTCTTTTCAAAACCATGATTAAATCTC

[0154] CCCGTACTCATGATTTTCCCCTCTGGCTCAGATCGGTTCCCTGTACTGGCAGGCCAGCCGAGGCCCGACTTGACCCTGGG

[0155] GCGTGTCCCCCTTGTCCCTGTGCTGGGGCTGCAGCCAGCGATGGGGCAGCAGCGCCGGCGTCTGGGTCTTGACGCCGGCA

[0156] GGGCGCTGACCGTGGTGCTGGGACCGAGGGCCGCCCTGCAAAGCGTATTCCTGCTCCTCCTACGCGTTCCTTATTGAATC

[0157] TCGAAATCTAAATGAAAGGGCTACATCCGCTTTTAAAGTACCGGTTCTCATTTACAAAGTGAAGATAACCACTTCTATTC

[0158] CGGACTGAGACGGCCTTTGCTTCAAGCCGGGTGTTGATGGTATAAAAAAGCTTCCTTTGGCGGGGGGGTGAGTGGGGCCC

[0159] AGAGGTGGGAGGAGGATGAGGAAAGACCAGTTGACGGTTCCAAAGGAAGGACTGTGCATAGGHR-Cssap-PCK1 vector nucleotide sequence (SEQ ID NO:3):

[0160] CCTTACGGTCAGATAACTCTCACGTGAGAGTTATCTGACCGTAAGGGCGGCCGCTTGAGCAGGTGTACGAGGACGAGCCC

[0161] AATTTCTCTATATTCCCACAGTCTTGAGTTTGTGTCACAAAATAATTATAGTGGGGTGGAGATGGGAAATGAGTCCAGGC

[0162] AACACCTAAGCCTGATTTTATGCATTGAGACTGCGTGTTATTACTAAAGATCTTTGTGTCGCAATTTCCTGATGAAGGGA

[0163] GATAGGTTAAAAAGCACGGATCTACTGAGTTTTACAGTCATCCCATTTGTAGACTTTTGCTACACCACCAAAGTATAGCA

[0164] TCTGAGATTAAATATTAATCTCCAAACCTTAGGCCCCCTCACTTGCATCCTTACCCTAGGTTAATTAAGACATTGATTAT

[0165] TGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACG

[0166] GTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCC

[0167] AATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATA

[0168] TGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGAC

[0169] TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTTAATTAACGTACGAGTAACCCGCTTACTCAG

[0170] GCGAGTGCAAGCTGCTGCTGTGTCCCCAAAGTGTACATGTATGGAGGCGGCCTTCCACCTGCCCCTCAGGTCTGTCCCTA

[0171] AGCCAATGAGAATTCAGCAGCACCAGAATGGCCTGGAGCTGCCCCATGGTGACAGTGGGGGAGTATTTAAAGTGGCCCAA

[0172] CCAGGGCTGAGAGCAGACATCTCCACTGGGCATCTCAGGGCCCATCTCCCCAAGGTGTGCTGGACTAGCTAGATCTCTGC

[0173] TAGCCTGTGAGTGTTGTGTTCATAGCACTTCATGTGTGCTTGAAGCCAATTAGAAAAGCTTGCTATAGGAGAGAAAAGAG

[0174] CTGCACTGAGCACCCTCCTTCCCCTTTAAATGTCAACAGATTAGGAGTCAGTGAATGACAGCACACCTCTTGCTACCTTA

[0175] GAGACCAAAATTTAAGCTACTCCCCTTAAGCTATAGCTAGAGTGCACCTGCCAGTGTCTTTAGTCCCCACTGATGGAACA

[0176] GGACCCAAGGTATTGAAGATGGAACATAGTTATTCATTCATCCTCTAATTTAAAAAGCTGGATATGCTGTACAGCAGAAA

[0177] TTGACGGAACAATGTAAATCAACTATAACAGAAGAAATAAAAACCTGGGGGGAAAGAAGCTGACTATGAAACCCCAGGAG

[0178] CTTTCTACATGGGCCTGGACTCACCAAACTCTTTATTTTGTAATGGACTTCTGACATTTTTAGGAAGGGCTGTCCTGATG

[0179] TGGGCTATAGAAGAGGGTTTCACATGCTTCTTCAAGAGGACCCACACTGTCCCAGTTGCTGAGTCCCACCACCAGATGCT

[0180] AGTGGCAGCTATTTGGGGAACACTTAGGCACTACAAAAAAATGAGTGATTCCATTCTGGCTCACACCATATCCCTGATGT

[0181] ACCCCTTAAAGCATGTCACTGAGTTCATCACAGAAAATTGTTTCCCCTGTGCCTTCCACAACAAGGTTAGAGCTGTCCTT

[0182] GGGGCCAGGGGAAGGGGGCAGGGAGTGAGAAGCACCAACTGGATAACCTCCTCTGACCCCCACTCCACCTTACCATAAGT

[0183] AGATCCAAATCCTTCTAGAAAATTAGGAAGGCATATCCCCATATATCAGCGATATAAATAGAACTGCTTCAGCGCTCTGG

[0184] TAGACGGTGACTCTCCAAGGTGGACTGGGAGGCAGCCTGGCCTTGGCTGGGCATCGTCCTCTAAATAGAAAGATGAACTT

[0185] GTTCAGCCTTTCCAGAAGGAAAACTGCTGCCCAGCCTACAGTGCAACGTCCTTGTCTTCCATCTGGAGGAAGCACGGGTG

[0186] ACATATCATCTAGTAAGGGCACCTCTCTGTTTCCACCTCCAGGTCGAGGGGTGTGACCCTTACTTCTCAGCCTCAAGGGA

[0187] GGGACACTCAACCCCCCAAAAAGACATGAGGGCGCTCAGCTCGGCCCACCGCACCCCGGACCGGAGCCGTCACCCCCCGA

[0188] AATTCACTCCCTTCACAAGCCCCCAAGCGCGTTCTCTGGTGCGGACTGCTCCGGGGCCCTGGCTTTGTGCCCAGCGTTGT

[0189] CAGAGCCACCGCCCTGAGCCTGTCCCCGGGAGCCCCGCGCCTCCTCCCACCGCTCCGCTCTCGCGCCCCGCGGCCAGTTG

[0190] TCTGCCCCGAGACAGCTGCGCGCCCTCCCGCTGCCGGTGGCCCTCTCCGGTGGGGGTGGGGACCGACAGGGTCAGCCCTC

[0191] CGGATCCGGGGCGCTCCGGGTAGCGGGGAGAAGTGATCGCTGGGGAGCTGGGGGAGGGGTCGCCTTCCTGCCCTACCCAG

[0192] GACTCCGGGTGCGACCGCTCCTCTATCTCTCCAGCCCACCACCACTCCACCACTTGGACACGTCTCCCTCCTCCCTGGAG

[0193] TCGCTCTAGAGGGTTTGGGGGTCTGAGTAAAGAACCCGAAGTAGGGATACAGTGTGGCGGCACCTTCCAGAGGCCCCGGG

[0194] CGCAGGGTAGACCGGGGCGGGGCGGCCCGCGGACAGGTGCAGCCCCAGGCGCAGGCGCACTCGCGCCTCCCGGCGCAGGCGGTGAA

[0195] CCTCGCCCCACCCCAGCCCCTCCGGGGGGCAGCTGGGCCGGGTCGGGAGGGGCCCACCAGCCCGGGAGACACTCCATATACGGCCAG

[0196] GCCCGCTTTACCTGGGCTCCGGCCAGGCCGCTCCTTCTTTGGTCAGCACAGGGGACCCGGGCGGGGG

[0197] CCCAGGCCGCTAACCCGCCGGGGGAGGGGGCTCCAGTGCCCAACACCCAAATATGGCTCGAGAAGGGGAGCGACATTCCT

[0198] GTGAGGCGGCTCGGGGGGAGAACCCGCGGGCTATATAAAACCTGAGCGTGGGGACCAGCGGCCACCGCAGCGGACAGCGC

[0199] CGAGAGAAGCCTCGCTTCCCTCCCGCGGCGACCAGGGCCCCAGCCGGAGAGCAGCAGCAATTGAAACCCGACGCCGGCGC

[0200] GCCACCATGCCTCCTCAGCTCTCAAACGGCCTCAACCACTCGGCCAAAGTCGTCCGGGGCACCCTGGACAGCCTGCCCCA

[0201] GGCCGTGCGGGATTTCGTGGAGAGCAGCGCCAAGCTGTGCCAGCCTGACCAAATCCACATCTGCGATGGCTCCGAGGAGG

[0202] AGAACCAGCAGCTGCTGAGCCACATGGAGGAAGAGGGTGTCATCAAGAGGCTGAAGAAGTATGACAACTGCTGGTTGGCT

[0203] CTCACAGACCCCAGGGACGTGGCCAGAATTGAAAGCAAGACGGTCATCATTACTCAGGAGCAAAGGGATGCGGTGCCCAT

[0204] CCCCAGAAGCGGCCTCAGCCAGCTGGGTCGCTGGATGTCCCCGGAGGACTTCGAGAAAGCCTTCAATGCCCGGTTTCCGG

[0205] GGTGCATGAAGGGTCGCACGATGTATGTCATCCCGTTCAGCATGGGGCCCCTGGGCTCGCCCCTGTCCAAGATTGGCATC

[0206] GAGCTGACGGACTCGCCCTACGTGGTGGCCAGCATGCGCATCATGACGCGGATGGGCTCGGCCGTCCTGGACGCGCTGGG

[0207] GGCGGGCGAGTTCATCAAGGGCCTCCACTCCGTGGGGTGCCCCCTGCCTTTGAAAAAGCCCTTGGTTAACAACTGGGCCT

[0208] GTAAC CCAGAGCTGACGCTCATTG CCCACCTGCC CGAC CGC CGAGAAATCATCTCCTTCGGGAGCGGGTACGGCG GGAAC

[0209] TCACTCCTTGGGAAGAAGTGCTTTGCCCTGAGGATGGCCAGCCGGCTGGCCAAGGAGGAGGGGTGGCTGGCAGAGCACAT

[0210] GCTGATCCTGGGCGTAAC CAACC CCGAGGGC CAGAAGAAATACTTTGCCGCAGCTTTCCCCAGCGCCTGCGGGAAGACCA

[0211] ACCTGGCTATGATGAACCC CACG CTCCC GGGCTGGAAAGTGGAGTGCGTGGGGGAC GACATCGCCTGGATGAAATTC GAC

[0212] CAACAAGGTAATTTACG GGCCATCAACCCAGAGAACGGGTTTTTTGG CGTTG CTCCC GGAACCTCTGTGAAGACAAACC C

[0213] CAATGCCATCAAAACCATCCAAAGCAACAC CATCTTCACCAACGTGGCCGAGACCAGCGACGGGGGCGTTTACTGGGAAG

[0214] GCATCGACCAGCCGCTGGC CCCGGGCATCAAGCTCACTTCCTGGAAGGGCACAGAGTGGGACCCCAAGGATGGGGAACCT

[0215] TGCGCTCACC CCAACTCGC GGTTCTGCACC CCTGC CAGC CAGTGC CCCATCATCGACCCTGCCTGGGAGGCC CCG GAAGG

[0216] CGTGCC CATTGAGG GCATCATCTTC GGAG GGC GCC GC CCTGCTGG CGTCCCTCTGGTGTACGAGGCTCTCAGCTG GCAGC

[0217] ACGGCGTGTTCGTGGGGGCGGCCATGAGGTCCGAGGCCACCGCGGCTGCGGAGCACAAGGGCAAAGTGATTATGCACGAC

[0218] CCCTTC GC CATG CG GCC GTTCTTC GG CTACAACTTC GG CAAATACCTGGCCCACTGGCTCAGCATG GC CCAG CACC CG GC

[0219] GGCCAAGCTGCCCAAGATCTTCCACGTCAACTGGTTC CGGAAGGACAAGGACGGCAGGTTCCTCTGGCC GGGCTTCGGCG

[0220] AGAACTGC CGGGTGCTGGCGTGGATGTTCGACCGC GTCCAGGGGAAGGGCGGCGCCAGGCTCAC GCCCATCGGCTAC GTC

[0221] CCC GAGGAGGC CGCCCTGGACCTGCGGGGCCTGGAGGC CATCCGCGTGAGCGAGCTCTTCCAGGTCTCCAAGGAGTTCTG

[0222] GGAGGAGGAGGCGGACGAGATCCACAAGTACCTGGAGGAGCAGGTGAACGCAGACCTGCCCTACGAAATCCAGAGCGAGGTCCTGG

[0223] CCCTG AA GCAGAG AAT CAGCCAG AT GTGAGGCGCGC CA GGCC TA CCCC ATT CC AA CAGCTGCGACTTCTCCTCA

[0224] GGACGAC GAATCTGCTCAAGTGCAGTGGCTGACCTCGAG CCAC CAAGCGGCAACTGCTCTC CCACCGCCTGC GTTGCTGC

[0225] TG CC CAAAC C GAC ACAAT GTTTATAAC GT GTACATACATTAACTTATTTAC CTCATTTT GTTATTTTTAAAAC GAAG CCC

[0226] TGTGGAAG GAAATG GAAAACTTGAAGCATTAAACTCAG C CATTCTGTTATGCTG CGTAAAATTGTCTG CTGT GTTTATTT

[0227] GCGGGGGTGAGGAGCGGCGGGGGTGGGGAGCGGCGGGGTCGGGAGGAAGGGACCCCCAGCTTTCTACCTCAGCGTCACTTTTCA

[0228] CA ATA CTCTGAACGA AT GCACTCTTTTCAAAACC AND GAT TA A AT CTCCCCG TA CTC AT G AT TTTCCCCTCTGG

[0229] CTCAGATCG GTTC CCTGTACTGGCAGG CCAGC CGAGGC CC GACTTGACC CTGG GGCGTGTCC CC CTTGTCC CTGTG CTGG

[0230] GGCTGCAGC CAGCGATGGGGCAGCAGCGCCGGCGTCTGGGTCTTGACGC CGGCAGGGCGCTGACCGTGGTGCTGGGACCG

[0231] AGG GC CGC CCTG CAAAGC GTATTC CTGCTC CTC CTAC G CGTTC CTTATTGAATCTCGAAATCTAAATGAAAGG G CTACAT

[0232] CC GCTTTTAAAGTAC CG GTTCTCATTTACAAAGT GAAGATAAC CACTTCTATTC CGGACTGAGAC GG C CTTTGCTTCAAG

[0233] CCGGGTGTTGATGGTATAAAAGCTTCCTTTGGCGGGGGGTGAGTGGGGCCCAGAGGTGGGAGGAGGAGGAAAGA

[0234] CCAGTTGAC G GTTC CAAAG GAAG GACTGT GCATAG GC CTATAACTTCGTATAGCATACATTATAC GAAGTTATGATC GAT

[0235] ATGAAGAATCTG CTTAG G GTTAG GATATC GC GTTTTGC G CTG CTTC G C GATGTAC GGG C CAGATATAC GC GTTGACATTG

[0236] ATTATT GAC TAGTTATTAATAG TAATC AATTAC G G G GT C ATTAG TT C ATAG C CCATATAT G GAG TT C C G C GTTAC ATAAC

[0237] TTAC GGTAAATGG C CC G C CT GG CTGACC G C CCAAC GAC C C CC G CCCATTGACGTCAATAATGACGTATGTTC C CATAGTA

[0238] AC G CCAATAG GGACTTTC CATTGAC GTCAATGGGTGGAGTATTTAC GGTAAACTG CCCACTTGG CAGTACATCAAGTGTA

[0239] TCATATG C CAAGTAC G C C C C CTATTGAC GTCAATGAC G GTAAAT GG C C C G CCTG G CATTATG C C CAGTACAT GAC CTTAT

[0240] GG GACTTTC CTAC TTG G CAGTACATCTAC GTATTAGTC ATC G CTATTAC CAT G GTGAT G C G GTTTTG G CAGTACATCAAT

[0241] GG GC GTGGATAGC GGTTTGACTCAC GG GGATTTCCAAGTCTC CAC CC CATTGACGTCAATGGGAGTTTGTTTTG GCACCA

[0242] AAATCAACGG GACTTTCCAAAATGTCGTAACAACTC CGCC CCATTGACGCAAATGGGCGGTAGGCGTGTAC GGTGGGAGG

[0243] TCTATATAAGCAGAGCT CTCTG G CTAACTAGAGAAC C CACTG CTTACTG G CTTATCGAAATTAATAC GACTCACTATAG G

[0244] GAGAC CCAAGCTGGCTAG CGTTTAAACTTAAG CTTGGTACC GAG CTC GGATCCACTAGTCCAGTGTGGTGGAATTC GC CA

[0245] CCATGG GATCGG CCATTGAACAAGATGGATTGCAC GCAGGTTCTC CGG CC GCTTGGGTGGAGAGG CTATTC GG CTATGAC

[0246] TGGGCACAACAGACAATCG GCTGCTCTGATGC CG CC GTGTTC CGGCTGTCAGC GCAGGGGCGC CC GGTTCTTTTTGTCAA

[0247] GACC GACCTGTC CGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTC CTT

[0248] GCGCAGCTGTGCTC GACGTTGTCACTGAAGC GGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTG

[0249] TCATCTCAC CTTG CT CCT GC C GAGAAAGTATC CATCATGG CTGATGCAATG C GG C GGCTG CATAC G CTTGATC CG G CTAC

[0250] CTGC CCATTCGACCACCAAGC GAAACATC GCATCGAGCGAGCAC GTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATG

[0251] ATCTGGACGAAGAGCATCAGGGGCTC GCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGATGAT

[0252] CTC GTC GTGAC C CATG GC GATGC CTG CTTGC C GAATATCATG GT GGAAAATGG C CGCTTTTCTG GATTCATC GACTGTGG

[0253] CC GGCTGGGTGTGG CGGACC GCTATCAGGACATAGCGTTGG CTACC CGTGATATTGCTGAAGAGCTTGGC GGC GAATGGG

[0254] CTGAC C GCTTC CTC GTG CTTTAC G GTATC GC C G CTC C C GATTC G CAG C GCATC G C CTT CTATC G C CTTCTTGAC GAGTTC

[0255] TTCGCCATGGTGAGCAAGGGCGAGGAGCTGTTCAC CGGGGTGGTGCC CATCCTGGTCGAGCTGGACGGCGACGTAAACGG

[0256] CCACAAGTTCAGCGTGTCC GGCGAGGGCGAGGGCGATGC CACCTAC GGCAAGCTGACCCTGAAGTTCATCTGCACCACCG

[0257] GCAAGCTGC CC GTGC CCTG GCC CAC CCTC GTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGC CGCTACC CC GACCAC

[0258] ATGAAG CAGCACGACTTCTTCAAGTCCG CCATGCCC GAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGAC GACGG

[0259] CAACTACAAGAC CCGCGCCGAGGTGAAGTTC GAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCA

[0260] AGGAGGACGGCAACATC CTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGC CGACAAGCAG

[0261] AAGAACGGCATCAAGGTGAACTTCAAGATC CGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCA

[0262] GAACACC CCCATCGGC GACGGCCCC GTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGC CCTGAGCAAAGACC

[0263] CCAACGAGAAGCG CGATCACATGGTC CTGCTGGAGTTCGTGAC CGCC GCC GGGATCACTCTCGGCATGGACGAGCTGTAC

[0264] AAGTAATCTAGAGG G CC C GTTTAAAC CC G CTGATCAGC CTC GACTGTG CCTTCTAGTTG C CAG CCATCTGTTGTTTGC C C

[0265] CTC C C CC GTG CCTT CCTTGAC CCTG GAAGGTG CCACTC C CACTGTC CTTTCCTAATAAAATGAGGAAATTG CATC G CATT

[0266] GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGG

[0267] CATG CT G GG GATG C G GT G GG C TCTATG G CTTCT GAAGATCTATAACTT CGTATAG CATACATTATAC GAAGTTATG GAT C

[0268] TGTC GACTC GAC G GTCAGATAACT CTCACTCATACTTTAAG C C CATTTTGTTTGTTGTACTTG CTCATC CAGTC C CAGTC

[0269] CCATTG GCTTTCTC CTCAC CTGTTTTAG GTAG CCAG CAAGTCATGAAATCAGATAAGTTCCAC CAC CAATTAACACTAC C

[0270] CAT C TT GAG CATA GGCCC AACA GT G CATTTATTC CT C ATTTAC T GATG TTC GT GAATATTTAC C TTG ATTTT CATTTTTT

[0271] TCTTTTT CTTAAG CTG G GATTTTACT C CTGAC C CTATTCACAGTCAGATGATCTTGACTAC CACTG C ​​GATTG GAC CT GAG

[0272] GTT CAG C AATAC TCCC CTTTAT GTC TTTT GAATAC TTTT CAATAAATC TG TTTGTATTTT CATTAGTTA GTAAC T GAG CT

[0273] CAGTTG CC GTAATG CTAATAG CTTC CAAACTAGTGTCT CTGT CTC CAGTATTCTGATAAATCTTAG GT GTT GCT GG GACAG

[0274] TTGTC CTAAAATTAAGATAAAGCATGAAAATAACTGACACAACTC CATTACTG GCT CCTAACTACTTAAACAATGCATTC

[0275] TATCTTCACAAATGTGAAAAAG GAGTTC C CTCAGTG GACTAAC CTTATCTTTT CTCAACACCTTTTT CTTTG CACAATTT

[0276] TC CACACATG CCTACAAAGGTACTTTT CT GCTCAAGTCACACTGAGTTGATTGCTATTTACCAAAATCAAAGTAACATT

[0277] ATCAGATCTCTGTAGG GTG GTTC CCTCTG GAATG CTAC C CTC CATAGT CCTTAC CCTTCAAGTAAAGAGCATGAAGACTG

[0278] AAATATCT CT GT GAT CTGT CATC CTTTAAG CC AGAATC CC CCATAAAAAAGTTAGTATTG CTTT CTC C TGATC CC ATAG C

[0279] AG GTTGAATC ATAGCACTTATCAG GTTGTTGTCATTG CTTG CTTAAATT CTCCTAACTATTTG GAG CTTCTTGAGG G CAC

[0280] AGGTTCTTGTTGAGTCTT GTAC CTAAG CAC CTAGTATAGTC CTTGATGTCTAG CCAACCCTAAATAAAATG CAGTGAGTG

[0281] ACAT GTAGAT GT CTTTATAAG GTTT GATA G GTT G GT CTC TC AAATAG TTCTTTTGTAT GTTTG G TAGTG C TC TAGATTAG

[0282] CAC TG GC CAGTATAACT CT GATGATG G AAAT GTT CTATAG C TAT G CT GTCTAATAT G GTAGT CAC TACTAAC ATATGTTA

[0283] CT GTTGAG C CTTG GAAATATG G CTTTT GT GACAAAAC TGAATTTTTCATGCTATGTAATTTAAGTCTAAATTG CTACT GT

[0284] GTACATTGT GG CT GTAG C CAC AAATTT GT G CT GTG GATT G CAGAATAATTAATATG GACATTGATAATTTT CTTTTCATA

[0285] CTAAG CAGTAAG GAAAGAAAAGTT GAAACT CTGTG GTC CATTTAGGTTATATGTGTATTTGTACTTGATT GGTTTGTTTG

[0286] AATAC CTATTTCTATACTTTAGCTGAGAGCTAAAGC CAACAAAC CAGTACTGTAGATAAC CTGCTTTGGACAACAATGTG

[0287] TT GAC TAGTT G GATTTCATCAAAGAATG C CTAATAAATTTTAAGAAAATGAGATTTCATTAAAC C ATAATACTGACATAA

[0288] GTTTAG GGAAGAATCAGACTATATCTGGTGTTTGTGAAACTAC C C CTGAATTTCAGTCCTACAAAGTTTTCAGTTTTG GA

[0289] AAAACTTTCATCAGAGAGGG CACTAAGTTACAG GAAGC CATCACAAAGTAAGTTTTCATCTGATGAATTATAAATTTAAG

[0290] ATATATTTTAATAC C AAAATTC TTTAT G GTTTATGT G C TAAC TTAAAATTT CT CCTTAAAATAT GAGAA CTAA GTAC ACA

[0291] ATTGTACTTG GCTGTTTAATG C GGATTC C CAGTC CCTCACACAGAGATTCTCAATTAAGATTG GAGAG CAG GG GTTACTA

[0292] GAATT CTTTTTC AG GTT C CTTATATG CTT CTGATTTG GTG G C CTAGAAATCACAATGCTAGTG CAG C C CTCATG G GG CTA

[0293] CAGTATAC GTATCT GAAACATGATTACATCAG G GAAACTGTATGT CTAATCTACTTTGTC CCTAAAG GAAG CATTTT GAA

[0294] AGG CAGAAAGTAATATGTGATAGTTTTTGAAACTTGTAG GTCACATTGTTTTTAAAAG G GATCCAAGTAAGTTTTTTTTT

[0295] CTTTTGAGG GCTACACCTGTGGCACATGGAGGTTCC CAGGCTAG GGGTTAAATCAGAACTG CAGCTGC CAGCCTATGC CA

[0296] GAG CCACAG CAATGCCAGATCTGAGCTGTGTCTGCAACTGTGTAG CTCACAGCAACGCTGGATCCTTAACC CAATGAG CA

[0297] AG GC CAGAGATTGAACCTACAAC CTC GTGGTTCCTAGTTAGATTTGTTTC CG CTGTGC CACGATG GGAACTC CAAGTAAT

[0298] TTTTTTTTGAG CAAG GAAGTTAC C TTTTTTGTCT GTTTTC C CACTAAATG CATT CCTCAAG GATTC C C AGTTT GTTCTTG

[0299] ATTC CTC AGTG C C TTAACACAGAC CTG G GTTC TCAGTAAATGTTGATTTTATTGATTTATAT GTGAAATTGTTTTT CAAA

[0300] TAATAGTTTTTAAGTC C ATAGAAACAAT G CTT CTTTTATG GAGATACTTTAG GATCATACTTGTAAC C CAAGTT G C CTAA

[0301] TACTC TGTT CATAAAGAAAACTCATG C C TCATG GTCT CTGAATAATACAT CTGT CTACCATTGAG CT CTT C CTTG G GTTT

[0302] CCT GTG CAAAC CATTG CAC TTAT C CT CTTC CT GTG C TATACTTC CT CAG GCTTTATTACAGTTTTTAAAATAAAC C AAC T

[0303] ATC TAT CT CT CTTT GAAGTAGAG C CATAATAATT G CATGAGAA CACT GAAGGTTTTTAG G CTTTAATTTTTT CTTTTTTT

[0304] TTAAGATATTCAAAATTTGGAGTTCTTGTTGTG GC GCAGTGGTTAACGAAC C CAACTAGGAACCATGAC GCTGCAG GTTC

[0305] GGTC C CTGG C CTC G CTCAGTGG GTTAAG GATC CAG C GTTG C CATGAGCTGTGGTATAGGTC CATATGTG GCTC GGATC CT

[0306] GCATTG CTGTG GCTGTGGCAG CAG CCACAG GTACGATTAGAC C CCTAGCCTGG GACCCTCCATATGCTGTGG GCG CG GTC

[0307] CTAGAAAAGAAAAAAAAAAAGAAAAGAAAAGAAAGATATACAAAATTTGAACTACGCATTGTTTCTCTTAACAGTTGTTA

[0308] TGTAT GGAG GAG GTTT GTTATAATTACAGTTTACAAC TCTTAATC CAGAATATGTTAG GGATC CACATTC C CAG G GTAAG

[0309] ACTAGTTTGTTTTAG GC CAGACTTAATT GTACAG C C CATTGTC CAG CCACATACTCAGGAGT CTCATACTTTG CAG G CTA

[0310] AAAATT CTT GATTTT GTTAC CTAGTAGT GTACTGTT CAT GTT G G G GAACTTTTTTCTC CAGAAAAGTTTATTATC CATTA

[0311] TC C T G C C T C CTTT TTATTTT C ATTTATTTATTTATTTATTTTT G CTTT TTTA GG G C C AC A CT T GT G G C ATAT G G AAATT C

[0312] CTGG GCTAGG GGTCAAATCAGG GCTTCAGCTGCTGGC CTATGC CACAACAACACGGGATCAGAGCTGCATCTGCAATCTA

[0313] TAC CACAGCTTTTG GCAACC C C GTATC CTTAAC C CAATGAATACTAGTTG GGTTCTTAAC C C GCTAAG C CATAATTG GAA

[0314] CTCC CATGATC C C CATTCTTGATGAAGTG GATATC GGATCC C GG GC C CGTC GACTGCAGAGG C CTG CATGCAAGCTTGG C

[0315] GTAATCATGGTCATAG CTGTTTC CTGTGTGAAATTGTTATC C G CT CACAATTCCACACAACATAC GAG C C GGAAG CATAA

[0316] AGTGTAAAG CCTGG GGTG CCTAATGAGTGAG CTAACTCACATTAATTGC GTTGC GCTCACTGCC C GCTTTC CAGTC GG GA

[0317] AACCTGTCGTG CCAGCTGCATTAATGAATC GGC CAAC GCG CG GGGAGAG GC GGTTTGCGTATTGGG CG CTCTTC CG CTTC

[0318] CTC G CTCACTGACTCG CTG CG CTC GGTCGTTC GG CTG CG GC GAG CG GTATCAGCTCACTCAAAGGC GGTAATAC GGTTAT

[0319] CCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTG

[0320] G C G T T TT T C C ATA G G C T C C G C C C C C C T G A C G A G C AT CA C AAAAAT C G A C G C T C AA G T C A G A G G T G G C G AAA C C C

[0321] GACAGGACTATAAAGATAC CAGG CGTTTCC C CCTGGAAGCTCC CTCGTGC GCTCTC CTGTTCCGAC CCTGC C GCTTACC G

[0322] GATAC CTGTC C GC CTTTCTC C CTTC GG GAAG C GTG G C GCTTT CTCATAG CTCACG CTGTAG GTATCTCAGTTC GGTGTAG

[0323] GTC GTTC GCTC CAAG CTG GG CTGTGTGCACGAACC C CC C GTTCAG CC C GACCGCTGC G CCTTATC C GGTAACTATC GTCT

[0324] TGAGTCCAACCC GGTAAGACAC GACTTATC GCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGC GAGGTATGTAG

[0325] GC GGTGCTACAGAGTTCTTGAAGTGGTG GC CTAACTAC GGCTACACTAGAAGAACAGTATTTGGTATCTG CG CTCTGCTG

[0326] AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGT

[0327] TTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT

[0328] GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAA

[0329] TGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTAT

[0330] CTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTAC

[0331] CATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCC

[0332] GGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGT

[0333] AAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA

[0334] TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCC

[0335] TTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCT

[0336] TACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGC

[0337] GACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGA

[0338] AAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAA

[0339] CTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAA

[0340] TAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTC

[0341] ATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACC

[0342] TGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

[0343] Nucleotide sequence of Cssap-PCK1 (SEQ ID NO:4):

[0344] GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT

[0345] ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC

[0346] CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC

[0347] AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATG

[0348] ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTTAATTAACGTACGAGTAAC

[0349] CCGCTTACTCAGGCGAGTGCAAGCTGCTGCTGTGTCCCCAAAGTGTACATGTATGGAGGCGGCCTTCCACCTGCCCCTCA

[0350] GGTCTGTCCCTAAGCCAATGAGAATTCAGCAGCACCAGAATGGCCTGGAGCTGCCCCATGGTGACAGTGGGGGAGTATTT

[0351] AAAGTGGCCCAACCAGGGCTGAGAGCAGACATCTCCACTGGGCATCTCAGGGCCCATCTCCCCAAGGTGTGCTGGACTAG

[0352] CTAGATCTCTGCTAGCCTGTGAGTGTTGTGTTCATAGCACTTCATGTGTGCTTGAAGCCAATTAGAAAAGCTTGCTATAG

[0353] GAGAGAAAAGAGCTGCACTGAGCACCCTCCTTCCCCTTTAAATGTCAACAGATTAGGAGTCAGTGAATGACAGCACACCT

[0354] CTTGCTACCTTAGAGACCAAAATTTAAGCTACTCCCCTTAAGCTATAGCTAGAGTGCACCTGCCAGTGTCTTTAGTCCCC

[0355] ACTGATGGAACAGGACCCAAGGTATTGAAGATGGAACATAGTTATTCATTCATCCTCTAATTTAAAAAGCTGGATATGCT

[0356] GTACAGCAGAAATTGACGGAACAATGTAAATCAACTATAACAGAAGAAATAAAAACCTGGGGGGAAAGAAGCTGACTATG

[0357] AAACCCCAGGAGCTTTCTACATGGGCCTGGACTCACCAAACTCTTTATTTTGTAATGGACTTCTGACATTTTTAGGAAGG

[0358] GCTGTCCTGATGTGGGCTATAGAAGAGGGTTTCACATGCTTCTTCAAGAGGACCCACACTGTCCCAGTTGCTGAGTCCCA

[0359] CCACCAGATGCTAGTGGCAGCTATTTGGGGAACACTTAGGCACTACAAAAAAATGAGTGATTCCATTCTGGCTCACACCA

[0360] TATCCCTGATGTACCCCTTAAAGCATGTCACTGAGTTCATCACAGAAAATTGTTTCCCCTGTGCCTTCCACAACAAGGTT

[0361] AGAGCTGTCCTTGGGGCCAGGGGAAGGGGGCAGGGAGTGAGAAGCACCAACTGGATAACCTCCTCTGACCCCCACTCCAC

[0362] CTTACCATAAGTAGATCCAAATCCTTCTAGAAAATTAGGAAGGCATATCCCCATATATCAGCGATATAAATAGAACTGCT

[0363] TCAGCGCTCTGGTAGACGGTGACTCTCCAAGGTGGACTGGGAGGCAGCCTGGCCTTGGCTGGGCATCGTCCTCTAAATAG

[0364] AAAGATGAACTTGTTCAGCCTTTCCAGAAGGAAAACTGCTGCCCAGCCTACAGTGCAACGTCCTTGTCTTCCATCTGGAG

[0365] GAAGCACGGGTGACATATCATCTAGTAAGGGCACCTCTCTGTTTCCACCTCCAGGTCGAGGGGTGTGACCCTTACTTCTC

[0366] AGCCTCAAGGGAGGGACACTCAACCCCCCAAAAAGACATGAGGGCGCTCAGCTCGGCCCACCGCACCCCGGACCGGAGCC

[0367] GTCACCCCCCGAAATTCACTCCCTTCACAAGCCCCCAAGCGCGTTCTCTGGTGCGGACTGCTCCGGGGCCCTGGCTTTGT

[0368] GCCCAGCGTTGTCAGAGCCACCGCCCTGAGCCTGTCCCCGGGAGCCCCGCGCCTCCTCCCACCGCTCCGCTCTCGCGCCC

[0369] CGCGGCCAGTTGTCTGCCCCGAGACAGCTGCGCGCCCTCCCGCTGCCGGTGGCCCTCTCCGGTGGGGGTGGGGACCGACA

[0370] GGGTCAGCCCTCCGGATCCGGGGCGCTCCGGGTAGCGGGGAGAAGTGATCGCTGGGGAGCTGGGGGAGGGGTCGCCTTCC

[0371] TGCCCTACCCAGGACTCCGGGTGCGACCGCTCCTCTATCTCTCCAGCCCACCACCACTCCACCACTTGGACACGTCTCCC

[0372] TCCTCCCTGGAGTCGCTCTAGAGGGTTTGGGGGTCTGAGTAAAGAACCCGAAGTAGGGATACAGTGTGGCGGCACCTTCC

[0373] AGAGGCCCCGGGCGCAGGGTAGACCGGGGCGGGGCGGCCCGCGGACAGGTGCAGCCCCAGGCGCAGGCGCACTCGCGCCT

[0374] CCCGGCGCAGGCGGTGAACCTCGCCCCACCCCAGCCCCTCCGGGGGGCAGCTGGGCCGGGTCGGGAGGGGCCCACCAGCC

[0375] CGGGAGACACTCCATATACGGCCAGGCCCGCTTTACCTGGGCTCCGGCCAGGCCGCTCCTTCTTTGGTCAGCACAGGGGA

[0376] CCCGGGCGGGGGCCCAGGCCGCTAACCCGCCGGGGGAGGGGGCTCCAGTGCCCAACACCCAAATATGGCTCGAGAAGGGG

[0377] AGCGACATTCCTGTGAGGCGGCTCGGGGGGAGAACCCGCGGGCTATATAAAACCTGAGCGTGGGGACCAGCGGCCACCGC

[0378] AGCGGACAGCGCCGAGAGAAGCCTCGCTTCCCTCCCGCGGCGACCAGGGCCCCAGCCGGAGAGCAGCAGCAATTGAAACC

[0379] CGACGCCGGCGCGCCACCATGCCTCCTCAGCTCTCAAACGGCCTCAACCACTCGGCCAAAGTCGTCCGGGGCACCCTGGA

[0380] CAGCCTGCCCCAGGCCGTGCGGGATTTCGTGGAGAGCAGCGCCAAGCTGTGCCAGCCTGACCAAATCCACATCTGCGATG

[0381] GCTCCGAGGAGGAGAACCAGCAGCTGCTGAGCCACATGGAGGAAGAGGGTGTCATCAAGAGGCTGAAGAAGTATGACAAC

[0382] TGCTGGTTGGCTCTCACAGACCCCAGGGACGTGGCCAGAATTGAAAGCAAGACGGTCATCATTACTCAGGAGCAAAGGGA

[0383] TGCGGTGCCCATCCCCAGAAGCGGCCTCAGCCAGCTGGGTCGCTGGATGTCCCCGGAGGACTTCGAGAAAGCCTTCAATG

[0384] CCCGGTTTCCGGGGTGCATGAAGGGTCGCACGATGTATGTCATCCCGTTCAGCATGGGGCCCCTGGGCTCGCCCCTGTCC

[0385] AAGATTGGCATCGAGCTGACGGACTCGCCCTACGTGGTGGCCAGCATGCGCATCATGACGCGGATGGGCTCGGCCGTCCT

[0386] GGACGCGCTGGGGGCGGGCGAGTTCATCAAGGGCCTCCACTCCGTGGGGTGCCCCCTGCCTTTGAAAAAGCCCTTGGTTA

[0387] ACAACTGGGCCTGTAACCCAGAGCTGACGCTCATTGCCCACCTGCCCGACCGCCGAGAAATCATCTCCTTCGGGAGCGGG

[0388] TACGGCGGGAACTCACTCCTTGGGAAGAAGTGCTTTGCCCTGAGGATGGCCAGCCGGCTGGCCAAGGAGGAGGGGTGGCT

[0389] GGCAGAGCACATGCTGATCCTGGGCGTAACCAACCCCGAGGGCCAGAAGAAATACTTTGCCGCAGCTTTCCCCAGCGCCT

[0390] GCGGGAAGACCAACCTGGCTATGATGAACCCCACGCTCCCGGGCTGGAAAGTGGAGTGCGTGGGGGACGACATCGCCTGG

[0391] ATGAAATTCGACCAACAAGGTAATTTACGGGCCATCAACCCAGAGAACGGGTTTTTTGGCGTTGCTCCCGGAACCTCTGT

[0392] GAAGACAAACCCCAATGCCATCAAAACCATCCAAAGCAACACCATCTTCACCAACGTGGCCGAGACCAGCGACGGGGGCG

[0393] TTTACTGGGAAGGCATCGACCAGCCGCTGGCCCCGGGCATCAAGCTCACTTCCTGGAAGGGCACAGAGTGGGACCCCAAG

[0394] GATGGGGAACCTTGCGCTCACCCCAACTCGCGGTTCTGCACCCCTGCCAGCCAGTGCCCCATCATCGACCCTGCCTGGGA

[0395] GGCCCCGGAAGGCGTGCCCATTGAGGGCATCATCTTCGGAGGGCGCCGCCCTGCTGGCGTCCCTCTGGTGTACGAGGCTC

[0396] TCAGCTGGCAGCACGGCGTGTTCGTGGGGGCGGCCATGAGGTCCGAGGCCACCGCGGCTGCGGAGCACAAGGGCAAAGTG

[0397] ATTATGCACGACCCCTTCGCCATGCGGCCGTTCTTCGGCTACAACTTCGGCAAATACCTGGCCCACTGGCTCAGCATGGC

[0398] CCAGCACCGGCGGCCAAGCTGCCCAAGATCTTCCACGTCAACTGGTTCCGGAAGGACAAGGACGGCAGGTTCCTCTGGC

[0399] CGGGCTTCGGCGAGAACTGCCGGGTGCTGGCGTGGATGTTCGACCGCGTCCAGGGGAAGGGCGGCGCCAGGCTCACGCCC

[0400] ATCGGCTACGTCCCCGAGGAGGCCGCCCTGGACCTGCGGGGCCTGGAGGCCATCCGCGTGAGCGAGCCTTCCAGGTCTC

[0401] CAAGGAGTTCTGGGGAGGAGGAGGCGGACGAGATCCCAAGTACCTGGAGGAGCAGGTGAACGCAGACCTGCCCTACGAAA

[0402] TCCAGAGCGAGGTCCTGGCCCTGAAGCAGAGAATCAGCCAGATGTGAGGCGCGCCAGGCCTACCCCATTCCAACAGCTGC

[0403] GACTTCTCCTCAGGACGACGAATCTGCTCAAGTGCAGTGGCTGACCTCGAGCCACCAAGCGGCAACTGCTCTCCCCACGGC

[0404] CTGCGTTGCTGCTGCCCAAACCGACCAATGTTTATAACGTGTACATACATTAACTTATTTACCTCATTTTGTTATTTTT

[0405] AAAACGAAGCCCTGTGGAAGGAAATGGAAAACTTGAAGCATTAAACTCAGCCATTCTGTTATGCTGCGTAAAATTGTCTG

[0406] CTGTGTTTATTTGCGGGGGTGAGGAGCGGCGGGGGTGGGGAGCGGCGGGGGTCGGGAGGAAGAGGGACCCCCAGCTTTTCT

[0407] ACCTCAGCGTCACTTTTCACAATACTCTGAACGAATGCACTCTTTTCAAAACCATGATTAAATCTCCCCGTACTCATGAT

[0408] TTTCCCCTCTGGCTCAGATCGGTTCCCTGTACTGGCAGGCCAGCCGAGGCCCGACTTGACCCTGGGGCGTGTCCCCCTTG

[0409] TCCCTGTGCTGGGGCTGCAGCCAGCGATGGGGCAGCAGCGCCGGCGTCTGGGTCTTGACGCCGGCAGGGCGCTGACCGTG

[0410] GTGCTGGGACCGAGGGCCGCCCTGCAAAGCGTATTCCTGCTCCTCCTACGCGTTCCTTATTGAATCTCGAAATCTAAATG

[0411] AAAGGGCTACATCCGCTTTTAAAGTACCGGTTCTCATTTACAAAGTGAAGATAACCACTTCTATTCCGGACTGAGACGGC

[0412] CTTTGCTTCAAGCCGGGTGTTGATGGTATAAAAAAGCTTCCTTTGGCGGGGGGGTGAGTGGGGCCCAGAGGTGGGAGGAG

[0413] G A T G A G G A A A G A C C A G T T G A C G G T T C C A A A G G A A GG A C T G T G C A T A G Nucleotide sequence of the GHR-Mssap-PCK1 vector (SEQ ID NO:5):

[0414] TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGAT

[0415] GCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGA

[0416] GCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCC

[0417] ATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGG

[0418] GGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATT

[0419] CGAGCTCGGTACCATGGTCGACCTGCAGCCTTACGGTCAGATAACTCTCACGTGAGAGTTATCTGACCGTAAGGGCGGCC

[0420] GCTTGAGCAGGTGTACGAGGACGAGCCCAATTTCTCTATATTCCCACAGTCTTGAGTTTGTGTCACAAAATAATTATAGT

[0421] GGGGTGGAGATGGGAAATGAGTCCAGGCAACACCTAAGCCTGATTTTATGCATTGAGACTGCGTGTTATTACTAAAGATC

[0422] TTTGTGTCGCAATTTCCTGATGAAGGGAGATAGGTTAAAAAGCACGGATCTACTGAGTTTTACAGTCATCCCATTTGTAG

[0423] ACTTTTGCTACACCACCAAAGTATAGCATCTGAGATTAAATATTAATCTCCAAACCTTAGGCCCCCTCACTTGCATCCTT

[0424] ACCCTAGGTTAATTAAATGAGAGCAGCCACTACGGGTCTAGGCTGCCCATGTAAGGAGGCAAGGCCTGGGGACACCCGAG

[0425] ATGCCTGGTTATAATTAACCCAGACATGTGGCTGCCCCCCCCCCCCCAACACCTGCTGCCTGAGCCTCACCCCCACCCCG

[0426] GTGCCTGGGTCTTAGGCTCTGTACACCATGGAGGAGAAGCTCGCTCTAAAAATAACCCTGTCCCTGGTGGATCCAGGGTG

[0427] AGGGGCAGGCTGAGGGCGGCCACTTCCCTCAGCCGCAGGTTTGTTTTCCCAAGAATGGTTTTAATTAACGTACGAGTAAC

[0428] CCGCTTACTCAGGCGAGTGCAAGCTGCTGCTGTGTCCCCAAAGTGTACATGTATGGAGGCGGCCTTCCACCTGCCCCTCA

[0429] GGTCTGTCCCTAAGCCAATGAGAATTCAGCAGCACCAGAATGGCCTGGAGCTGCCCCATGGTGACAGTGGGGGAGTATTT

[0430] AAAGTGGCCCAACCAGGGCTGAGAGCAGACATCTCCACTGGGCATCTCAGGGCCCATCTCCCCAAGGTGTGCTGGACTAG

[0431] CTAGATCTCTGCTAGCCTGTGAGTGTTGTGTTCATAGCACTTCATGTGTGCTTGAAGCCAATTAGAAAAGCTTGCTATAG

[0432] GAGAGAAAAGAGCTGCACTGAGCACCCTCCTTCCCCTTTAAATGTCAACAGATTAGGAGTCAGTGAATGACAGCACACCT

[0433] CTTGCTACCTTAGAGACCAAAATTTAAGCTACTCCCCTTAAGCTATAGCTAGAGTGCACCTGCCAGTGTCTTTAGTCCCC

[0434] ACTGATGGAACAGGACCCAAGGTATTGAAGATGGAACATAGTTATTCATTCATCCTCTAATTTAAAGCTGGATATGCT

[0435] GTCAGCAGAAATTGACGGAACAATGTAAATCAACTATAACAAGAAATAAAACCTGGGGGGAAAGAAGCTGACTATG

[0436] AAACCCCAGGAGCTTTCTACATGGGCCTGGACTCACCAAACTCTTTATTTTGTAATGGACTCTGACATTTTTAGGAAGG

[0437] GCTGTCCTGATGTGGGCTATAGAAGAGGGTTTCACATGCTTCTTCAAGAGGACCCACTGTCCCAGTTGCTGAGTCCCA

[0438] CCACCAGATGCTAGTGGCAGCTATTTGGGGAACACTTAGGCACTACAAAAAAATGAGTGATTCCATCTGGCTCACACCA

[0439] TATCCCTGATGTACCCCTTAAAGCATGTCACTGAGTTCATCACAGAAAATTGTTTCCCCTGTGCCTTCCACAAAAGGTT

[0440] AGAGCTGTCCTTGGGGCCAGGGGAAGGGGGCAGGGAGTGAGAAGCACCAACTGGATAACCTCCTCTGACCCCCACTCCAC

[0441] CT TA CC ATA AGTAG AT CCAA AT CCTTCTAGAAA AT TA GG AAGGC ATAT CCCC ATATAT CAGCG ATATA A AT AGAACTGCT

[0442] T C A G C G C T C T G G TA G A C G G T G A C T C T C C A A G G T G GA C T G G G A G G C A G C C T G G C C T T G G C T G G G C AT C G T C C T CTAAATAG

[0443] AA A G AT G A A C T T G T T C A G C C T T T C C A G A A G G A A A A CT G C T G C C C A G C C TA C A G T G C A A C G T C C T T G T C T T C C AT C TG GAG

[0444] GA A G C A C G G G T G A C ATAT C AT C TA G TA A G G G C A C C T C TC T G T T T C C A C C T C C A G G T C G A G G G G T G T G A C C C T TA C T TC T C

[0445] AGC C T C A AG G GA G GG AC AC T C A AC C C C C C A AA A AG AC AT GAG GG C G C T C A G C T C GG C C C A C C G C AC C C C G GA C C G GA G C C

[0446] GT C A C C C C C C G A A AT T C A C T C C C T T C A C A A G C C C C CA A G C G C G T T C T C T G G T G C G G A C T G C T C C G G G G C C C T G G CT T T GT

[0447] GC C C A G C G T T G T C A G A G C C A C C G C C C T G A G C C T G TC C C C G G G A G C C C C G C G C C T C C T C C C A C C G C T C C G C T C T CGC G C C C

[0448] CG C G G C C A GT T G T C T G C C C C G A G AC A GC T GC G C G C C CT C C C GC T GC C G G T GG C C C T C T C C G G T G G G G G T G G G G A C C GAC A

[0449] GGG T C A G C C C T C C GG AT C C G G GG C G C T C C GG G TA G C G GGG A G AA G T GAT C G C T G GG G A GC T G G GG G A GG G G T C GC C T T C C

[0450] T G C C C TA C C C A G G A C T C C G G G T G C G A C C G C T C C T CT AT C T C T C C A G C C C A C C A C C A C T C C A C C A C T T G G A C A C GT C T C C C

[0451] T C C T C C C T G G A G T C G C T C TA G A G G G T T T G G G G G T CT G A G TA A A G A A C C C G A AG TA G G G ATA C A G T G T G G C G G C AC C TT C C

[0452] AG A GG C C C C GG GC G C AG GG TA GA C C GG GG C GG GG C GG C C C GC GG AC A GG T G C AG C C C C AG GC G C AG GC GC A C T C G C GC C T

[0453] CC C G G C GC AG G C GG T GA A C C T C GC C C C AC C C C A GC C C C TC C GG G GG G C AG C T GG G C C G GG T C GG G AG G GG C C C A C C A G C C

[0454] C G G G A G A C A C T C C ATATA C G G C C A G G C C C G C T T TA C CT G G G C T C C G G C C A G G C C G C T C C T T C T T T G G T C A G C A C A GG G GA

[0455] CC C G G GC GG G GG C C C A G GC C GC TA AC C C GC C GG G GG A GG GGG C T C C A GT G C C C A A C AC C C A AATAT GG C T C G AG A AG G GG

[0456] AG C G AC AT T C C T GT G A G GC GG C T C GG G G G GA G A A C C C GCGG G C TATATA AA A C C T G A G C G T G GG G A C C A G C G G C C AC C G C

[0457] AGC G G AC A G C GC C GA GA G AA G C C T C GC T T C C C T C C C GC GGC G AC C A G GG C C C C A G C C G GA GA G C AG C A GC AAT T G AA A C C

[0458] CG A C G C C G G C G C G C C A C C AT G C C T C C T C A G C T C T C AA A C G G C C T C A A C C AC T C G G C C AA A G T C G T C C G G GG C AC C C TGGA

[0459] CA G C C T G C C C C A G G C C G T GC G G G AT T T C G T G G A G A GCA G C G C C A A G C T G T G C C A G C C T G A C C A A AT C C A C AT C T G CGAT G

[0460] GC T C C GA GG A GG AG A AC C AG C AG C T GC T G AG C C A C AT G GA GGA AG A GG G T G T C AT C AA G AG GC T G AA G AA G TAT G AC A A C

[0461] T G C T G GT T G G C T C T C A C A G A C C C C A G G GA C GT G G C CAG A AT T G A A A G C A A GA C G G T C AT C AT TA C T C A GG A G C A A A GGGA

[0462] T G C G G T G C C C AT C C C C A G A A GC G G C C T C A G C C A G C TG G G T C G C T G G AT G T C C C C GG A G G A C T T C G A G A AA G C C T T CAAT G

[0463] C C C G G T T T C C G G G G T G C AT G A A G G G T C G C A C G AT GTAT G T C AT C C C G T T C A G C AT G G G G C C C C T G G G C T C G C C C C TG T C C

[0464] AA G AT T G G C AT C G A G C T G A C G G A C T C G C C C TA C G T G GT G G C C A G C AT G C G C AT C AT G A C G C G G AT G G G C T C G G C C G T CC T

[0465] GG A C G C G C T G G GG G C G G GC G A G T T C AT C A A G G G C C T CC A C T C C G T G G G G T G C C C C C T G C C T T T G A A A A A G C C C T T GG T TA

[0466] AC A A C T G G G C C T G TA A C C C A G A G C T G A C GC T C AT T G CC C A C C T G C C C G A C C G C C G A G A A AT C AT C T C C T T C G G G A G CGGG

[0467] TAC GG C G G GA A C T C A C T C C T T G GG A A GA A G T G C T T T GCC C T G AG G AT G G C C A G C C G G C T GG C C AA G G AG G A GG G G T G G C T

[0468] GG C A G A G C A C AT G C T G AT C C T G G G C G TA A C C A A C C C CG A G G G C C A G A AG A A ATA C T T T G C C G C A G C T T T C C C C A G C GCC T

[0469] GC G G GA A G AC C A AC C T GG C TAT GAT G AA C C C C A C G C T C C CG GG C T G G AA A G T GG A G T G C G T G GG G G AC GA C AT C G C C T G G

[0470] AT G A A AT T C G A C C A A C A A G G TA AT T TA C G G G C C AT C A AC C C A G A G A A C G G G T T T T T T G G C G T T G C T C C C G G A A C C T CT GT

[0471] GAA G A C A AA C C C C A AT G C C AT C A A AA C C AT C C AA A GC AA CA C C AT C T T C A C C A A C G T G G C C G A G AC C AG C G A C G G GG G C G

[0472] T T TA C T G G G AA G G C AT C G A C C AG C C G C T G GC C C C G GG CAT C A A G C T C A C T T C C T GG A A G G GC A C A G AG T G G GA C C C C AAG

[0473] GAT G G G G A A C C T T G C G C T C A C C C C A A C T C G C G G T T CT G C A C C C C T G C C A G C C A G T G C C C C AT C AT C G A C C C T G C C TG GGA

[0474] GG C C C C G G A A G G C G T G C C C AT T G A G G G C AT C AT C T T CG G A G G G C G C C G C C C T G C T G G C G T C C C T C T G G T G TA C GAGGCT C

[0475] T C AG C T GG C A GC A C G GC GT GT T C GT G GG GG C G GC C AT G AGGT C C G A GG C C A C C G C GG C T GC G G AG C A C AA G GG C A AA GT G

[0476] AT TAT G C A C G A C C C C T T C G C C AT G C G G C C G T T C T T C GG C TA C A A C T T C G G C A A ATA C C T G G C C C A C T G G C T C A G C AT GG C

[0477] CC AG C AC C C G G C G G C C A A G C T G C C C A AG AT C T T C C A CGT C A A C T G G T T C C G G AA G G A C A A GG A C G G C A G G T T C C T C TG G C

[0478] CG G GC T T C G G C G AG A AC T G C C GG G T G C T G GC GT G G AT G TT C G A C C G C G T C C A GG G G AA G GG C G G C G C C A G GC T C A C G C C C

[0479] AT C G G C TA C G T C C C C G A G G A G G C C G C C C T G G A C C TGC G G G G C C T G G A G G C C AT C C G C G T G A G C G A G C T C T T C C AGGT C T C

[0480] CA AG GA GT T C T G GG AG G AG GA GG C G GA C GA GAT C C AC A AG TA CC T GG AG GA G C AG GT GA A C GC A GA C C T GC C C TAC G A AA

[0481] T C C AG A G C G A G G T C C T G G C C C T G A AG C A GA G A AT C A GC C A G AT G T GA G G C G C GC C A G G C C TA C C C C AT T C C A AC A G C T GC

[0482] GA C T T C T C C T C A GG A C G A C G A AT C T G C T C A A G T GC A GT G GC T G A C C T C G A G C C AC C A A G C GG C A A C T G C T C T C C C A CC G C

[0483] C T G C G T T G C T G C T G C C C A A A C C G A C A C A AT G T T T ATA A C G T G TA C AT A C AT TA A C T T AT T T A C C T C AT T T T G T T AT T TT T

[0484] AA A A C G A A G C C C T G T G G A A G G A A AT G G A A A A C T T G AA G C AT TA A A C T C A G C C AT T C T G T TAT G C T G C G TA A A AT T GT C TG

[0485] CT G T G T T TAT T T G C G G GG G T G A G GA G C G GC G G GG G T GG GGA G C G GC G G G GG T C GG G A GG A AG A GG G AC C C C C A G C T T T C T

[0486] AC C T C A G C G T C A C T T T T C A C A ATA C T C T G A A C G A AT GC A C T C T T T T C A A A A C C AT G AT T A A AT C T C C C C G TA C T C AT GAT

[0487] T T T C C C C T C T G G C T C A G AT C G G T T C C C T G TA C T G G CA G G C C A G C C G A G G C C C G A C T T G A C C C T G G G G C G T G T C C CC C T T G

[0488] T C C C T G T G C T G GG G C T G C A G C C A G C G AT GG G G C A G CA G C G C C G G C G T C T G GG T C T T G AC GC C G G C A G GG C G C T G A C CGT G

[0489] GT G C T G G G A C C G A G G G C C G C C C T G C A A A G C G TAT T CC T G C T C C T C C TA C G C G T T C C T TAT T G A AT C T C G A A AT C TA AAT G

[0490] AA A G G G C TA C AT C C G C T T T TA A A G T A C C G G T T C T C ATT TA C A A A G T G A A G ATA A C C A C T T C TAT T C C G G A C T G A G A C GG C

[0491] CT T T G C T T C A A G C C GG G T G T T G AT G G TATA A A A A AG C TT C C T T T G GC G G GG G G G T G AG T G G G G C C C A G AG G T G G G A GGAG

[0492] GAT G A G G A A A G A C C A G T T G A C G G T T C C A A A G G A A G GA C T G T G C ATA G G C C TATA A C T T C G TATA G C ATA C AT TATA C G AA

[0493] G T T AT G AT C G ATAT G A A G A AT C T G C T TA G G G T T A G G ATATC G C G T T T T G C G C T G C T T C G C G AT G TA C G G G C C A G ATATA C

[0494] G C G T T G A C AT T G AT T AT T G A C TA G T T AT T A AT A G T A ATC A AT TA C G G G G T C AT T A G T T C ATA G C C C AT ATAT G G A G T T C C

[0495] GC G T TA C ATA A C T TA C G G TA A AT G G C C C G C C T G G C T G AC C G C C C A A C G A C C C C C G C C C AT T G A C G T C A ATA AT G A C G TAT

[0496] GT T C C C ATA G T A A C G C C A ATA G G G A C T T T C C AT T G A C GT C A AT G G G T G G A G TAT T T A C G G TA A A C T G C C C A C T T G G C A GT

[0497] AC AT C A A G T G T AT C ATAT G C C A A G TA C G C C C C C TAT T G AC G T C A AT G A C G G T A A AT G G C C C G C C T G G C AT TAT G C C C A G T

[0498] A C AT G A C C T TAT G G G A C T T T C C T A C T T G G C A G TA C ATC TA C G T AT TA G T C AT C G C T AT T A C C AT G G T G AT G C G G T T T T GG

[0499] C A G TA C AT C A AT G G G C G T G G ATA G C G G T T T G A C T C A CG G G G AT T T C C A A G T C T C C A C C C C AT T G A C G T C A AT G G G A GT T T

[0500] GT T T T G G C A C C A A A AT C A A C G G G A C T T T C C A A A AT GT C G TA A C A A C T C C G C C C C AT T G A C G C A A AT G G G C G G TA G G CGT G

[0501] TA C G G T G G G A G G T C TATATA A G C A G A G C T C T C T G G C TAA C TA G A G A A C C C A C T G C T T A C T G G C T TAT C G A A AT TA ATA C G

[0502] AC T C A C TATA G G G A G A C C C A A G C T G G C TA G C G T T TA A AC T TA A G C T T G G TA C C G A G C T C G G AT C C A C TA G T C C A G T G TGG

[0503] T G G A AT T C G C C A C C AT G G G AT C G G C C AT T G A A C A A GAT G G AT T G C A C G C A G G T T C T C C G G C C G C T T G G G T G G A G A GG C TA

[0504] T T C G G C TAT G A C T G G G C A C A A C A G A C A AT C G G C T G CT C T G AT G C C G C C G T G T T C C G G C T G T C A G C G C A G G G G C G CC C GGT

[0505] T C T T T T T G T C A A G A C C G A C C T G T C C G G T G C C C T GAAT G A A C T G C A G G A C G A G G C A G C G C G G C TAT C G T G G C T G G CC A C GA

[0506] CG G G C G T T C C T T G C G C A GC T G T G C T C G A C G T T G T CAC T GA A G C G G G A A G GG A C T G G C T G C TAT T G G GC G AA G T G C C GGGG

[0507] C A G G AT C T C C T G T C AT C T C A C C T T G C T C C T G C C G A GA A A G TAT C C AT C AT G G C T G AT G C A AT G C G G C G G C T G C AT A C GC T

[0508] T G AT C C G G C TA C C T G C C C AT T C G A C C A C C A A G C G A AA C AT C G C AT C G A G C G A G C A C G TA C T C G G AT G G A A G C C G GT CT T G

[0509] T C G AT C A G G AT G AT C T G G A C G A A G A G C AT C A G G G G C TC G C G C C AG C C G A A C T G T T C G C C A G G C T C AA G G C GC G C AT GCC C

[0510] GA C G G C G AT G AT C T C G T C G T G A C C C AT G G C G AT G C C TG C T T G C C G A ATAT C AT G G T G G A A A AT G G C C G C T T T T C T G G ATT

[0511] C AT C G A C T G T G G C C G G C T G G G T G T G G C G G A C C G CTAT C A G G A C ATA G C G T T G G C TA C C C G T G ATAT T G C T G A A G A G CT T G

[0512] GC G G C G A AT G G G C T G A C C G C T T C C T C G T G C T T TA C GG T AT C G C C G C T C C C G AT T C G C A G C G C AT C G C C T T C TAT C G CC T T

[0513] CT T G A C G A G T T C T T C G C C AT G GT G A G C A A G G G C G A GG A G C T G T T C A C C G GG G T G G T G C C C AT C C T G G T C G A G C T GGAC GG

[0514] CG A C G TA A A C G GC C A C A AG T T C A GC GT G T C C G G C G AG GG C G AG G G C G AT G C C A C C TA C G G C A A GC T G A C C C T G A A GT T CA

[0515] T C T G C A C C A C C G G C A A G C T G C C C G T G C C C T G G C C CA C C C T C G T G A C C A C C C T G A C C TA C G G C G T G C A G T G C T T CA G C C G C

[0516] TA C C C C G A C C A C AT G A A G C A G C A C G A C T T C T T C A A GT C C G C C AT G C C C G A A G G C TA C G T C C A G G A G C G C A C C AT C TT C T T

[0517] CA AG GA C G AC G G C AA C TA C A AG AC C C GC G C C G AG G T G AA GT T C GA G GG C GA C A C C C T GG T G A AC C G C AT C G AG C T GA A GG

[0518] GC AT C G A C T T C A A G G A G G A C G G C A A C AT C C T G G G G CA C A A G C T G G A G TA C A A C TA C A A C A G C C A C A A C G T C TATAT CAT G

[0519] GC C G AC A A GC AG A AG A AC G G C AT C A A GG T GA A C T T C A AGAT C C G C C A C AA C AT C GA G GA C G GC AG C G T G C A G C T C GC C GA

[0520] CC A C TA C C A G C A G A A C A C C C C C AT C G G C G A C G G C C CC G T G C T G C T G C C C G A C A A C C A C TA C C T G A G C A C C C A G T CC G C C C

[0521] T G A G C A A A G A C C C C A A C G A G A A G C G C G AT C A C AT G GT C C T G C T GG A G T T C G T G A C C G C C GC C G G G AT C A C T C T CGGCAT G

[0522] GA C G A G C T G TA C A A G TA AT C TA G A G G G C C C G T T TA A A CC C G C T G AT C A G C C T C G A C T G T G C C T T C TA G T T G C C A G C CAT C

[0523] T G T T G T T T G C C C C T C C C C C G T G C C T T C C T T G A C C CT G G A A G G T G C C A C T C C C A C T G T C C T T T C C TA ATA A A AT G A GG A A A

[0524] T T G C AT C G C AT T G T C T G A G TA G G T G T C AT T C TAT T C T GG G G G G T G G G G T G G G G C A G G A C A G C A AG G G G G A G G AT T GGGAA

[0525] GA C A AT A G C A G G C AT G C T G G G G AT G C G G T G G G C T C TATG G C T T C T G A A G AT C T AT A A C T T C G T AT A G C AT A C AT T AT A C G

[0526] A A G T T AT G G AT C T G T C G A C G G T C A G AT A A C T C T C A CT C ATA C T T T A A G C C C AT T T T G T T T G T T G T A C T T G C T C AT C CA G T

[0527] C C C A G T C C C AT T G G C T T T C T C C T C A C C T G T T T TA G GTA G C C A G C A A G T C AT G A A AT C A G AT A A G T T C C A C C A C C A ATTA A

[0528] C A C T A C C C AT C T T G A G C AT A G G C C C A A C A G T G C AT TT AT T C C T C AT T T A C T G AT G T T C G T G A AT AT T T A C C T T G AT T TT C

[0529] AT T T T T T T C T T T T T C T T A A G C T G G G AT T T T A C T C C TG A C C C TAT T C A C A G T C A G AT G AT C T T G A C T A C C A C T G C G ATT G G

[0530] A C C T G A G G T T C A G C A AT A C T C C C C T T T AT G T C T T T TG A ATA C T T T T C A AT A A AT C T G T T T G T AT T T T C AT T A G T T A GTA A

[0531] C T G A G C T C A G T T G C C G TA AT G C T A ATA G C T T C C A A A CTA G T G T C T C T G T C T C C A G T AT C T G ATA A AT C T T A G G T G T T GC T

[0532] GG G A C A G T T G T C C TA A A AT TA A G ATA A A G C AT G A A A ATAA C T G A C A C A A C T C C AT TA C T G G C T C C TA A C TA C T TA A A C A A

[0533] T G C AT T C TAT C T T C A C A A AT G T G A A A A A G G A G T T C C CT C A G T G G A C TA A C C T TAT C T T T T C T C A A C A C C T T T T T C T TT G C

[0534] AC A AT T T T C C A C A C AT G C C TA C A A A A A G TA C T T T T C TG C T C A A G T C A C A C T G A G T T G AT T G C TAT T TA C C A A A AT C A AA G

[0535] TA A C AT T AT C A G AT C T C T G T A G G G T G G T T C C C T C T G GA AT G C TA C C C T C C ATA G T C C T TA C C C T T C A A G TA A A G A G C ATG

[0536] A A G A C T G A A AT AT C T C T G T G AT C T G T C AT C C T T T A A GC C A G A AT C C C C C AT A A A A A A G T T A G TAT T G C T T T C T C C T GAT C

[0537] C C AT A G C A G G T T G A AT C AT A G C A C T T AT C A G G T T G T TG T C AT T G C T T G C T T A A AT T C T C C TA A C TAT T T G G A G C T T C TT G

[0538] AG G G C A C A G G T T C T T G T T G A G T C T T G TA C C TA A G C AC C TA G TATA G T C C T T G AT G T C T A G C C A A C C C T A A ATA A A AT G CA

[0539] G T G A G T G A C AT G TA G AT G T C T T TAT A A G G T T T G ATA G GT T G G T C T C T C A A ATA G T T C T T T T G TAT G T T T G G TA G T G C T CT

[0540] A G AT TA G C A C T G G C C A G TATA A C T C T G AT G AT G G A A AT GT T C T AT A G C T AT G C T G T C TA ATAT G G TA G T C A C TA C T A A C A

[0541] TAT G T T A C T G T T G A G C C T T G G A A AT AT G G C T T T T G T GA C A A A A C T G A AT T T T T C AT G C TAT G T A AT T TA A G T C T A A AT TG

[0542] C T A C T G T G T A C AT T G T G G C T G TA G C C A C A A AT T T G TG C T G T G G AT T G C A G A ATA AT TA AT AT G G A C AT T G AT A AT T T T C T

[0543] T T T C ATA C TA A G C A G TA A G G A A A G A A A A G T T G A A A C TC T G T G G T C C AT T TA G G T T AT AT G T G TAT T T G TA C T T G AT T G G T

[0544] T T G T T T G A ATA C C T AT T T C T ATA C T T TA G C T G A G A G C TA A A G C C A A C A A A C C A G TA C T G TA G ATA A C C T G C T T T G G A C AA

[0545] C A AT G T G T T G A C T A G T T G G AT T T C AT C A A A G A AT G C CT A AT A A AT T T T A A G A A A AT G A G AT T T C AT T A A A C C AT A ATA C T

[0546] GA C ATA A G T T TA G G G A A G A AT C A G A C T ATAT C T G G T G T TT G T G A A A C TA C C C C T G A AT T T C A G T C C TA C A A A G T T T T C AG

[0547] T T T T G G A A A A A C T T T C AT C A G A G A G G G C A C TA A G TTA C A G G A A G C C AT C A C A A A G TA A G T T T T C AT C T G AT G A ATTATA A

[0548] AT T T A A G AT AT AT T T T A AT A C C A A A AT T C T T T AT G G T TT AT G T G C T A A C T T A A A AT T T C T C C T T A A A AT AT G A G A A C T AA

[0549] GTA C A C A AT T G TA C T T G G C T G T T TA AT G C G G AT T C C C AG T C C C T C A C A C A G A G AT T C T C A AT TA A G AT T G G A G A G C A G GG

[0550] G T T A C TA G A AT T C T T T T T C A G G T T C C T T ATAT G C T T CT G AT T T G G T G G C C TA G A A AT C A C A AT G C T A G T G C A G C C C T CAT

[0551] GG G G C TA C A G TATA C G TAT C T G A A A C AT G AT TA C AT C A G GG A A A C T G TAT G T C TA AT C TA C T T T G T C C C TA A A G G A A G C A

[0552] T T T T G A A A G G C A G A A A G TA AT AT G T G ATA G T T T T T G AA A C T T G TA G G T C A C AT T G T T T T T A A A A G G G AT C C A A G TA A GT T

[0553] T T T T T T T C T T T T G A G G G C TA C A C C T G T G G C A C AT G GA G G T T C C C A G G C TA G G G G T TA A AT C A G A A C T G C A G C T G C CA G C C

[0554] TAT G C C A G A G C C A C A G C A AT G C C A G AT C T G A G C T G T GT C T G C A A C T G T G TA G C T C A C A G C A A C G C T G G AT C C T TA A CC C A

[0555] AT G A G C A A G G C C A G A G AT T G A A C C TA C A A C C T C G T GG T T C C TA G T TA G AT T T G T T T C C G C T G T G C C A C G AT G G G A A CT C C

[0556] A A G TA AT T T T T T T T T G A G C A A G G A A G T T A C C T T T T TT G T C T G T T T T C C C A C TA A AT G C AT T C C T C A A G G AT T C C C A GT T T

[0557] G T T C T T G AT T C C T C A G T G C C T TA A C A C A G A C C T G G GT T C T C A G T A A AT G T T G AT T T TAT T G AT T T ATAT G T G A A AT T G TT

[0558] T T T C A A AT A AT A G T T T T T A A G T C C ATA G A A A C A AT G CT T C T T T TAT G G A G AT A C T T TA G G AT C ATA C T T G T A A C C C A A GT

[0559] TGCCTAATACTCTGTTCATAAAGAAAACTCATGCCTCATGGTCTCTGAATAATACATCTGTCTACCATTGAGCTCTTCCT

[0560] TGGGTTTCCTGTGCAAACCATTGCACTTATCCTCTTCCTGTGCTATACTTCCTCAGGCTTTATTACAGTTTTTAAAATAA

[0561] ACCAACTATCTATCTCTCTTTGAAGTAGAGCCATAATAATTGCATGAGAACACTGAAGGTTTTTAGGCTTTAATTTTTTC

[0562] TTTTTTTTTAAGATATTCAAAATTTGGAGTTCTTGTTGTGGCGCAGTGGTTAACGAACCCAACTAGGAACCATGACGCTG

[0563] CAGGTTCGGTCCCTGGCCTCGCTCAGTGGGTTAAGGATCCAGCGTTGCCATGAGCTGTGGTATAGGTCCATATGTGGCTC

[0564] GGATCCTGCATTGCTGTGGCTGTGGCAGCAGCCACAGGTACGATTAGACCCCTAGCCTGGGACCCTCCATATGCTGTGGG

[0565] CGCGGTCCTAGAAAAGAAAAAAAAAAAGAAAAGAAAAGAAAGATATACAAAATTTGAACTACGCATTGTTTCTCTTAACA

[0566] GTTGTTATGTATGGAGGAGGTTTGTTATAATTACAGTTTACAACTCTTAATCCAGAATATGTTAGGGATCCACATTCCCA

[0567] GGGTAAGACTAGTTTGTTTTAGGCCAGACTTAATTGTACAGCCCATTGTCCAGCCACATACTCAGGAGTCTCATACTTTG

[0568] CAGGCTAAAAATTCTTGATTTTGTTACCTAGTAGTGTACTGTTCATGTTGGGGAACTTTTTTCTCCAGAAAAGTTTATTA

[0569] TCCATTATCCTGCCTCCTTTTTATTTTCATTTATTTATTTATTTATTTTTGCTTTTTTAGGGCCACACTTGTGGCATATG

[0570] GAAATTCCTGGGCTAGGGGTCAAATCAGGGCTTCAGCTGCTGGCCTATGCCACAACAACACGGGATCAGAGCTGCATCTG

[0571] CAATCTATACCACAGCTTTTGGCAACCCCGTATCCTTAACCCAATGAATACTAGTTGGGTTCTTAACCCGCTAAGCCATA

[0572] ATTGGAACTCCCATGATCCCCATTCTTGATGAAGTGGATATCGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAA

[0573] GCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA

[0574] AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCA

[0575] GTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTT

[0576] CCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA

[0577] CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA

[0578] GGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGC

[0579] GAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG

[0580] CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTC

[0581] GGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACT

[0582] ATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGG

[0583] TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGC

[0584] TCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTT

[0585] TTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGAC

[0586] GCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAA

[0587] TTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG

[0588] CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAG

[0589] GGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCA

[0590] GCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAG

[0591] CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG

[0592] TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGT

[0593] TAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATA

[0594] ATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGT

[0595] ATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCAT

[0596] CATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTG

[0597] CACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAA

[0598] AAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTA

[0599] TTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAG

[0600] TGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

[0601] Nucleotide sequence of the HR-Ssap-PCK1 vector (SEQ ID NO: 6):

[0602] tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagc

[0603] gggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgca

[0604] tcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgg

[0605] gtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattcgagctcggtaccatggtcgacctgcagccttacggtcagataactctcacgtgagagttatctga

[0606] ccgtaagggcggccgcttgagcaggtgtacgaggacgagcccaatttctctatattcccacagtcttgagtttgtgtcacaaaataattatagtggggtggagatgggaaatgagtcca

[0607] ggcaacacctaagcctgattttatgcattgagactgcgtgttattactaaagatctttgtgtcgcaatttcctgatgaagggagataggttaaaaagcacggatctactgagttttacagtca

[0608] tcccatttgtagacttttgctacaccaccaaagtatagcatctgagattaaatattaatctccaaaccttaggccccctcacttgcatccttaccctaggttaattaaaattaacgtacgagta

[0609] acccgcttactcaggcgagtgcaagctgctgctgtgtccccaaagtgtacatgtatggaggcggccttccacctgcccctcaggtctgtccctaagccaatgagaattcagcagcacc

[0610] agaatggcctggagctgccccatggtgacagtgggggagtatttaaagtggcccaaccagggctgagagcagacatctccactgggcatctcagggcccatctccccaaggtgtg

[0611] ctggactagctagatctctgctagcctgtgagtgttgtgttcatagcacttcatgtgtgcttgaagccaattagaaaagcttgctataggagagaaaagagctgcactgagcaccctcctt

[0612] cccctttaaatgtcaacagattaggagtcagtgaatgacagcacacctcttgctaccttagagaccaaaatttaagctactccccttaagctatagctagagtgcacctgccagtgtcttta

[0613] gtccccactgatggaacaggacccaaggtattgaagatggaacatagttattcattcatcctctaatttaaaaagctggatatgctgtacagcagaaattgacggaacaatgtaaatcaa

[0614] ctataacagaagaaataaaaacctggggggaaagaagctgactatgaaaccccaggagctttctacatgggcctggactcaccaaactctttattttgtaatggacttctgacatttttag

[0615] gaagggctgtcctgatgtgggctatagaagagggtttcacatgcttcttcaagaggacccacactgtcccagttgctgagtcccaccaccagatgctagtggcagctatttggggaac

[0616] acttaggcactacaaaaaaatgagtgattccattctggctcacaccatatccctgatgtaccccttaaagcatgtcactgagttcatcacagaaaattgtttcccctgtgccttccacaaca

[0617] aggttagagctgtccttggggccaggggaagggggcagggagtgagaagcaccaactggataacctcctctgacccccactccaccttaccataagtagatccaaatccttctaga

[0618] aaattaggaaggcatatccccatatatcagcgatataaatagaactgcttcagcgctctggtagacggtgactctccaaggtggactgggaggcagcctggccttggctgggcatcgt

[0619] cctctaaatagaaagatgaacttgttcagcctttccagaaggaaaactgctgcccagcctacagtgcaacgtccttgtcttccatctggaggaagcacgggtgacatatcatctagtaag

[0620] ggcacctctctgtttccacctccaggtcgaggggtgtgacccttacttctcagcctcaagggagggacactcaaccccccaaaaagacatgagggcgctcagctcggcccaccgca

[0621] ccccggaccggagccgtcaccccccgaaattcactcccttcacaagcccccaagcgcgttctctggtgcggactgctccggggccctggctttgtgcccagcgttgtcagagccac

[0622] cgccctgagcctgtccccgggagccccgcgcctcctcccaccgctccgctctcgcgccccgcggccagttgtctgccccgagacagctgcgcgccctcccgctgccggtggccc

[0623] tctccggtgggggtggggaccgacagggtcagccctccggatccggggcgctccgggtagcggggagaagtgatcgctggggagctgggggaggggtcgccttcctgcccta

[0624] cccaggactccgggtgcgaccgctcctctatctctccagcccaccaccactccaccacttggacacgtctccctcctccctggagtcgctctagagggtttgggggtctgagtaaaga

[0625] acccgaagtagggatacagtgtggcggcaccttccagaggccccgggcgcagggtagaccggggcggggcggcccgcggacaggtgcagccccaggcgcaggcgcactcg

[0626] cgcctcccggcgcaggcggtgaacctcgccccaccccagcccctccggggggcagctgggccgggtcgggaggggcccaccagcccgggagacactccatatacggccagg

[0627] cccgctttacctgggctccggccaggccgctccttctttggtcagcacaggggacccgggcgggggcccaggccgctaacccgccgggggagggggctccagtgcccaacacc

[0628] caaatatggctcgagaaggggagcgacattcctgtgaggcggctcggggggagaacccgcgggctatataaaacctgagcgtggggaccagcggccaccgcagcggacagcg

[0629] ccgagagaagcctcgcttccctcccgcggcgaccagggccccagccggagagcagcagcaattgaaacccgacgccggcgcgccaccatgcctcctcagctctcaaacggcct

[0630] caaccactcggccaaagtcgtccggggcaccctggacagcctgccccaggccgtgcgggatttcgtggagagcagcgccaagctgtgccagcctgaccaaatccacatctgcga

[0631] tggctccgaggaggagaaccagcagctgctgagccacatggaggaagagggtgtcatcaagaggctgaagaagtatgacaactgctggttggctctcacagaccccagggacgt

[0632] ggccagaattgaaagcaagacggtcatcattactcaggagcaaagggatgcggtgcccatccccagaagcggcctcagccagctgggtcgctggatgtccccggaggacttcga

[0633] gaaagccttcaatgcccggtttccggggtgcatgaagggtcgcacgatgtatgtcatcccgttcagcatggggcccctgggctcgcccctgtccaagattggcatcgagctgacgga

[0634] ctcgccctacgtggtggccagcatgcgcatcatgacgcggatgggctcggccgtcctggacgcgctgggggcgggcgagttcatcaagggcctccactccgtggggtgccccct

[0635] gcctttgaaaaagcccttggttaacaactgggcctgtaacccagagctgacgctcattgcccacctgcccgaccgccgagaaatcatctccttcgggagcgggtacggcgggaact

[0636] cactccttgggaagaagtgctttgccctgaggatggccagccggctggccaaggaggaggggtggctggcagagcacatgctgatcctgggcgtaaccaaccccgagggccag

[0637] aagaaatactttgccgcagctttccccagcgcctgcgggaagaccaacctggctatgatgaaccccacgctcccgggctggaaagtggagtgcgtgggggacgacatcgcctgga

[0638] tgaaattcgaccaacaaggtaatttacgggccatcaacccagagaacgggttttttggcgttgctcccggaacctctgtgaagacaaaccccaatgccatcaaaaccatccaaagcaa

[0639] caccatcttcaccaacgtggccgagaccagcgacgggggcgtttactgggaaggcatcgaccagccgctggccccgggcatcaagctcacttcctggaagggcacagagtggga

[0640] ccccaaggatggggaaccttgcgctcaccccaactcgcggttctgcacccctgccagccagtgccccatcatcgaccctgcctgggaggccccggaaggcgtgcccattgaggg

[0641] catcatcttcggagggcgccgccctgctggcgtccctctggtgtacgaggctctcagctggcagcacggcgtgttcgtgggggcggccatgaggtccgaggccaccgcggctgc

[0642] ggagcacaagggcaaagtgattatgcacgaccccttcgccatgcggccgttcttcggctacaacttcggcaaatacctggcccactggctcagcatggcccagcacccggcggcc

[0643] aagctgcccaagatcttccacgtcaactggttccggaaggacaaggacggcaggttcctctggccgggcttcggcgagaactgccgggtgctggcgtggatgttcgaccgcgtcc

[0644] aggggaagggcggcgccaggctcacgcccatcggctacgtccccgaggaggccgccctggacctgcggggcctggaggccatccgcgtgagcgagctcttccaggtctccaa

[0645] ggagttctgggaggaggaggcggacgagatccacaagtacctggaggagcaggtgaacgcagacctgccctacgaaatccagagcgaggtcctggccctgaagcagagaatc

[0646] agccagatgtgaggcgcgccaggcctaccccattccaacagctgcgacttctcctcaggacgacgaatctgctcaagtgcagtggctgacctcgagccaccaagcggcaactgct

[0647] ctcccaccgcctgcgttgctgctgcccaaaccgacacaatgtttataacgtgtacatacattaacttatttacctcattttgttatttttaaaacgaagccctgtggaaggaaatggaaaactt

[0648] gaagcattaaactcagccattctgttatgctgcgtaaaattgtctgctgtgtttatttgcgggggtgaggagcggcgggggtggggagcggcgggggtcgggaggaagagggaccc

[0649] ccagctttctacctcagcgtcacttttcacaatactctgaacgaatgcactcttttcaaaaccatgattaaatctccccgtactcatgattttcccctctggctcagatcggttccctgtactgg

[0650] caggccagccgaggcccgacttgaccctggggcgtgtcccccttgtccctgtgctggggctgcagccagcgatggggcagcagcgccggcgtctgggtcttgacgccggcagg

[0651] gcgctgaccgtggtgctgggaccgagggccgccctgcaaagcgtattcctgctcctcctacgcgttccttattgaatctcgaaatctaaatgaaagggctacatccgcttttaaagtacc

[0652] ggttctcatttacaaagtgaagataaccacttctattccggactgagacggcctttgcttcaagccgggtgttgatggtataaaaaagcttcctttggcgggggggtgagtggggccca

[0653] gaggtgggaggaggatgaggaaagaccagttgacggttccaaaggaaggactgtgcataggcctataacttcgtatagcatacattatacgaagttatgatcgatatgaagaatctgc

[0654] ttagggttaggatatcgcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccata

[0655] tatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactt

[0656] tccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggc

[0657] attatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttg

[0658] actcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggc

[0659] ggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggcta

[0660] gcgtttaaacttaagcttggtaccgagctcggatccactagtccagtgtggtggaattcgccaccatgggatcggccattgaacaagatggattgcacgcaggttctccggccgcttgg

[0661] gtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtcc

[0662] ggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctg

[0663] ctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcc

[0664] cattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaact

[0665] gttcgccaggctcaaggcgcgcatgcccgacggcgatgatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgt

[0666] ggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgct

[0667] cccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttcgccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgac

[0668] gtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccacc

[0669] ctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccat

[0670] cttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacat

[0671] cctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacgg

[0672] cagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagacccca

[0673] acgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaatctagagggcccgtttaaacccgctgatcagcc

[0674] tcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgt

[0675] ctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaag

[0676] atctataacttcgtatagcatacattatacgaagttatggatctgtcgacGgtcagataactctcactcatactttaagcccattttgtttgttgtacttgctcatccagtcccagTCCcattg

[0677] gctttctcctcacctgttttaggtagccagcaagtcatgaaatcagataagttccaccaccaattaacactacccatcttgagcataggcccaacagtgcatttattcctcatttactgatgtt

[0678] cgtgaatatttaccttgattttcatttttttctttttcttaagctgggattttactcctgaccctattcacagtcagatgatcttgactaccactgcgattggacctgaggttcagcaatactcccctt

[0679] tatgtcttttgaatacttttcaataaatctgtttgtattttcattagttagtaactgagctcagttgccgtaatgctaatagcttccaaactagtgtctctgtctccagtatctgataaatcttaggtgt

[0680] tgctgggacagttgtcctaaaattaagataaagcatgaaaataactgacacaactccattactggctcctaactacttaaacaatgcattctatcttcacaaatgtgaaaaaggagttccct

[0681] cagtggactaaccttatcttttctcaacacctttttctttgcacaattttccacacatgcctacaaaaagtacttttctgctcaagtcacactgagttgattgctatttaccaaaatcaaagtaac

[0682] attcagatctctgtagggtggttccctctggaatgctaccctccatagtccttacccttcaagtaaagagcatgaagactgaaatatctctgtgatctgtcatcctttaagccagaatccc

[0683] ccataaaaaagttagtattgctttctcctgatcccatagcaggttgaatcatagcacttatcaggttgttgtcattgcttgcttaaattctcctaactatttggagcttcttgagggcacaggttc

[0684] ttgttgagtcttgtacctaagcacctagtatagtccttgatgtctagccaaccctaaataaaatgcagtgagtgacatgtagatgtctttataaggtttgataggttggtctctcaaatagttctt

[0685] ttgtatgtttggtagtgctctatagttagcactggccagtataactctgatgatggaaatgttctatagctatgctgtctaatatggtagtcactactaacatatgttactgttgagccttggaaat

[0686] atggcttttgtgacaaaactgaatttttcatgctatgtaatttaagtctaaattgctactgtgtacattgtggctgtagccacaaatttgtgctgtggattgcagaataattaattggacattgat

[0687] aattttcttttcatactaagcagtaaggaaagaaaagttgaaactctgtggtccatttaggttatatgtgtatttgtacttgattggtttgtttgaatacctatttctatactttagctgagagctaa

[0688] agccaacaaaccagtactgtagataacctgctttggacaacaatgtgttgactagttggatttcaTcaaagaatgcctaataaattttaagaaaatgagatttcattaaaccataatactga

[0689] cataagttgggaagaatcagactatatctggtgtttgtgaaactacccctgaatttcagtcctacaaagttttcagttttggaaaaactttcatcagagagggcactaagttacaggaag

[0690] ccatcacaaagtaagttttcatctgatgaattataaatttaagatatattttaataccaaaattctttatggtttatgtgctaacttaaaatttctccttaaaatatgagaactaagtacacaattgta

[0691] cttggctgtttaaatgcggattcccagtccctcacacagagattctcaattaagattggagagcaggggttactagaattctttttcaggttccttatatgcttctgatttggtggcctagaaatc

[0692] acaatgctagtgcagccctcatggggctacagtatacgtatctgaaacatgattacatcagggaaactgtatgtctaatctactttgtccctaaaggaagcattttgaaaggcagaaagta

[0693] atatgtgatagtttttgaaacttgtaggtcacattgtttttaaaagggatccaagtaagtttttttttcttttgagggctacacctgtggcacatggaggttcccaggctaggggttaaatcaga

[0694] actgcagctgccagcctatgccagagccacagcaatgccagatctgagctgtgtctgcaactgtgtagctcacagcaacgctggatccttaacccaatgagcaaggccagagattga

[0695] acctacaacctcgtggttcctagttagatttgtttccgctgtgccacgatgggaactccaagtaatttttttttgagcaaggaagttaccttttttgtctgttttcccactaaatgcattcctcaag

[0696] gattcccagtttgttcttgattcctcagtgccttaacacagacctgggttctcagtaaatgttgattttattgatttatatgtgaaattgtttttcaaataatagtttttaagtccatagaaacaatgc

[0697] ttcttttatggagatactttaggatcatacttgtaacccaagttgcctaatactctgttcataaagaaaactcatgcctcatggtctctgaataatacatctgtctaccattgagctcttccttgg

[0698] gtttcctgtgcaaaccattgcacttatcctcttcctgtgctatacttcctcaggctttattacagtttttaaaataaaccaactatctatctctctttgaagtagagccataataattgcatgagaa

[0699] cactgaaggtttttaggctttaattttttctttttttttaagatattcaaaatttggagttcttgttgtggcgcagtggttaacgaacccaactaggaaccatgacgctgcaggttcggtccctgg

[0700] cctcgctcagtgggttaaggatccagcgttgccatgagctgtggtataggtccatatgtggctcggatcctgcattgctgtggctgtggcagcagccacaggtacgattagacccctag

[0701] cctgggaccctccatatgctgtgggcgcggtcctagaaaagaaaaaaaaaaagaaaagaaaagaaagatatacaaaatttgaactacgcattgtttctcttaacagttgttatgtatgga

[0702] ggaggtttgttataattacagtttacaactcttaatccagaatatgttagggatccacattcccagggtaagactagtttgttttaggccagacttaattgtacagcccattgtccagccacat

[0703] actcaggagtctcatactttgcaggctaaaaattcttgattttgttacctagtagtgtactgttcatgttggggaacttttttctccagaaaagtttattatccattatcctgcctcctttttattttca

[0704] tttatttatttatttatttttgcttttttagggccacacttgtggcatatggaaattcctgggctaggggtcaaatcagggcttcagctgctggcctatgccacaacaacacgggatcagagct

[0705] gcatctgcaatctataccacagcttttggcaaccccgtatccttaacccaatgaatactagttgggttcttaacccgctaagccataattggaactcccatgatccccattcttgatgaagtg

[0706] gatatcggatcccgggcccgtcgactgcagaggcctgcatgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagc

[0707] cggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaa

[0708] tcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaagg

[0709] cggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccatag

[0710] gctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctc

[0711] ctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctg

[0712] ggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaac

[0713] aggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttacctt

[0714] cggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttga

[0715] tcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaat

[0716] ctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataact

[0717] acgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgc

[0718] agaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgt

[0719] ggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgtt

[0720] gtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattct

[0721] gagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaa

[0722] actctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaa

[0723] aatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgta

[0724] tttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccct

[0725] ttcgtc

Claims

1. A DNA fragment, wherein: The sequence of the DNA fragment is shown in SEQ ID NO:

4.

2. The DNA fragment of claim 1 is used to prepare a broadly ectopically expressed PCK1 The application of gene editing on pigs, among which, The extensive ectopic expression refers to ectopic expression in any one or more tissues of the testicles, kidneys, lungs, spleen, brain, intestines, stomach, heart, and skeletal muscles of gene-edited pigs.

3. A vector comprising the DNA fragment according to claim 1, wherein The nucleotide sequence of the vector is shown in SEQ ID NO:

3.

4. A vector, wherein The nucleotide sequence of the vector is shown in SEQ ID NO:

5.

5. A method for preparing a lean gene-edited pig, wherein: The method is to transfect cells with the vector described in claim 3 to obtain a cell that can be widely ectopically expressed in tissues. PCK1 Genetically modified pigs with high-quality lean meat, wherein the tissues include any one or more tissues of testicles, kidneys, lungs, spleen, brain, intestines, stomach, heart, and skeletal muscles; Or by transfecting cells with the vector described in claim 4, a cell can be obtained that can be ectopically expressed in tissues PCK1 Genetically modified pigs with high-quality lean meat.

6. A method for breeding a new breed of lean pigs, wherein: The method comprises the following steps: S1: First, synthesize the DNA fragment described in claim 1, and then add homologous arms to both ends of the obtained DNA fragment to construct a homologous recombination vector, wherein the homologous arms can undergo homologous recombination with the sequence between 65754555-65758570 bp of chromosome 13 of the pig genome Sscrofa11.1 version; or directly construct and synthesize the vector described in claim 3 or 4; S2: Transfect the obtained vector into pig cells to obtain site-specific integration pig positive cell lines; S3: Use the obtained pig positive cell line as the donor cell for nuclear transplantation to obtain primary gene-edited pigs; S4: The obtained primary gene-edited pigs can be bred and bred to obtain a new breed of lean meat high-quality pigs.

7. Use of the DNA fragment described in claim 1 or the vector described in claim 3 or 4 in increasing the intramuscular fat content of lean pigs.

8. The DNA fragment of claim 1 or the vector of claim 3 is used to improve the production of lean meat pigs PCK1 Application of genes in extensive ectopic expression in various tissues, among which, The tissues include any one or more of testicles, kidneys, lungs, spleen, brain, intestines, stomach, heart, and skeletal muscles.

9. Use of the DNA fragment described in claim 1 or the vector described in claim 3 or 4 in breeding new lean meat pig breeds.

10. Use of the DNA fragment of claim 1 or the vector of claim 3 or 4 in improving the intramuscular fat, later growth rate and reproductive performance of lean pigs.

Images