CN115322993B - Safety site for site-directed integration of exogenous genes in pig genome and method for constructing pig breeding group by using safety site - Google Patents
Safety site for site-directed integration of exogenous genes in pig genome and method for constructing pig breeding group by using safety site Download PDFInfo
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Abstract
The invention discloses a novel safe site gene fragment PSPD of a pig genome for site-directed integration of exogenous genes and a technical method for constructing a novel transgenic pig breeding material and a transgenic breeding population by using the same, wherein the sequence of the safe site gene fragment is located as chromosome 36,699,989 ～ 36,736,679 of pig No. 17. The site is positioned in the middle open chromatin region between the BPIFA2 (PSP) gene and the BPIFA3 gene, and is favorable for cutting various editing tools and fixed-point insertion of large fragment genes; the integration efficiency is high, and the expression of exogenous genes is facilitated; the transgenic pig constructed by adopting the PSPD safety site has high cloning efficiency, no adverse effect on pig and embryo development, and higher safety. The invention also discloses a method for quickly constructing a grain-saving environment-friendly pig breeding population with completely consistent genotypes by utilizing PSPD safety sites, and the method lays a foundation for the cultivation of various new transgenic pig varieties/strains.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a safe site for site-directed integration of exogenous genes in pig genome and a method for constructing pig breeding groups by using the safe site.
Background
Since the first world example of the production of transgenic mice by microinjection technology in 1980 (Gordon 1980), transgenic animal breeding technology has been developed for more than 30 years, and prokaryotic injection technology, cytoplasmic injection (ISCI) technology, somatic cloning transgene technology (SCNT) and the like have been developed at the level of genetic manipulation; the gene transfer technology comprises a calcium phosphate precipitation method, an electrotransfer method and a liposome transfection method, retrovirus-mediated gene transfer, an original germ cell technology and an active integration transposon system technology, but the mediated transgene integration mode is random, and different families with consistent genetic background are difficult to obtain, so that the method is unfavorable for the cultivation of new transgenic lines.
In recent years, along with the rapid progress of ZFNs, TALENs, CRISPR/Cas9 and other gene editing technologies, the efficiency of gene homologous recombination is improved in gene replacement, repair, increasing precision and high efficiency, and the editing technologies are integrated with the traditional transgenic technology to form a fixed-point integration technology, so that the efficiency of fixed-point integration of transgenes is improved, and the construction of new varieties with consistent transgene integration sites is facilitated. Site-directed integration transgenic techniques are techniques in which after a DNA Double Strand Break (DSB) is generated at a target site in an animal genome, exogenous genes are integrated into the specific target site by homologous recombination repair (HDR) mechanisms, or micro-homologous recombination mechanisms (MMEJ), or single-stranded oligonucleotide (SSODN) -mediated recombination repair mechanisms. Although the gene editing tools provide DSB conditions for targeted insertion of foreign genes, the subsequent HDR efficiencies are still quite low, particularly in porcine primary cells (Li Guoling, zhong Cuili, mo Jianxin, etc.. Animal genome targeted integration transgene technology research progress [ J ]. Genetics, 2017,039 (002): 98-109.). In addition, common exogenous expression vectors, such as a mammary gland bioreactor (> 25 kb), a salivary gland bioreactor (> 20 kb) and a polygenic coexpression vector, have very large DNA fragments to be integrated, and still have the problem of low homologous recombination efficiency. In addition, the gene editing technology also increases the unknown off-target risk, which leads to embryo development disorder, low pregnancy rate, less birth, malformation and increased stillbirth, and the obtained transgenic animal has unknown cause of growth retardation, thus increasing breeding difficulty and cost.
In addition to the risk of off-target of gene editing tools, the insertion of foreign genes can also affect the expression of target site genes, flanking neighboring genes, and chromatin status, thereby adversely affecting embryo development or individual health. For the safety and normal expression of the exogenous gene insertion, the development of a safety site on the genome of an animal is an important content, and the safety site provides a Safe and reliable Harbor for the exogenous gene, so that the exogenous gene can be stably and efficiently expressed, and has no side effects on cell growth, embryo development and individual health. A number of genomic Safe harbors available for mammalian foreign gene insertion have been discovered and applied to human, mouse, rat, rabbit-regulatable or reversible gene overexpression, for example: AAVS1, CCR5, HPRT, H11, col1a1, TIGRE, etc.
The Rosa26 site and the H11 site are also used for a pig genome safety site, but the inventor research team finds that the site can be used for fixed-point insertion of most genes and regulatory regions thereof when researching the pig Rosa26 site, however, when the large fragment genes are integrated by using the site, the site is easy to break or recombine, and transgenic animals carrying complete transgenic fragments cannot be obtained. The H11 locus is located in a narrow interval (5 kb) between Eif4 end 1 and Drg1 genes, the risk of influencing endogenous gene expression after exogenous gene insertion is small, and the exogenous gene insertion which is 9kb in length can be supported on pigs and can be expressed in various tissues of cells, embryos and animals. It is not known whether the loading is sufficient for integration of larger fragments. In addition, the study shows that (PMID: 32789000) AAVS1 safe site insertion reporter gene has larger variation in expression and silencing among different hiPSC cell clones, and the stability of gene expression is affected.
The low grain consumption and the low emission are all the targets pursued by the cumin of the pig industry, and the national grain safety and ecological safety are concerned, so that the cultivation of new grain-saving environment-friendly pig varieties has important strategic value for sustainable development of the pig industry in China. In the earlier stage study, the earlier stage inventor research team has successfully prepared a BEXA gene-transferred pig breeding model of co-expressed glucanase-xylanase-phytase by using a piggybac transposon mediated transgenic technology, the fragment is integrated in a pig CEP112 gene intron, and compared with a common pig, the same production performance is obtained, and the content of boar manure nitrogen, manure phosphorus and manure calcium is respectively reduced by 17.17%, 51.95% and 72.65%; the contents of the manure nitrogen, the manure phosphorus and the manure calcium of the sow are respectively reduced by 15.10 percent, 52.42 percent and 67.15 percent, and the environmental pressure of pig raising is greatly relieved. The environment-friendly pigs have higher ecological value and are expected to bring great economic benefit to farmers. The study shows that compared with the common pigs fed with the same feed, the daily gain (ADG) of the pigs is improved by about 20 percent, the Feed Conversion Rate (FCR) is reduced by about 15 percent, the feeding period is shortened by about 30 days, the average feed per head can be saved by 26-33 kg, and the feed cost is saved by 78-99 yuan per head. The environment-friendly pig accords with the idea of advocating low carbon, green and environment protection by the government, and has very broad market prospect.
The invention relates to grain-saving environment-friendly transgenic breeding production application, which also needs to construct 6-8 ancestral lines which are integrated on the same genetic locus and have no blood relationship, and is used for constructing a breeding population, in order to solve the technical problem of rapid construction of grain-saving environment-friendly pig population, the inventor research team also establishes a large-fragment gene fixed-point integration technology, utilizes a CRISPR-Cas9 system to integrate the transgenic fragments into Cep112 sites respectively, PSP-TC terminal stop codon (termination codon) sites and Rosa26 sites (first introns), and early test results show that: the Cep112 locus can insert the transgene into pig genome and express normally, but has the disadvantages of low cloning efficiency, less birth and difficult survival, and the cost for constructing breeding population by using the locus is high; although the PSP-C terminal locus has high cloning efficiency and healthy and normal piglets, the target gene cannot be expressed; the Rosa26 locus is a safety locus accepted by the academic community, but the grain-saving environment-friendly pig homologous recombinant vector constructed by the Rosa26 locus is easy to break, and has unknown reasons of deletion in the gene integration process. The prior art integrates polycistronic BEXA (bg 17-eg 1314-xynB-appA) expressing glucanase (bg 17A and eg 1314), xylanase (xynB) and phytase (APPA) into the pig parotid protein gene PSP (parotid secretory protein, PSP) upstream of the stop codon, and allows the expression of exogenous and endogenous genes in tandem. The results show that editing at the stop codon of the PSP gene, although the heterozygote or homozygote mutation does not affect the survival of transgenic pig cells and individuals, the cloning efficiency is low and is only 0.87% (the number of cloned piglets/embryo transfer is obtained), and the cloning efficiency is only equivalent to the cloning efficiency of Rosa26 locus of 0.81%, so that the marketization of the transgenic pigs is seriously affected. There is a need to develop a new independent intellectual property safety site for constructing new grain-saving and environment-friendly pig breeder groups.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a safe site for site-directed integration of exogenous genes in a pig genome and a method for constructing a pig breeding group by using the safe site, develops a novel safe site PSPD on the pig genome, has larger bearing capacity, can keep the independence of an exogenous gene regulation region to play a role and enable the efficient expression of exogenous inserted genes, has higher targeting efficiency, site-directed integration efficiency and cloning efficiency, has higher safety on the exogenous genes and animals, and can be used for breeding novel transgenic animals. The method is mainly used for biological breeding and breeding new transgenic animal varieties with great economic value.
The invention discloses a novel pig genome safety site for site-specific integration of exogenous large fragment genes and application thereof. The site interval is located in pig Chromosome 17 (Chromosome 17:36,699, 989-36,736,679), 500bp downstream of the BPIFA2 (PSP) stop codon to 3000bp upstream of the BPIFA3 gene start codon. The locus is positioned in an open chromatin region, no other genes exist in the region, no other transcripts exist, the interval region is 36690bp, the GC content of the whole region is 44.72%, and the locus is very beneficial to cutting by various editing tools and homologous recombination insertion and expression of exogenous genes. The invention screens 128 ideal sgRNA targeting sites in the interval. Experiments prove that the 11 sgRNAs have higher cleavage efficiency on pig genome, 9 sgRNAs have higher cleavage activity, wherein the targeting cleavage efficiency of 2 sgRNAs is up to 62.8% and 63.1% respectively, then, one of the sites is selected, the flanking homologous sequences of the sites are cloned to construct a large-fragment transgenic homologous targeting donor plasmid, the large-fragment transgenic 23.66kb is successfully integrated into pig genome, the site-directed integration efficiency is up to 27.27% -33.3%, and is far higher than rosa26 (7.69%), cep112 (12.94%) and H11 (25%), and the average value of the sites is 2.5-3.1 times of that of the prior art. The fluorescent protein marker gene at the cellular level of the site is well expressed. The pregnancy rate of the transgenic pig transplantation receptor sow prepared by the site reaches 80%, the cloning efficiency of the transgenic pig is up to 2.15% (the average value of transgenic pig/transplantation embryo number in the prior art is 0.06%), and 4 exogenous genes expressed by tandem polycistronic in primary transgenic pig gram Long Zhu (foundation) pig can be expressed efficiently, and strict tissue specificity is presented. Compared with the prior safe site, the safe site has high site-directed integration efficiency and cloning efficiency, can be used for inserting and expressing large fragment genes, has no adverse effect on pig and embryo development, has higher safety, and is an ideal safe site for pig biological breeding. The invention uses PSPD safe site and combines large fragment transgene fixed point integration technology to successfully construct grain-saving environment-friendly transgene pig breeding population, compared with traditional variety/strain cultivation method (decades) and line ancestor construction method (inbreeding is difficult to control), the invention adopts transgene breeding population construction method to construct line ancestor blood margin more widely, can effectively slow down population inbreeding increment, has shorter breeding time (only 2 generations and 2 years), and can save a great amount of breeding cost.
It is a first object of the present invention to provide a safe site gene fragment.
The second object of the invention is to provide the application of the safe site gene fragment in the fixed-point integration of exogenous genes in pig genome.
A third object of the present invention is to provide a homology arm for a homologous recombination vector for site-directed integration of a foreign gene into a mammalian genome.
A fourth object of the present invention is to provide a homologous recombination vector.
The fifth object of the invention is the application of the homology arm and/or the homologous recombination vector in the application of site-directed integration exogenous genes of mammalian genome or preparation of a kit for site-directed integration exogenous genes of pig genome.
It is a sixth object of the present invention to provide an sgRNA targeting said safe site gene fragment.
It is a seventh object of the present invention to provide a CRISPR/Cas9 expression vector.
An eighth object of the present invention is to provide a recombinant cell for integrating exogenous genes into porcine genome.
It is a ninth object of the present invention to provide a composition for integration of exogenous genes into the genome of swine.
The tenth object of the invention is to provide the application of one or more of the safe site gene fragment, the homology arm, the homologous recombination vector, the sgRNA, the CRISPR/Cas9 expression vector or the recombination cell in the application of the pig genome site-specific integration exogenous gene or the preparation of the pig genome site-specific integration exogenous gene kit.
An eleventh object of the present invention is to provide a transgenic method for site-directed integration of exogenous genes into porcine genome.
The twelfth object of the invention is to provide a construction method of the grain-saving environment-friendly pig.
The thirteenth object of the invention is to provide a method for constructing a grain-saving environment-friendly pig breeding group.
In order to achieve the above object, the present invention is realized by the following means:
a safe site gene fragment for site-directed integration of a foreign gene in a porcine genome, the sequence of which is porcine chromosome 17 36,699,989 ～ 36,736,679 (porcine genome sequencing batch (SGSC) Sscoffa 11.1 (NCBI project 13421, GCF_000003025.6)) (SEQ ID NO: 159), which is located 500bp downstream of the stop codon of BPIFA2 (PSP) to 3000bp upstream of the start codon of the BPIFA3 gene. The locus is positioned in an open chromatin region, no other genes exist in the region, no other transcripts exist, the interval region is 36690bp, the GC content of the whole region is 44.72%, and the locus is very beneficial to cutting by various editing tools and homologous recombination insertion and expression of exogenous genes.
Preferably, the safety site gene fragment is located on chromosome 17 of swine 36,704,009 ～ 36,736,679.
Preferably, the gene fragment is as set forth in SEQ ID NO: 1-2, SEQ ID NO:9 to 124, SEQ ID NO: 129-139, SEQ ID NO: 3-4, or a combination of one or more of the sequences shown in figures.
The homology arm of the homologous recombination vector for site-directed integration of exogenous genes in mammalian genome is designed based on the safety site gene fragment, and the nucleotide sequence of the homology arm is shown as SEQ ID NO:1 and 2, said integration of the exogenous gene being based on homologous recombination
A homologous recombination vector comprising: an upstream homology arm, an insert, and a downstream homology arm, the upstream homology arm having a nucleotide sequence as set forth in SEQ ID NO:1, the nucleotide sequence of the downstream homology arm is shown as SEQ ID NO: 2. as shown in the drawing,
preferably, the homologous recombination vector is a homologous recombination vector containing a Cre-LoxP recombinase system, and the homologous recombination vector further contains a selectable marker gene, wherein the selectable marker gene can be deleted by using Cre recombinase.
Preferably, the selectable marker gene is EGFP and/or NEO.
The homology arm and/or the homologous recombination vector are/is applied to site-directed integration of exogenous genes in a mammalian genome or preparation of a kit for site-directed integration of exogenous genes in the mammalian genome.
The sgRNA of the safe site gene fragment is targeted, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO:9 to 124, SEQ ID NO:129 to 139.
A CRISPR/Cas9 expression vector comprising said sgRNA sequence.
Preferably, the coding and CRISPR structural sequences of the Cas9 protein are also contained.
A recombinant cell of pig genome integrated exogenous gene, transformed or transfected with the CRISPR/Cas9 expression vector and/or the homologous recombinant vector.
A composition for integration of a foreign gene into a porcine genome, comprising one or more of said homology arm, said homologous recombination vector, said sgRNA, said CRISPR/Cas9 expression vector, or said recombinant cell.
The application of one or more of the safe site gene fragment, the homology arm, the homology recombination vector, the sgRNA, the CRISPR/Cas9 expression vector or the recombination cell in pig specific site-specific integration gene editing or the application of preparing pig specific site-specific integration gene encoding kit.
A method for transgene of pig genome site-directed integration exogenous gene, which uses one or more of the safe site gene fragment, the homology arm, the homologous recombination vector, the sgRNA, the CRISPR/Cas9 expression vector, or the recombination cell.
A grain-saving environment-friendly pig breeding group construction method utilizes one or more of the safe site gene fragment, the homology arm, the target gene homologous recombination vector, the sgRNA, the CRISPR/Cas9 expression vector and the recombination cell, and the transgenic heterozygote or homozygote subgroup construction method.
The construction method of the grain-saving environment-friendly pig utilizes one or more of the homologous arm, the homologous recombination vector, the sgRNA, the CRISPR/Cas9 expression vector or the recombination cell to integrate polycistronic BEXA into a pig genome, and the nucleotide sequence of the polycistronic BEXA is shown as SEQ ID NO: shown at 158.
Preferably, the CRISPR/Cas9 expression vector and the homologous recombination vector are co-transfected and resuscitated pig embryo fibroblasts, the transfected cells are subjected to limiting dilution and plating, positive clone cell clusters are screened after 12 to 15 days of culture, cells after screening marker genes are removed as nuclear transfer donor cells, a transgenic embryo is constructed by utilizing a somatic cloning transfer technology, the reconstructed embryo is transplanted into a surrogate sow body through operation, and the transgenic cloned pig is prepared, namely the grain-saving environment-friendly pig, and the insertion fragment of the homologous recombination vector is the polycistronic BEXA.
More preferably, the screening of positive clonal cell populations comprises screening marker gene expression positives and polycistronic BEXA insertion positives;
more preferably, transfection is performed using the A-033 procedure in a LONZA electroporation transfection system.
More preferably, a nucleotide sequence as set forth in SEQ ID NO: the primers 144 to 149 were subjected to a positive selection of polycistronic BEXA.
More preferably, the homologous recombination vector is a homologous recombination vector containing a Cre-LoxP recombinase system, and the homologous recombination vector further contains a selection marker gene, wherein the selection marker gene can be deleted by using Cre recombinase, and after the integration of polycistronic BEXA is identified, the Cre recombinase is deleted by using Cre recombinase.
A grain-saving environment-friendly pig breeding group construction method utilizes one or more of the safe site gene fragment, the homology arm, the homologous recombination vector, the sgRNA, the CRISPR/Cas9 expression vector or the recombination cells.
Preferably, the method comprises the following steps:
s1, selecting a boar without a common ancestor within no less than 6 generations as a boar ancestor; selecting sows with the same number of first three or less generations and without common ancestor from sows as sow progenitors, and taking the selected sow progenitors as mating objects of the above boar progenitors;
S2, selecting the sow progenitors and the boar progenitors one by one, wherein no common ancestor exists in 3 generations between the sow progenitors and the boar progenitors to be bred, and taking out embryos when the embryos are pregnant for 25 to 35 days old;
s3, after three times of washing by DMEM, digesting the tissue to be fluffy, adding serum for plating overnight, adding the tissue to a high-sugar DMEM culture solution containing 10 to 14% (V/V) FBS for maintenance culture for 2 to 3 days, obtaining embryo fibroblasts (PEF) of offspring of each pair of sow-based progenitors and boar-based progenitors, and identifying the sex;
s4, constructing grain-saving and environment-friendly pigs by embryo fibroblasts of offspring of each pair of sow progenitors and boar progenitors according to the construction method of claim 12 or 13, and producing transgenic cloned boars and sows with no less than 6 families as F0 transgenic pig groups;
s5, mating the F0 transgenic cloned boars and sows of each family with F0 transgenic boars or sows of other families respectively, propagating to obtain F1 transgenic grain-saving environment-friendly transgenic pigs, selecting transgenic pig homozygotes (+/+) boars and sows, and constructing to obtain grain-saving environment-friendly pig breeding core groups.
Preferably, in step S3, a nucleotide sequence as set forth in SEQ ID NO:154 to 157.
Preferably, in step S3, after overnight, the culture is maintained by adding the medium to a high-sugar DMEM medium containing 12% (V/V) FBS for 2 to 3 days.
Preferably, in step S4, the female and male embryos are mixed according to 1:1, and transplanting the mixture into oviducts of 4-6 pregnant sows.
Preferably, in step S4, the boar ancestor and the sow ancestor are each 6
Preferably, in step S5, a nucleotide sequence as set forth in SEQ ID NO:150 to 153, wherein the transgenic pig homozygote is selected, and only the nucleotide sequence is shown as SEQ ID NO:152 to 153, is a transgenic pig homozygote
Compared with the prior art, the invention has the following beneficial effects:
1) The invention discovers that the PSPD safety site gene fragment is positioned in the open chromatin region between the BPIFA2 (PSP) gene and the BPIFA3 gene, the region interval is as long as 37kb (the H11 site is only provided with a 5kb interval region, the rosa26 site and the cep112 site are positioned in an intron), and compared with the existing reporting safety site, the PSPD safety site fragment is very favorable for cutting various editing tools; facilitating the fixed-point insertion of large fragment genes (including HDR, MMEJ, SSA, NHEJ and other technologies); the safe site is favorable for the expression of exogenous genes and keeps the tissue specificity of the exogenous genes from being influenced by endogenous genes.
2) Compared with the prior art, the PSPD safety site obtained by the invention has higher site-specific integration efficiency (the efficiency is higher than Rosa26, cep112 and H11), the difficulty of screening the site-specific integration cell line is obviously reduced, and the time and cost of cell screening are saved.
3) Compared with the prior art, the safe site obtained by the invention has higher cloning efficiency, the cloned animal prepared by the safe site has higher pregnancy rate, and the litter size and the healthy young are all the same, which shows that the safe site is not negatively affected by off-target and the like, and is very beneficial to constructing a transgenic new breed population.
4) Compared with the prior art, the safety site obtained by the invention can ensure the integrity of the inserted fragment gene in the homologous recombination process with the inserted fragment, and the rosa26 site is easier to break and recombine.
Compared with the prior safety sites (Rosa 26, cep112 and H11), the PSPD safety site has high site-directed integration efficiency and cloning efficiency, can be used for large fragment gene insertion and expression, has no adverse effect on pig and embryo development, has higher safety, and is an ideal safety site for pig biological breeding.
5) Compared with the existing breeding method, the method for breeding the transgenic new variety overcomes the defects of long time consumption and slow progress of the traditional breeding, introduces effective exogenous genes into the existing pig breeding population, and constructs a new pig breeding population with special effects, wherein more than 6 families and new transgenic homozygote populations with completely consistent genotypes can be obtained only by 2 generations. .
Drawings
FIG. 1 is a predicted position of the PSPD site at the safety site and the chromosomal environment in which it is located.
FIG. 2 shows the prediction of the ability of PSPD to form nucleosomes in the region of PSPD.
FIG. 3 shows the GC% analysis results of PSP gene and PSPD region.
FIG. 4 is a schematic of the predicted promoter and enhancer regions of PSPD locus regions using softberry.
FIG. 5 is a schematic diagram showing the prediction of the suspected Promoter region of the PSPD site (downstream of the 3' UTR of the PSP gene) by using the Promoter-2.0 online software.
FIG. 6 shows the predicted results of the PSPD site region cis-regulatory elements polyT and polyA.
FIG. 7 shows PSPD site regions (chromoname 17:36,699, 989-36,736,679) for insertion of foreign genes into potential sgRNA sites.
FIG. 8 is a T7EI cleavage analysis of PSPD-sgRNA-Cas9 target site mutation efficiency.
FIG. 9 is a scheme of KI-PSPD-npsp-BEXA vector construction.
FIG. 10 is an electrophoretogram of PSPD-LA and PSPD-RA amplification; m is DNA Marker, PSPD-LA is 350bp, and PSPD-RA is 3511bp.
FIG. 11 is an inf-PSPD-LA, inf-PSPD-RA amplification electrophoretogram; m is DNA Marker, inf-PSPD-LA is 380bp, inf-PSPD-RA is 3541bp.
FIG. 12 is a screening marker (neoGFP) amplification electrophoretogram; m is DNA Marker, and PCR target band is 2627bp.
FIG. 13 is a screening marker homology arm (inf-neoGFP) amplification electrophoresis pattern; m is DNA Marker, and PCR target band is 2638bp.
FIG. 14 is a double cleavage of the CEP112-npsp-BEXA vector (supercoiled donor plasmid CEP112-LA340RA3219, chinese patent CN 201711477805.5) FastDigest NotI and FastDigest SmaI; m is a DNA Marker, NC is a cep112-npsp-BEXA vector (26310 bp), sample is a cep112-npsp-BEXA double-restriction enzyme vector, and two sections after restriction enzyme are 25941bp and 373bp respectively.
FIG. 15 is a PCR identification of PSPD-LA-npsp-BEXA bacterial fluids; m is DNA Marker, and PCR target band is 1076bp.
FIG. 16 is a FastDigest SpeI cleavage of the PSPD-LA-npsp-BEXA vector; m1 is DNA Marker 15K, M2 is DNA Marker 2K, NC is PSPD-LA-npsp-BEXA vector (26291 bp), ZT is PSPD-LA-npsp-BEXA digestion vector, and two sections after digestion are 25605bp and 690bp respectively.
FIG. 17 PCR identification of PSPD-LA neoGFP-npsp-BEXA bacterial liquid; m is DNA Marker, and PCR target band is 1564bp.
FIG. 18 is a FastDigest XhoI cleavage of the PSPD-LA neoGFP-npsp-BEXA vector; m is DNA Marker 15K, NC is PSPD LA neoGFP-npsp-BEXA vector (28252 bp), ZT is PSPD-LA-npsp-BEXA digestion vector, and two sections after digestion are 23152bp and 5103bp respectively.
FIG. 19 is a PCR identification of KI-PSPD-npsp-BEXA bacterial fluids; m is DNA Marker, and PCR target band is 984bp.
FIG. 20 shows the cleavage of KI-PSPD-npsp-BEXA vector; m1 is DNAMmarker 15K, M2 is DNA Marker 8K, M2 is 5Kb DNA Marker,M2 is 2Kb DNA Marker,NC1, NC 2 is KI-PSPD-npsp-BEXA vector (26687 bp), S1 and S2 are KI-PSPD-npsp-BEXA digestion vectors, and the lengths of target fragments when digested with FastDiget SgsI, fastDiget SnaBI and FastDiget SmaI are respectively: 13007bp, 8735bp, 4103bp and 842bp. When FastDiget Kpn2I, fastDiget BshTI, fastDiget SmaI are used for digestion, the lengths of the target fragments are respectively: 10576bp, 7644bp, 6978bp and 1499bp.
FIG. 21 is a map of KI-PSPD-nPSP-BEXA vector.
FIG. 22 is a fluorescent screen of transgenic monoclonal cell lines with PSPD site-directed integration of BEXA.
FIG. 23 is a PCR map of cell screening LA and RA integration assays for PSPD site-directed integration of transgenes of BEXA; and (3) injection: the upper panel shows the selected Duroc boar cell line 5F1 and the lower panel shows the selected Duroc sow cell line AB17-046400.
FIG. 24 shows the sequencing results of PSPD site integration exogenous fragments.
FIG. 25 is a transgenic pig with PSPD site-directed integration of BEXA.
FIG. 26 shows PSPD site cloning pig PCR detection.
FIG. 27 shows the relative expression levels of PSPD site KI transgenic cloned pig target genes in various tissues.
FIG. 28 shows cloned pig parotid and submaxillary glucanase activity.
FIG. 29 shows the activity of cloned pig parotid and submaxillary glands.
FIG. 30 shows cloned pig parotid and submaxillary gland xylanase activity.
FIG. 31 shows cloned pig parotid and submaxillary phytase activity.
FIG. 32 shows a scheme for H11-npsp-BEXA vector construction.
FIG. 33 shows the sex determination results of Duroc pig PEF cell line.
FIG. 34 shows the results of transgenic cell line selection and identification.
FIG. 35 is a diagram of a method of constructing and breeding a grain-saving and environmentally friendly pig breeding herd.
FIG. 36 shows the genotyping results (partial porcine identification) of the F1 transgenic pigs.
FIG. 37 is a F1 generation grain-saving environment-friendly pig homozygous breeding population
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 determination of PSPD safety site Gene fragments
1. PSPD site
PSP genes were searched on NCBI (pig genome sequencing Consortium (SGSC) Sscoffa 11.1 (NCBI project 13421, GCF_000003025.6)) and NCBI database, respectively, and PSP gene promoters (3000 bp upstream) from 168bp downstream to BPIFA3 gene stop codon on Chromosome 17 of pig were found (chromosomer 17:36,699, 989-36,736,679), designated PSPD sites, see FIG. 1, nucleotide sequences as shown in SEQ ID NO: 159.
No other genes were found in the PSPD site region, nor were other transcripts found to be present, the spacer was 36690bp long.
2. Chromatin accessibility analysis of PSPD sites
1. Experimental method
Chromatin accessibility (Chromatin accessibility) is the degree to which an intranuclear macromolecule can physically contact chromatin DNA, reflects the overall regulatory potential of transcription factor TF binding and genetic loci, and has strong ability to form closed chromatin (closed chromomatrix) nucleosomes, high nucleosome occupancy, while loose chromatin (permissive chromatin) nucleosomes occupancy is low, facilitating Transcription Factor (TF) binding, regulating gene transcription.
Nucleosome occupancy capacity prediction was performed on PSPD site regions (pig chromosome17:36, 699,989-36,736,679) using Recon program (http:// wwmgs. Bionet. Nsc. Ru/mgs/programs/Recon /) on-line software.
2. Experimental results
Chromosome accessibility is predicted through nucleosome formation ability, and the chromatin opening degree in a PSPD locus region is estimated, and as shown in a result of fig. 2, a weak region occupied by a more polynuclear corpuscle exists in the PSPD locus region, and the PSPD locus is presumed to be positioned in the more open chromatin region, so that gene editing, insertion and transcription of exogenous genes are facilitated.
3. Predicting PSPD site GC content and distribution
1. Experimental method
GenSmart was provided using gold Style TM Codon Optimization software, predicting PSPD site GC content and distribution.
2. Experimental results
As a result, as shown in FIG. 3, it was predicted that the GC content 44.72% in the whole PSPD site region was almost not present in more than 70%, which is very advantageous for the homologous recombination operation of the foreign gene. GC content <30%, GC content <30% was distributed only 0-4020 bp downstream of 3' UTR under PSP gene, giving knockout, the remaining region was (chromoname 17:36,704, 009-36,736,679).
3. PSPD site promoter and enhancer and prediction of transcription initiation site
1. Experimental method
The present invention uses software on-line to predict the possible presence of promoter and enhancer regulatory sequences in PSPD regions (http:// linux1. Software. Com/berry. Phtmtropic = fprom & group = program & subgrouping = promoter).
The transcription initiation site of the vertebrate PolII Promoter was also predicted using Promoter-2.0 on-line software (https:// services. Healthcare. Dtu. Dk/services. PhpPromoter-2.0).
2. Experimental results
As shown in FIG. 4, the result is predicted by software on line of softberry, and the potential promoter and the enhancer are mainly distributed in 7005 bp-35112 bp region (downstream of PSP gene 3 'UTR) of PSPD site, in order to avoid the influence of potential regulatory sequences on inserted genes, the invention selects the PSPD 4020-7005 bp region (downstream of PSP gene 3' UTR) as the foreign gene inserted safety region.
As shown in FIG. 5, the prediction of Promoter 2.0 shows that the 2400 bp-15000 bp interval of PSPD site (downstream of 3' UTR of PSP gene) does not predict the initiation site with high credibility, which indicates that the interval has no transcription region and the risk of the insertion gene interfering with endogenous gene is small.
4. Prediction of PSPD site cis-acting elements ployA and ployT
1. Experimental method
ployA and ployT were predicted using the gold-srey GenSmart Codon Optimization on-line cis-acting element.
2. Experimental results
Since the RNA polymerase III promoter (pol III) is usually a ployT terminator, the region 600bp upstream of ployT should be avoided so as not to disrupt transcription of endogenous miRNA or siRNA, and generally the downstream region is not affected. The prediction results are shown in fig. 6. The safe site area is further reduced, and the PSPD site area is further reduced to 4020 bp-5000 bp downstream of the PSP gene 3'UTR (Chromosome 17:36,704, 009-36,704,989) and 5637-7005 bp downstream of the PSP gene 3' UTR (Chromosome 17:36,705, 626-36,706,994).
Based on the above results, the PSP gene 3' UTR downstream 4020-5000 bp (namely, chromes 17:36,704, 009-36,704,989, the nucleotide sequence is shown as SEQ ID NO: 3) and PSP gene 3' UTR downstream 5637-7005 bp (chromes 17:36,705,626-36,706,994, the nucleotide sequence is shown as SEQ ID NO: 4) are selected as PSPD safety site gene fragments, and due to poor prediction accuracy of promoters and regulatory elements, the PSP gene 3' UTR downstream 168 bp-36858 bp (namely, chromes 17:36,699, 989-36,736,679, the nucleotide sequence is shown as SEQ ID NO: 159) is incorporated into the PSPD safety site gene fragment, and in the range, some high-efficiency sgRNA targets (SEQ ID NO: 9-124 and SEQ ID NO: 129-139) are found, part of which can be used for inserting a potential sgRNA site in FIG. 7. Further, flanking the PSPD region, some high-efficiency sgRNA targets (SEQ ID NOS: 5-8 and SEQ ID NOS: 125-128) were still found, without excluding their potential to still serve as safety sites, part of which could be used for insertion of foreign genes into potential sgRNA site segments, see FIG. 7. All the above targets and sequences for insertion are shown in Table 1.
Table 1:
example 2 efficient targeting of parotid secretory protein PSPD sgRNA acquisition
1. Experimental method
1. CRISPR/Cas9 expression system construction
1) Pig PSPD gene region target site selection
Genomic sequences of porcine parotid secretion protein PSP were downloaded from database GenBank, and sgRNAs with higher scores were used as candidate sgRNA sequences for 4020 to 5000bp downstream of the PSP gene 3'UTR (i.e., chromoname 17:36,704,009-36,704,989) and 5637 to 7005bp downstream of the PSP gene 3' UTR (chromoname 17:36,705,626-36,706,994), using the sequence information of candidate sgRNAs in the wire mesh station http:// crispr. Mit. Edu/pick-up score as candidate sgRNA sequences, see Table 2 below.
TABLE 2 sgRNA sequence information for porcine PSPD loci
2) sgRNA oligo annealing
The designed subsequent sgrnas and corresponding complementary sequences were added with the cohesive ends complementary to the Bbs I linearized PX330 plasmid, respectively, to synthesize the corresponding oligos, the oligo sequences are shown in table 3.
TABLE 3 Oligo sequence of pig PSPD site sgRNA
Preparing an oligo annealing system: 4.5. Mu.l of each forward and reverse primer was added with 1. Mu.l of 10 XT 4 Ligation buffer.
The annealing procedure is as follows: 5min at 95℃and 1min at 10 ℃; 5min at 95℃and 1min at 10 ℃; the double-stranded gRNA obtained at 95 ℃ for 5min and 10 ℃ for 3min can be stored in a refrigerator at-20 ℃ for standby.
3) Construction of CRISPR/Cas9 expression vectors
The PX330 carriers were linearized according to the cleavage reaction system of table 4 below. The digested product was separated by electrophoresis on a 1% agarose gel at 37℃for 1 hour, and the band at 8462bp was excised, and the kit E.Z.N.A was recovered on a DNA agarose gel. R Recovery of Tissue DNA Kit linearized Cas9 expression scaffold was stored in-20 ℃ refrigerator for use.
Table 4 PX330 carrier backbone cleavage reaction system
A ligation reaction system (Table 5) was prepared with reference to the T4 DNA library instruction, and ligated for 30min on a 16℃hot plate. Mu.l of the ligation product was added to the Trans T1-competent cells and gently mixed. Then ice-bath for 30min, heat-shock for 30s at 42℃and addition of 500. Mu.l of LB medium without antibiotics, resuscitation for 1h on a constant temperature shaker at 37℃and 220 rpm. 200. Mu.L of the bacterial liquid is smeared on a LA solid culture dish containing ampicillin, and the culture is carried out in a constant temperature incubator at 37 ℃ overnight (12-16 h).
TABLE 5 T4 DNA library reaction System
After the colony grows up, the monoclonal bacteria are selected and inoculated into 500 mu L of corresponding resistant LB culture medium, and shake cultivation is carried out for 2 hours. The upstream primer hU6-F was used: GAGGGCCTATTTCCCATGATT and downstream corresponding sgRNA reverse primer are subjected to bacterial liquid PCR to identify positive colonies containing the sgRNA sequence, 50 μl of the selected positive colonies are taken and sent to be tested and the hU6-F is selected as an upstream primer for sequencing verification, and the colonies with correct sequencing are subjected to amplification culture, and the endotoxin removal plasmid extraction kit E.Z.N.A is utilized. TM And (3) extracting plasmids by Endo-Free Plasmid Mini Kit II, respectively preparing PSPD-sgRNA1-Cas9 to PSPD-sgRNA11-Cas9, and storing in a refrigerator at-20 ℃ for later use.
2. Efficient sgRNA screening
1) Resuscitating and culturing of Duroc boar embryo fibroblasts
The frozen Duroc boar embryo fibroblast cells are taken out from a liquid nitrogen tank and quickly thawed in a water bath at 37 ℃. The thawed cell suspension was added to 9mL of preheated DMEM complete medium containing 15% FBS, shaken well, inoculated into 10cm cell culture plates, and placed at 38.5℃in 5% CO 2 And (5) culturing the cells in a cell culture box. After 6 hours, when the cells are attached, the upper layer culture solution is discarded, new 10ml of DMEM complete medium containing 15% FBS is added again, and the cells are placed in an incubator for continuous culture. When the cell confluence reaches 80-90%, the DMEM culture medium is removed, 2mL of DPBS is used for cleaning for 2 times, 1mL of 0.05% trypsin digestion solution is added for digesting the cells, the cells can be placed in an incubator for digestion for 1-2 min, observation is carried out under a microscope, after the cells retract and become bright, 2mL of complete culture medium is added for stopping digestion, the cells are gently blown down by a pipette gun and transferred into a 15mL centrifuge tube, the centrifuge is centrifuged for 5min at 90G, the upper culture solution is discarded, 4mL of complete culture medium is added for resuspension of the cells, 1mL of each cell is taken out and inoculated into a new 10cm culture dish, 9mL of complete culture medium is added for continuous culture in the cell incubator, and electroporation transfection is carried out until the cell round reaches 80%.
(2) Cell electrotransfection
Cell confluenceWhen the degree reaches 80% -90%, 0.05% Trysin-EDTA is used for digestion. Cells terminating digestion were counted and aspirated to contain 1X 10 cells 6 The individual cell suspensions were centrifuged at 90g for 5min in a new centrifuge tube, the upper culture medium was discarded, and 1mLOpti-MEM was added TM I Reduced Serum Medium cells were resuspended, centrifuged at 90g for 5min, the upper culture medium was discarded, and electrotransfer solutions were prepared with reference to Amaxa basic nucleofector kit for primary mammalian fibroblasts, 82. Mu.l of Basic Nucleofector per reaction TM Solutions and 18. Mu.L of support were mixed and transferred into an electrotransfer cup for electrotransfer, using an electrotransfer apparatus Nuclelector 2b, with transfection procedure A-033, followed by addition of 12. Mu.g of PSPD-sgRNA-Cas9 plasmid (PSPD-sgRNA 1-Cas9 to PSPD-sgRNA11-Cas 9). Immediately after electrotransfer, all cells in the electrotransfer cup were transferred to 6 well plates (15% FBS complete media 2 mL/well), 37℃at 5% CO 2 Culturing in an incubator. After 48h, the cells were digested and the DNA extraction kit E.Z.N.A was used. RT Tissue DNA kit extracts the genomic DNA of the cells and stores the genomic DNA in a refrigerator at-20 ℃ for later use.
(3) Amplification of PSPD sgRNA target site DNA fragments
The corresponding primers were designed according to the corresponding sgRNA target sites as shown in table 6 below.
TABLE 6 PSPD-sgRNA target site detection primer sequences
The reaction system of Table 7 was prepared according to the instructions of 2X Rapid Taq Master Mix PCR enzyme, and the PCR amplification procedure was: 3min at 95 ℃; 15s at 95 ℃, 15s at 60 ℃, 30s at 72 ℃ and 36 cycles; 72 ℃ for 5min;4 ℃. Electrophoresis of PCR product with 1.5% agarose gel, cutting off target band on blue light gel cutting instrument, and recovering kit E.Z.N.A TM Gel Extraction Kit, the target DNA band is recovered and stored at-20℃for further use.
Table 7 2X Rapid Taq Master Mix PCR enzyme reaction System
(4) Cleavage efficiency of PSPD-sgRNA-Cas9 by T7EI cleavage
The DNA recovered above was subjected to the cleavage reaction system and the procedure shown in Table 8 according to the instructions of T7 endonuclease I.
Table 8T 7E1 cleavage reaction System and reaction procedure
The cleavage products were electrophoresed on a 1.5% agarose gel and the efficiency of 11 sgrnas mediated cleavage was estimated by band grey scale using the gel imaging analysis software BioCaptMw.
2. Experimental results
The results are shown in FIG. 8, which shows that targeting efficiencies of sgRNA2 and sgRNA11 were as high as 62.8% and 63.1%, respectively.
EXAMPLE 3 construction of homologous targeting vector KI-PSPD-npsp-BEXA for PSPD safety site
The sgRNA11 locus is selected for further verification, and an upstream RA 350bp homology arm (the nucleotide sequence is shown as SEQ ID NO: 1) and a downstream LA3511bp homology arm (the nucleotide sequence is shown as SEQ ID NO: 2) are cloned to construct a large fragment transgenic homologous targeting donor plasmid KI-PSPD-npsp-BEXA. The KI-PSPD-npsp-BEXA vector construction scheme is shown in FIG. 9. The preparation method comprises the steps of amplifying three fragments Inf-LA, inf-RA and Inf-neoGFP carrying a 15bp recombinant linker, then cutting the Inf-LA after double-enzyme cutting the vector by NotI and SmaI on the basis of a CEP112-npsp-BEXA (which is a supercoiled donor plasmid CEP112-LA340RA3219 of China patent CN 201711477805.5) to obtain a PSPD-LA-npsp-BEXA vector, cutting the PSPD-LA-npsp-BEXA vector by SpeI, connecting the Inf-neoGFP to obtain a PSPD-LA-neoGFP-npsp-BEXA vector, finally cutting the PSPD-LA-neoGFP-npsp-BEXA vector by XhoI, and connecting the Inf-RA to obtain the KI-PSPD-npsp-BEXA.
1. Amplification of the left and right arms of the integration site
1. Experimental method
Left and right arm PCR amplification primers were designed based on the PSPD gene sequence of the pig on NCBI, and the Left (LA) Right (RA) arm was amplified using 2X Rapid Taq Master Mix enzyme using Duroc pig embryo fibroblast genomic DNA as template. The amplification primers are shown in Table 9 below.
Table 9:
the PCR amplification procedure was: 3min at 95 ℃;95℃15s,60℃15s,72℃15s/kb,36 cycles; 72 ℃ for 5min; 4 ℃. And (3) gel electrophoresis is carried out on the PCR product, then gel cutting is carried out, the gel is recovered, and the PCR product is stored in a refrigerator at the temperature of minus 20 ℃ for standby.
2. Experimental results
The result of gel electrophoresis of the PCR product is shown in FIG. 10, and shows that the target band PSPD-LA is 350bp, the PSPD-RA is 3511bp, and the size of the target band meets the expectations.
2. Amplification of the left and right homology arms of the integration site
1. Experimental method
In order to ligate LA and RA to the CEP112-npsp-BEXA vector (which is the supercoiled donor plasmid CEP112-LA340RA3219 of Chinese patent CN 201711477805.5) using a seamless cloning enzyme, primers were designed according to the cleavage sites at the corresponding positions on the vector. The amplification primers are shown in Table 10 below.
Table 10:
note that: thickening the recombinant sequences
LA and RA obtained by the above one-step amplification are used as templates to amplify Inf-LA (left homology arm) and Inf-RA (right homology arm) with 15bp homology joints respectively by using high-fidelity enzyme Prime STAR.
The LA and RA amplified in the previous step are used as templates and are amplified by using high-fidelity enzyme Prime STAR, and Inf-LA-F, inf-LA-R and Inf-RA-F, inf-RA-R carrying homology arms are respectively used for amplifying Inf-LA (left homology arm) and Inf-RA (right homology arm) carrying homology arms.
The PCR amplification procedure was: 98 ℃ for 5min;98℃10s,55℃10s,72℃5s/kb,36 cycles; 72 ℃ for 5min; 4 ℃. The amplified product is purified and recovered by gel electrophoresis, and is preserved in a refrigerator at the temperature of minus 20 ℃ for standby. The amplification primers are shown in Table 9.
2. Experimental results
The result of gel electrophoresis of the PCR product is shown in FIG. 11, and shows that the target band inf-PSPD-LA is 380bp, inf-PSPD-RA is 3541bp, and the size of the electrophoresis band meets the expectations.
3. Amplification of the screening marker (neoGFP)
1. Experimental method
PCR amplification primers for the selection marker (neoGFP) were designed based on the sequence information of the plasmid cep112-nPSP-BEXA, and the selection marker was amplified using the cep112-nPSP-BEXA vector as a template and a 2X Rapid Taq Master Mix enzyme, the primers being as shown in Table 11
TABLE 11 neoGFP amplification primers
And then, gel electrophoresis gel cutting, purifying and recycling the PCR product.
2. Experimental results
The result of agarose gel electrophoresis is shown in FIG. 12, and the result shows that the target band neoGFP is 2627bp, and the electrophoresis result meets the expectations.
4. Amplification of the screening marker homology arm (Inf-neoGFP)
1. Experimental method
The screening mark (neoGFP) recovered by gel cutting purification is used as a template, the screening mark with the homology arm is amplified by using high-fidelity enzyme Prime STAR, and the PCR product is subjected to agarose gel electrophoresis and then is subjected to gel cutting purification recovery, and is preserved in a refrigerator at the temperature of minus 20 ℃ for standby. The amplification primers are shown in Table 12.
TABLE 12 Inf-neoGFP amplification primers
2. Experimental results
The result of agarose gel electrophoresis is shown in FIG. 13, and the result shows that the target band inf-neoGFP is 2638bp, and the target band is cut, purified, recovered and stored for standby.
5. Double cleavage of cep112-npsp-BEXA vector
1. Experimental method
The cep112-npsp-BEXA vector was double digested with FastDiget NotI and FastDiget SmaI, digested for 1h in a 37℃thermostat water bath, the digestion system is shown in Table 13 below.
Table 13:
and (3) carrying out electrophoresis on the product after enzyme digestion by using 1% agarose gel, and carrying out gel cutting, purification and recovery on the target strip and storing the target strip in a refrigerator at the temperature of minus 20 ℃ for standby.
2. Experimental results
The result of electrophoresis of the agarose gel cut by the cep112-npsp-BEXA vector FastDiget NotI and FastDiget SmaI is shown in FIG. 14, and the result shows that the two sections after the enzyme cutting are 25941bp and 373bp respectively, the electrophoresis result meets the expectations, and the target band 25941bp cut gel is purified and recovered.
6. Attachment of Inf-LA to cep112-npsp-BEXA vector
1. Experimental method
According to the seamless cloning kitHD cloning kit) specification table 14 was attached to the reaction system, and after air-blow mixing, attached at 50 ℃ for 15min.
Table 14:
sucking 4 mu L of the connection product into 50 mu L of the Trans stbI3 competent cells melted on ice, gently blowing and mixing by a pipetting gun, ice-bathing for 30min, water-bathing for 45s at 42 ℃ and ice-bathing for 2min, adding 500 mu L of LB liquid medium, resuscitating for 1h by 220rpm in a shaking table at 37 ℃, sucking 200 mu L of resuscitated bacterial liquid, coating the bacterial liquid in an LA solid culture plate containing ampicillin, uniformly spreading, placing in a culture box at 37 ℃ for overnight culture, picking up monoclonal bacteria to inoculate in 500 mu L of corresponding resistant LB medium when obvious white colonies grow up, performing shaking table culture for 2h, and performing bacterial liquid PCR identification, wherein the bacterial liquid PCR identification primers are shown in the table 15 below.
Table 15 bacterial liquid PCR identification primers:
and (3) sending the positive result of the identification to the detection, carrying out amplification culture on the bacterial liquid with correct sequencing, and utilizing a plasmid extraction kit E.Z.N.A. TM Plasmid PSPD-LA-npsp-BEXA obtained by extraction of Plasmid Mini Kit I is stored in a refrigerator at-20 ℃ for standby.
2. Experimental results
The result of electrophoresis of the agarose gel identified by PSPD-LA-npsp-BEXA bacterial liquid PCR is shown in figure 15, and the result shows that the target band identified by bacterial liquid PCR is 1076bp, meets the expectation, and the bacterial liquid which meets the target band is selected for amplification culture.
7. Cleavage of the PSPD-LA-npsp-BEXA plasmid
1. Experimental method
PSPD-LA-npsp-BEXA plasmids were digested with FastDiget SpeI and digested for 1h in a 37℃thermostat water bath, the digestion system being shown in Table 16 below. And (3) carrying out electrophoresis on the product after enzyme digestion by using 1% agarose gel, and carrying out gel cutting, purification and recovery on the target strip and storing the target strip in a refrigerator at the temperature of minus 20 ℃ for standby.
Table 16 cleavage reaction System:
2. experimental results
The agarose gel of the PSPD LA-npsp-BEXA plasmid digested product is subjected to electrophoresis as shown in FIG. 16, and the result shows that two sections after digestion are 25605bp and 690bp respectively, and the target band 25605bp is purified and recovered for later use.
7. Inf-neoGFP-linked PSPD-LA-npsp-BEXA plasmid
1. Experimental method
According to the seamless cloning kitHD cloning kit) instruction manual the ligation reaction system of table 17 was prepared and, after blowing and mixing, was ligated for 15min at 50 ℃.
Table 17 seamless cloning reaction system:
sucking 4 mu L of the connection product into 50 mu L of the Trans stbI3 competent cells melted on ice, gently blowing and mixing by a pipetting gun, ice-bathing for 30min, water-bathing for 45s at 42 ℃ and ice-bathing for 2min, adding 500 mu L of LB liquid medium, resuscitating for 1h by 220rpm in a shaking table at 37 ℃, sucking 200 mu L of resuscitated bacterial liquid, coating the bacterial liquid in an LA solid culture plate containing ampicillin, uniformly spreading, placing in a culture box at 37 ℃ for overnight culture, picking up monoclonal bacteria to inoculate in 500 mu L of corresponding resistant LB medium when obvious white colonies grow up, performing shaking table culture for 2h, and performing bacterial liquid PCR identification, wherein the bacterial liquid PCR identification primers are shown in the table 18 below.
Table 18 bacterial liquid PCR identification primers:
to be authenticatedAnd (3) sending positive results to a detection device, and performing amplification culture on bacterial liquid with correct sequencing by using a plasmid extraction kit E.Z.N.A. TM Plasmid PSPD-LA neoGFP-npsp-BEXA prepared by extraction of Plasmid Mini Kit I. Storing in a refrigerator at-20deg.C for use. .
2. Experimental results
The agarose gel identified by the bacterial liquid PCR is subjected to electrophoresis as shown in FIG. 17, and the result shows that the target band identified by the bacterial liquid PCR is 1564bp, and the electrophoresis result meets the expectations.
8. Cleavage of the PSPD-LA-neoGFP-npsp-BEXA plasmid
1. Experimental method
PSPD-LA neoGFP-npsp-BEXA plasmid was digested with FastDiget XhoI, digested in a 37℃thermostat water bath for 1h, and digested system is shown in Table 19 below.
Table 19 cleavage reaction System
And (3) carrying out electrophoresis on the product after enzyme digestion by using 1% agarose gel, and carrying out gel cutting, purification and recovery on the target strip and storing the target strip in a refrigerator at the temperature of minus 20 ℃ for standby.
2. Experimental results
The agarose gel of the enzyme-digested product is subjected to electrophoresis as shown in FIG. 18, and the result shows that two sections after enzyme digestion are 23152bp and 5103bp respectively, the electrophoresis result meets the expectations, and the target band 23152bp is purified and recovered by gel-digested.
9. Ligation of Inf-RA into PSPD-LA-neoGFP-npsp-BEXA plasmid
1. Experimental method
According to the seamless cloning kitHD cloning kit) instruction manual the connection reaction system of table 20 was prepared, and after blowing and mixing, was connected at 50 ℃ for 15min.
TABLE 20 seamless cloning reaction system
Sucking 4 mu L of the connection product into 50 mu L of the Trans stbI3 competent cells melted on ice, gently blowing and mixing by a pipetting gun, ice-bathing for 30min, water-bathing for 45s at 42 ℃ and ice-bathing for 2min, adding 500 mu L of LB liquid medium, resuscitating for 1h by 220rpm in a shaking table at 37 ℃, sucking 200 mu L of resuscitated bacterial liquid, coating the bacterial liquid in an LA solid culture plate containing ampicillin, uniformly spreading, placing in a culture box at 37 ℃ for overnight culture, picking up monoclonal bacteria to inoculate in 500 mu L of corresponding resistant LB medium when obvious white colonies grow up, performing shaking table culture for 2h, and performing bacterial liquid PCR identification, wherein bacterial liquid PCR identification primers are shown in a table 21 below.
Table 21:
finally, the KI-PSPD-npsp-BEXA plasmid prepared by extracting the endotoxin removal particle extraction kit E.Z.N.A.TM Endo-Free Plasmid Mini Kit II is stored in a refrigerator at the temperature of minus 20 ℃ for standby.
2. Experimental results
The agarose gel identified by the bacterial liquid PCR is subjected to electrophoresis as shown in figure 19, and the result shows that the target band identified by the bacterial liquid PCR is 984bp, the electrophoresis result meets the expectations, and the bacterial liquid which meets the target band is selected for amplification culture.
10. Restriction enzyme identification of KI-PSPD-npsp-BEXA vector
1. Experimental method
PSPD homologous targeting vector KI-PSPD-npsp-BEXA is constructed, and enzyme digestion identification is carried out on the PSPD homologous targeting vector KI-PSPD-npsp-BEXA. The cleavage reaction system was prepared according to tables 22 and 238 below, and the cleavage reaction was carried out in a water bath at 37℃for 30 minutes, and the cleavage product was separated by electrophoresis using 1% agarose gel.
Table 22 KI-PSPD-npsp-BEXA vector cleavage reaction System:
table 23 KI-PSPD-npsp-BEXA vector cleavage reaction System:
2. experimental results
The cleavage results are shown in FIG. 20, and the results show that when the KI-PSPD-npsp-BEXA vector successfully constructed is cleaved by FastDiget SgsI, fastDiget SnaBI and FastDiget SmaI, the lengths of target fragments are respectively: 13007bp, 8735bp, 4103bp and 842bp. When FastDiget Kpn2I, fastDiget BshTI, fastDiget SmaI are used for digestion, the lengths of the target fragments are respectively: 10576bp, 7644bp, 6978bp and 1499bp.
And (3) selecting bacterial liquid with correct enzyme digestion strips, delivering the bacterial liquid to test, and when the sequencing result is correct, successfully constructing the PSPD homologous targeting vector KI-PSPD-npsp-BEXA, wherein a vector diagram is shown in figure 21.
EXAMPLE 4 selection of transgenic cells with site-directed integration of BEXA
1. Experimental method
1. Example 3 Co-transfection of Duroc embryo fibroblasts with the PSPD-sgRNA11-Cas9 expression plasmid and the homologous targeting vector KI-PSPD-npsp-BEXA
Resuscitating Duroc boar embryo fibroblast 5F 1 When the cells are converged to 80% -90%, the culture medium is discarded, 2ml of DPBS is added for cleaning once, the cells are digested from the culture dish by 0.05% Trysin-EDTA, and the cells are sucked to contain 1×10 6 The individual cell suspensions were centrifuged at 90g for 5min in a new centrifuge tube, the upper culture medium was discarded, and 1mLOpti-MEM was added TM I Reduced Serum Medium cells were suspended, centrifuged at 90g for 5min, the upper culture solution was discarded, 100. Mu.l of electrotransfer solution was added, and a cotransfection reaction system was prepared according to Table 24 below.
TABLE 24 cotransfection reaction System
Using electrokinetic transducer TM 2b, transfection procedure A-033. The cells after the electrotransformation were diluted to 25ml in a gradient and then spread on 20 10CM cell culture dishes, each dish being filled with 1ml of the electrotransformed cells and 9ml of DMEM medium containing 15% FBS at 37℃with 5% CO 2 Culturing in incubator, and replacing culture medium after 3 days.
2. Screening of transgenic cells for site-directed integration of BEXA
When the cells grow into better monoclonal cell clusters, the clone clusters are found out under a bright microscope, and then the fluorescent observation is switched to, if the fluorescent light exists and the cell state is good, mark is made by a mark pen, and otherwise, the mark is discarded. When the fluorescent monoclonal cell mass is relatively close to the non-fluorescent monoclonal cell mass, the non-fluorescent cells are carefully scraped by a gun head, so that the purity of the fluorescent cells is ensured. The upper medium was discarded and 3ml DPBS was added to wash 2 times to avoid floating cells on the monoclonal cell pellet. Then the cloning ring is washed 2 times with 75% alcohol and 2 times with DPBS, after a little air-drying, the cloning ring is put on the marked monoclonal cell mass, 30 mu l of 0.05% Trysin-EDTA is added to digest the cells, a proper amount of complete culture medium is added, the cells are transferred into a 48-well plate, and the cells are cultured in a cell incubator, and the culture is continued after 24 hours. After the cells are grown up, 100 μl of 0.05% Trysin-EDTA is added to digest the cells, 400 μl of complete medium is added to stop the cell, the cells are observed under a fluorescence microscope, the cells are transferred to a 24-well plate for culture with good fluorescence purity, and the culture is continued after 24 hours. After the cells grow fully, adding 200 mu l of 0.05% Trysin-EDTA to digest the cells, adding 800 mu l of complete culture medium to stop, observing under a fluorescence microscope, transferring the cells into a 12-well plate for culture with better fluorescence purity, and changing the liquid for continuous culture after 24 hours.
After the cells grow fully, adding 200 mu l of 0.05% Trysin-EDTA to digest the cells, adding 800 mu l of complete culture medium to stop, observing under a fluorescence microscope, performing PSPD site monoclonal cell line fluorescence screening, taking out 900 mu l of cells for freezing and storing for later use.
The remaining 100. Mu.l cells were further cultured by adding 900. Mu.l complete medium, and after confluence, the cells were digested and genomic DNA was extracted using DNA extraction Kit E.Z.N.A.RTissue DNA Kit, the single clone cell line was subjected to PCR identification of the integration site, the integrity of the integrated into the genomic vector was further subjected to PCR identification, 8 pairs of primers were used to cover all the fragments, and the gene integrity was subsequently identified by sequencing, and the identification primers were as shown in Table 25 below.
Table 25 transgenic cell identification primer sequences for targeted integration of BEXA:
and (3) carrying out agarose gel electrophoresis on the PCR product, cutting and recovering after the correct strip is determined, and delivering and identifying the recovered product to ensure that the gene sequence is correct.
2. Experimental results
The results of fluorescent screening of PSPD site-directed integration of BEXA transgenic monoclonal cell lines are shown in FIG. 22, the results of LA and RA detection of PSPD site-directed integration of BEXA transgenic monoclonal cell lines are shown in FIG. 23, and the results of sequencing of PSPD site-directed integration of BEXA transgenic monoclonal cells are shown in FIG. 24.
The result shows that the 23656bp transgenic fragment is successfully integrated into the pig genome, wherein the fixed-point integration efficiency of the boar cells reaches 33.3%, which is 3.1 times of the average value of the fixed-point integration efficiency of each position tested, and the fixed-point integration efficiency of the sow cells is 2.5 times of the average value.
EXAMPLE 5 transgenic cell selection marker deletion and cloning pig construction for site-directed integration of BEXA
1. Transgenic cell screening marker deletion and clone pig construction of site-directed integration BEXA
Resuscitates the successfully identified cells of example 4 onto a 24-well plate, and after the cells are full, takes out 100 mu l of each cell with the best fluorescence purity, transfers the cells into a 12-well plate, mixes the cells uniformly, cultures the cells in an incubator, and the rest cells continue to freeze. After the cells had grown, they were digested by adding 200. Mu.l of 0.05% Trysin-EDTA and terminated by adding 800. Mu.l of complete medium. 600 μl of extracted cell genomic DNA was removed and re-identified. Transferring the rest 400 mu l to a new hole, adding 600 mu l of complete culture medium, discarding the upper culture solution after 6h, adding 200 mu l of Cre recombinase to delete the screening Marker neoEGFP between two LoxP sequences, repeating the steps one to two times if the deletion is possible, and finally obtaining the Marker-free transgenic cells.
2. Preparation of Point-integrated transgenic pig
1. Experimental method
Transgenic cloned pigs were prepared using somatic cell nuclear transfer (Somatic cell Nuclear Transfer, SCNT) techniques using the deleted-tagged cells as nuclear transfer donor cells (nuclear donor). The specific method comprises the following steps:
(1) Collection and in vitro maturation culture of porcine oocyte-granulosa cell complexes (COCs)
Pig ovaries were collected from a pig slaughter house (Tianhe meat company, guangdong province), placed in 28-37 ℃ physiological saline containing 1% double antibody (double antibody is a life technology product: penicillin-Streptomycin-Glutamine) and returned to the laboratory within 4 hours. Oocytes in follicles with diameters of 2 to 6mm were collected by washing with physiological saline at 37℃and then taking a 10mL syringe with an 18-gauge needle. Selecting cumulus cell-oocyte complex (Cumulus oocyte complexes, COCs) with uniform cytoplasm, dense cumulus and more than 3 layers under microscope, washing with M199 mature culture solution, transferring into CO 2 Incubating in a four-well plate containing 500 μLM199 culture solution at 39deg.C and 5% CO in an incubator for more than 4 hr 2 Culturing in a saturated humidity incubator for 42-44 h.
(2) Granulosa cell removal and mature egg selection on COCs after maturation culture
After oocyte maturation, COCs were transferred to a centrifuge tube containing hyaluronidase, and after pipetting, the liquid was transferred to a 30mm dish, and the oophore removed from the egg was picked up with a port pipette. After washing, oocytes of the first polar body are selected under a solid microscope for discharge.
(3) Preparation of Nuclear donor cells
After digestion with pancreatin, the pellet was washed by centrifugation, resuspended in HN working fluid (calcium-free H-NCSU-23 micromanipulation fluid) and blown down to homogeneity for use as a nuclear donor.
The HN operating fluid formulations are shown in Table 26 (all reagents were of analytical grade, available from Kanlong Biotechnology Co., ltd.).
Table 26:
(4) Enucleation and enucleation of oocytes
Selecting oocytes which are discharged from the first polar body and have good shapes, and enucleating the oocytes by a blind aspiration method by using a fixed needle with the outer diameter of 100-120 mu m and an enucleating needle with the inner diameter of 15-20 mu m: approximately 50. Mu.L of the operating drop was added to a 65mm sterile culture dish and covered with paraffin oil, and then about 30 oocytes and an appropriate amount of somatic cells were transferred thereto. The oocyte is held with a holding needle and the enucleated needle is used to dial the oocyte with the polar body at about 5 o' clock. The embryo reconstruction process is completed by inserting a denucleated needle along the 3 o' clock position, removing the polar body and the cytoplasm nearby, withdrawing the needle, spitting out the polar body and the cytoplasm, and selecting a cell of the body to be injected into the periegg space. The reconstructed embryo is put into embryo culture solution for recovery culture for 1h.
(5) Fusion and activation of oocytes and somatic cells
Transferring the reconstructed eggs in batches to fusion solution for balancing for 2min, washing 3 times with fusion/activation solution, placing 5-8 eggs in each batch into fusion tank filled with fusion solution, stirring the reconstructed eggs with solid glass needle to make cell membrane contact surface of donor-acceptor eggs parallel to electrode, applying 120v/mm,100 μs,2DC direct current pulse to induce fusion while activating reconstructed embryo, washing 3 times with embryo culture solution, immediately transferring into mineral oil covered embryo culture solution, placing at 39deg.C, 5% CO 2 The fusion was determined under a stereomicroscope after 4 hours in an incubator with saturated humidity. Washing the fused reconstructed embryo with embryo culture solution for 5 times, and transferring into pre-preparationThe embryo culture solution is placed at 39deg.C, saturated humidity, and low oxygen (5% O) 2 +5％CO 2 +90％N 2 ) Is cultured under the condition of (2).
(6) Transgenic pig produced by surgical transplanting clone embryo
The recipient sow is a premium sow from the stock of Hua Nongwen, guangdong province. In this example, the oviduct transplantation method was used, and embryo development was carried out at 2-cell or 4-cell stage. The sow was fasted on the day of surgery, and the sow was fixed and given general intravenous anaesthesia before surgery. The surgical site is selected at the middle part of the penultimate nipple, the surgical site and the periphery are firstly cleaned by clean water, and then are firstly disinfected in a large range by iodine wine after being wiped dry, and then are deiodinated by 75% alcohol. Covering the surgical drape while exposing the surgical site, incising the skin and subcutaneous muscle along the midline of the abdomen, then separating subcutaneous fat and peritoneum, and slowly pulling the hand into the abdominal cavity to pull out the uterus and fallopian tube, and checking the ovulation condition. Embryo suction tubes containing embryos are inserted from the tubal ostium and the embryos are carefully blown in. The uterus and fallopian tubes are then restored into the abdominal cavity. Conventional surgical suturing followed by antibiotic anti-inflammatory injections for 4 consecutive days after surgery. Thus obtaining the transgenic pig with the exogenous DNA integrated at fixed points.
2. Experimental results
The monoclonal cells after deleting the markers are used as nuclear donor cells to prepare somatic cell cloned embryos, and then the somatic cell cloned embryos are transplanted into uterus oviduct of 5-generation pregnant sows, wherein four sows are pregnant, the pregnancy rate reaches 80%, 20 live piglets are born, and the transgenic piglets reach 4 live piglets/litter (figure 25), and 8 live piglets are born by one sow.
The transgenic pig has normal appearance and development, and the site has high animal safety by inserting exogenous gene.
EXAMPLE 6 identification of PSPD site-directed integration BEXA transgenic pigs
1. PCR identification of PSPD site-directed integration BEXA transgenic pigs
1. Experimental method
The transgenic clone produced in example 5 was used to extract DNA from the porcine ear skin tissue, and the promoter and target gene were amplified by PCR. The primers are shown in Table 27.
The PCR amplification procedure was: 98 ℃ for 5min;98 ℃ for 10s,60 ℃ for 10s,72 ℃ for 30s,32 cycles; 72 ℃ for 5min;4 ℃.
Table 27 PCR detection primers for site-directed integration transgenic BEXA cloned pigs
2. Experimental results
The results are shown in fig. 26, and the results show that all piglets are correctly integrated with the complete fragment of the target gene, and the nucleotide sequence of the complete fragment is shown as SEQ ID NO: shown at 158.
2. Transgenic pig mRNA level detection of PSPD site-directed integration BEXA
1. Experimental method
Transgenic cloned pigs produced in example 5 were selected to detect the expression level of the target gene BEXA mRNA.
Transgenic piglets of 13 days old and 18 days old were selected, and after euthanasia, various tissue samples were taken, such as heart, liver, spleen, lung, kidney, stomach, small intestine, large intestine, anterior leg muscle, posterior leg muscle, eye muscle, skin, subcutaneous fat, brain, parotid gland, submandibular gland, sublingual gland. And respectively extracting the RNA of the labeled Tissue sample by using a Tissue RNA extraction kit Tissue RNAkit, wherein the RNA degradation is avoided in the extraction process.
Reverse transcription kit using one-step methodII One Step RT-PCR Kit (Dye Plus) reverse transcribes the extracted RNA into cDNA. RT-qPCR experiments were then performed using the reverse transcribed cDNA as a template, and the relative expression of the integrated exogenous gene RNA levels was detected and analyzed based on the results of the experiments, and the quantitative primers are shown in Table 28.
Table 28 transgenic gram Long Zhu RT-qPCR detection primer for site-directed integration of BgEgXyAp
2. Experimental results
The results are shown in fig. 27, which shows that transgenic pigs were expressed only in three salivary glands (parotid, sublingual, submandibular) and not in other tissues.
3. Transgenic cloned pig glandular tissue non-amylopolysaccharase enzyme activity (NSPases) detection of PSPD site-directed integration BEXA
1. Experimental method
Collecting parotid gland, submaxillary gland and sublingual gland tissues for enzyme activity detection of beta-glucanase, cellulase, xylanase and phytase.
2. Experimental results
(1) Beta-glucanase and cellulase activity assay
As shown in FIG. 28, the results of the beta-glucanase activity assays showed that the parotid beta-glucanase activities of transgenic pigs 903405, 903409 and 903507 were 233.60U/g, 217.50U/g and 212.78U/g, respectively. The activity of the submaxillary gland beta-glucanase is 215.90U/g, 210.58U/g and 156.97U/g in sequence.
As shown in FIG. 29, the results of the cellulase activity assays showed that the parotid cellulase activities of transgenic pigs 903405, 903409, 903507 were 1.61U/g, 1.25U/g, 2.04U/g in this order. The submaxillary gland cellulase activity is 2.02U/g, 1.12U/g and 0.59U/g in sequence.
(2) Xylanase activity assay
The results of xylanase activity detection are shown in FIG. 30, and the parotid xylanase activities of transgenic pigs 903405, 903409 and 903507 are 36.76U/g, 30.87U/g and 39.43U/g in sequence. The activity of submaxillary gland xylanase is 41.00U/g, 14.96U/g and 2.60U/g in sequence.
(3) Phytase Activity assay
The phytase activity test shows that the parotid phytase activities of transgenic pigs 903405, 903409 and 903507 are 107.82U/g, 104.80U/g and 108.64U/g in sequence as shown in FIG. 31. The enzyme activities of submaxillary gland phytase are 108.00U/g, 87.82U/g and 51.90U/g in sequence.
In summary, 4 exogenous genes which can be efficiently expressed by tandem glucanase (bg 17A and eg 1314) -xylanase (xynB) -phytase (APPA) polycistronic gene (BEXA) carried by the primary transgenic cloned pig are utilized, and due to the fact that salivary gland specific promoters are adopted, target genes are expressed only in parotid gland, submandibular gland and sublingual gland, and are not expressed in other residual tissues, so that strict tissue specificity is presented; the target protein in the salivary gland of the transgenic pig expresses the enzyme activity as high as 40-200U/g.
Comparative example 1 comparison of cloning efficiency of different site transgenes/edited pigs
1. Experimental method
1. Construction method of H11 site targeting vector
For the safety site H11, the complementary sequences of the sgRNAs (Table 29) were designed according to the reported sequence GTTCCTGGAAGTTTAGATCA GGG (PMID: 26381350) of the sgRNAs of the H11 site, respectively, with the addition of the cohesive ends complementary to the Bbs I linearized PX330 plasmid, to synthesize the corresponding oligo.
The synthesized sgRNA oligo was subjected to an annealing procedure: 95℃for 5min,10℃for 1min,2 cycles; 95 ℃ for 5min and 10 ℃ for 3min.
The annealed product was ligated to linearized PX330 backbone reference T4 DNA library instructions. The ligation product was added to Trans5α competent cells and gently mixed. Then ice bath is carried out for 20-30 min, heat shock is carried out for 45s at 42 ℃, and LB culture medium which is balanced to room temperature is added for rejuvenation for 30min. 200 mu L of bacterial liquid is coated on a plate, after overnight culture, monoclonal bacteria are selected and inoculated in 500 mu L of corresponding resistant LB culture medium, and shaking culture is carried out for 6 hours at a constant temperature of 200rpm and 37 ℃.
Using hU6-F: GAGGGCCTATTTCCCATGATT sequencing and verifying, namely preserving 20% glycerol of a colony with correct sequencing for standby, and completing construction of the H11-sgRNA-Cas9 targeting vector.
TABLE 29 efficient targeting site sgRNA Synthesis
2. Construction of the donor vector H11-npsp-BEXA
The technical route for constructing the donor vector H11-npsp-BEXA is shown in FIG. 32.
1) Amplification of the H11 site LA and RA arms
LA and RA arm PCR amplification primers are designed according to the gene sequence of the pig H11 on NCBI, the genome DNA of the Duroc embryo fibroblast is used as a template, the 2X Rapid Taq Master Mix enzyme is used for amplifying the left homologous arm H11-LA and the right homologous arm H11-RA respectively, and gel electrophoresis is carried out on PCR products, and then gel cutting and recovery are carried out. Next, H11-sgRNA targets were added to the 5' end of H11-LA for intracellular linearization of the loop vectors and the amplification primers are shown in Table 30.
TABLE 30 left homology arm H11-LA and right homology arm H11-RA amplification primers
2) Cloning and construction of the LA homology arm H11-LA-npsp-BEXA
Using H11-LA as a template, using high-fidelity enzyme PrimeSTAR to amplify by using primers LA+sgRNA-F and LA+sgRNA-R, adding an H11sgRNA target GTTCCTGGAAGTTTAGATCA GGG into the H11-LA 5 end to obtain sgRNA-H11-LA, using sgRNA-H11-LA as a template, and amplifying by using primers Inf-H11-LA-F and Inf-H11-LA-R to obtain Inf-H11-LA (381 bp) carrying a recombinant joint for later use;
Firstly, using FastDiget NotI and FastDiget SmaI to digest the carrier CEP112-npsp-BEXA (which is the supercoiled donor plasmid CEP112-LA340RA3219 of Chinese patent CN 201711477805.5), removing the CEP112 locus LA carried by the original carrier, and cutting and purifying to obtain NotI-CEP112-npsp-BEXA-SmaI; inf-H11-LA was then obtained by adding recombinant linkers between H11-LA and NotI-cep112-npsp-BEXA-SmaI fragments using the primers Inf-H11-LA-F and Inf-H11-LA-R (Table 31) and H11-LA as templates.
TABLE 31 Inf-H11-LA amplification primers
Then using seamless cloned enzymeHD cloning kit) H11-LA was cloned into NotI-cep112-npsp-BEXA-SmaI vector to obtain H11-LA-npsp-BEXA. The connection system is shown in Table 32.
Table 32 seamless cloning reaction system
The ligation product was added to Trans5α competent cells and gently mixed. Then ice bath is carried out for 20-30 min, heat shock is carried out for 45s at 42 ℃, and LB culture medium which is balanced to room temperature is added for rejuvenation for 30min. 200 mu L of bacterial liquid is coated on a plate, after overnight culture, monoclonal bacteria are selected and inoculated in 500 mu L of corresponding resistant LB culture medium, and shaking culture is carried out for 6 hours at a constant temperature of 200rpm and 37 ℃. Primer sequencing using Table 33 verifies that the correct colony was sequenced and 20% glycerol was saved for later use.
TABLE 33 PCR identification primers for bacterial liquids.
3) RA homology arm cloning and construction of homologous targeting vector H11-npsp-BEXA
Because the 3 '-end of the H11-LA-npsp-BEXA carrying cep112-RA homology arm only carries one XhoI site, the H11-LA-npsp-BEXA carrier RA homology arm cannot be completely excised, an XhoI site is introduced into the 5' -end marker gene of the H11-LA-npsp-BEXA carrier RA homology arm, and then the XhoI site is excised.
The method comprises the following steps: the SpeI-CMV-NeoGFP-SpeI selection marker gene partial sequence on the H11-LA-npsp-BEXA vector was excised with Spel, and then the 2X Rapid Taq Master Mix enzyme was used with the Cep112-npsp-BEXA (which is the supercoiled donor plasmid CEP112-LA340RA3219 of China patent CN 201711477805.5) vector as template, with the primer Inf-neoGFP-F: TTGATTATTGACTAGCTTAGGGTTAGGCGTTTTGC; inf-neoGFP-R: ACCACACTGGACTAGCTCGAGCGACAGATCCATAAC after addition of XhoI cleavage site (CTCGAG) at its end, the complete CMV-NeoGFP-polyA-loxp was amplified and re-linked to H11-LA-npsp-BEXA vector (Spel after cleavage) using seamless ligation technique, the PCR amplification procedure was: 98 ℃ for 5min;98 ℃ for 5s,60 ℃ for 10s, 72 ℃ for 30, 35 cycles; 72 ℃ for 5min;4 ℃. And after the reaction is finished, performing gel electrophoresis separation, purification and recovery on the PCR product, and using the PCR product for subsequent connection. The intermediate vector H11-laneoGFP-npsp-BEXA-cep112-RA with partially overlapping marker genes was obtained, and the ligation system is shown in Table 34.
Table 34 seamless cloning reaction System
Next, the H11-LA-neoGFP-npsp-BEXA-cep112-RA was digested with FastDiget XhoI, the cep112-RA homology arm and the remaining selectable marker gene were removed, and XhoI-H11-LA-neoGFP-npsp-BEXA-XhoI was recovered by gel cutting and purification.
Inf-H11-RA was then obtained using Inf-H11-RA-F and Inf-H11-RA-R primers (Table 35), using H11-RA as a template, adding H11-RA and XhoI-H11-LA-neoGFP-npsp-BEXA-XhoI, and cloning it into XhoI-H11-LA-neoGFP-npsp-BEXA-XhoI to obtain the H11 site homology targeting vector H11-npsp-BEXA.
Table 35 Inf-H11-RA amplification primers
The homologous targeting vector H11-npsp-BEXA was added to the Trans5α competent cells, and gently mixed. Then ice bath is carried out for 20-30 min, heat shock is carried out for 45s at 42 ℃, and LB culture medium which is balanced to room temperature is added for rejuvenation for 30min. 200 mu L of bacterial liquid is coated on a plate, after overnight culture, monoclonal bacteria are selected and inoculated in 500 mu L of corresponding resistant LB culture medium, and shaking culture is carried out for 6 hours at a constant temperature of 200rpm and 37 ℃. Primer sequencing using table 36 was used to verify that the correct colony was sequenced and 20% glycerol was stored for later use.
Table 36 bacterial liquid PCR identification primer
The cleavage reaction system was prepared and identified in accordance with Table 37 below. When FastDiget SgsI, fastDiget SnaBI and FastDiget AfeI are used for cleavage, the expected target fragment lengths are: 17340bp, 4386bp, 4102bp and 842bp. And (3) selecting bacterial liquid with correct enzyme cutting strips, sending the bacterial liquid to a Huada gene (Guangzhou) for sequencing verification, and when the sequencing result is correct, successfully constructing an H11 homologous targeting vector H11-npsp-BEXA.
TABLE 37H 11-npsp-BEXA vector cleavage reaction System
3. Screening and identification of transgenic cells with BEXA integration at H11 site
1) Screening of transgenic cells with BEXA integration at H11 site
Resuscitates Duroc boar embryo fibroblast, when the cell is converged to 80% -90%, discarding the culture medium, adding 2ml DPBS, cleaning once, digesting the cell from the culture dish with 0.05% Trysin-EDTA, and sucking 1×10 cell 6 The individual cell suspensions were centrifuged at 90g for 5min in a new centrifuge tube, the upper culture medium was discarded, and 1mLOpti-MEM was added TM I Reduced Serum Medium the cells were washed, centrifuged at 90g for 5min, the upper culture solution was discarded, 100. Mu.l of electrotransfer solution was added, and a spot transfection reaction system was prepared according to Table 6 below. Using an electrotransfection apparatus N.mu.cleofectro.2b, the transfection procedure was A-033.
The targeting plasmid H11-sgRNA-Cas9 was co-transfected with donor plasmid H11-npsp-BEXA into Duroc embryonic fibroblasts according to the system shown in Table 38. The cells after the electrotransformation were diluted to 25ml in a gradient and then spread on 20 10CM cell culture dishes, each dish being filled with 1ml of the electrotransformed cells and 9ml of DMEM medium containing 15% FBS at 37℃with 5% CO 2 Culturing in incubator, and replacing culture medium after 7 days.
Table 38 cotransfection reaction System
2) Identification of transgenic cells with H11 site-directed integration of BEXA
Consistent with the screening method of PSPD site-directed integration BEXA transgenic cells. The positive transgenic cell clone was digested with 0.25% pancreatin, genomic DNA was extracted with DNA extraction Kit e.z.n.a.rtissue DNA Kit, the single clone cell line was subjected to PCR identification of integration sites, and the integrity of integration into the genomic vector was further subjected to PCR identification. The PCR products were sent to the Huada gene (Guangzhou) for sequencing and identification to ensure correct gene sequence, and the identification primers are shown in Table 39 below.
Table 39 transgenic cell identification primers for site-directed integration of BEXA
2. Experimental results
The results are shown in tables 40 and 41. The cloning of the transgenic pig of example 5 was up to 2.15% (knock in (KI) transgenic pig head number/total number of transferred embryos) 3.5 times the average cloning efficiency of 0.62% obtained at other sites and techniques.
Table 40 comparison of different bit position fix point integration efficiencies
Annotation: cep112 and Rosa26 site integration efficiency data were derived from this team patent CN201711477805.5 and published literature (PMID: 31818875), PSPD and H11 site integration efficiencies were obtained for example 5 and this comparative example; KI efficiency, using limiting dilution method, transgenic site-directed integration of cell clusters/selection of total number of fluorescent-labeled clusters carrying green fluorescent protein-labeled EGFP.
Table 41 comparison of different site transgene/editing pig cloning efficiency:
remarks: PDPD data was obtained for example 5, and other transgenic site data was derived from the historical data accumulated by this team, see in part published papers (PMID: 31818875 and PMID: 29784082).
And the batch 2 is the F1 generation transgenic embryo cell line propagated in the batch 1, and is directly cloned after being treated by cre enzyme and deleting NeoGFP marks. KI (knock in): site-directed integration techniques (transgene + editing); piggybac: piggybac transposon mediated transgene technology, random integration;
cloning efficiency = total number of pups/embryo total.
Example 7 construction of grain-saving Environment-friendly pig breeding herd Using PSPD loci
The construction method of the transgenic pig breeding group is suitable for breeding new varieties of transgenic animals, and is different from the method of breeding genetically edited animals, and the method needs to combine a transgenic fixed-point integration technology and a safety site, integrate effective transgenic fragments into the same genome safety site of pigs, construct more than 6 ancestor-free ancestor progenitors and construct the transgenic animal breeding group. By utilizing the technology, the pig variety construction time only needs 2 generations to obtain a new breeding population formed by homozygous transgenic animals in a certain scale, and compared with the traditional new variety or strain for decades of breeding period, the method provided by the invention can be completed only by 2 years, and has important economic value in transgenic animal breeding.
1. Experimental method
1. Duroc pig progenitor PEF cell obtaining method
Based on Duroc pig Wenshi S21 strain, 6 tertiary or 1 grade boar progenitors without common ancestors are screened to ensure the universality of blood margin. And 6 sow progenitors without common ancestors within three generations are selected from the first-class sow of the core group and used as mating objects of the boar progenitors. The 6 boars and the 6 sows are selected one by one, and no common ancestor exists between the sows to be bred and the boars within 3 generations. Taking out embryo when sow is pregnant for 25-35 days old, washing with DMEM three times, adding digestion medium (DMEM containing collagenase IV, 5% FBS and 1% penicillin with working concentration) for digestion for 2-4 hours until tissue is fluffy, adding 1ml serum (containing 1% penicillin with diabody) for plating, and adding high sugar DMEM (GlutaMAX) containing 12% (V/V) FBS and penicillin with diabody the next day TM Additive) for 2-3 days, female and male embryonic fibroblasts (PEF) of the offspring of each pair of line progenitors were obtained, followed by sexing with the primers in table 42, followed by F0 transgenic pigs, both male and female by cloning.
SRY gene fragments in the embryo DNA of different Duroc pig families were amplified separately using 2 XTaq Master Mix (Vazyme). PCR reaction procedure: 95 ℃ for 1min; 15s at 95℃and 15s at 60 ℃;72 ℃,60s;35 cycles; 72 ℃ for 2min and 4 ℃ for 1h. The reaction system was subjected to 1% agarose electrophoresis after completion of the reaction by referring to Vazyme 2×Taq Master Mix instructions. beta-actin-F and beta-actin-R are amplification primers of internal reference genes, SRY-F and SRY-R are Y chromosome specific gene SRY amplification primers, SRY amplification positive is boar, and no band is sow.
Table 42 embryo sex identification primers
2. Grain-saving environment-friendly method for transferring BEXA gene into Duroc pig group
Resuscitate the female and male embryonic fibroblasts (PEF cells) of the 6 Duroc pig families, then premix 4. Mu.g of the targeting vector PSPD-sgRNA11-Cas9 plasmid DNA prepared in example 3 with 10. Mu.g of the donor vector KI-PSPD-npsp-BEXA plasmid DNA prepared in example 3, place on ice, transfect the premix plasmid DNA into PEF cells of each Duroc pig family using the A-033 procedure in the LONZA electroporation transfection system, dilute the transfected cells to 20-50 cells/ml by limiting dilution, plate them into DMEM complete medium containing 12% (v/v) fetal bovine serum, place on CO 2 Continuously culturing in a cell culture incubator at 5% and 38.5 ℃ for 14 days, changing a complete culture medium once a week, taking an oily pen to mark the position of a cell clone, taking 8X 8mm cell clone rings by sterile forceps, carefully sleeving the cell clone rings, adding 0.25% pancreatin into the clone rings for digestion, transferring the digested cells into a 48-hole culture plate by a pipetting gun for expansion culture, and identifying the integration condition of a target gene by using primers in a table 43.
Table 43 site-directed integration PSPD site-transferred BEXA Gene cell identification primer sequences:
wherein PSPD-JDT A-F and PSPD-JDT A-R are detection primers for homology arm LA, PSPD-JDT A-F and PSPD-JDT A-R are detection primers for homology arm RA, bg17-F and eg1314-R are detection primers for target gene BEXA.
The transgenic pig cells (female and male) of each Duroc pig family were then deleted for neoGFP cell selection markers following the Cre treatment method mentioned in example 4 and then used for clone embryo construction.
Next, female and male embryos of each Duroc pig family were constructed according to the cloning embryo construction method in example 5, respectively, at a ratio of 1:1, transplanting the mixed mixture into oviducts of 4-6 pregnant sows, producing transgenic cloned boars and sows of 6 Duroc pig families, constructing an F0-generation grain-saving environment-friendly transgenic pig breeding population (heterozygote+/-, namely, each PSPD allele locus only contains 1 transgene), and carrying out transgenic pig genotype identification by using a transgenic genotype identification primer shown in a table 44.
Table 44 transgene genotype identifying primer
Wild-type swine PSPD (if no transgene is integrated, the target band is 909bp, if transgene integration occurs, the normal Taq enzyme has no amplification product) and the gene fragment of the site-directed integration transgenic BEA pig (KI-TG, transgenic pig amplification product 1278bp, negative pig has no amplification) are amplified separately using swine genomic DNA as templates and two pairs of primers in Table 42, using 2 XTaq Master Mix (Vazyme Co.).
PCR reaction procedure: 95 ℃ for 1min; 15s at 95℃and 15s at 60 ℃;72 ℃ for 60-90 s;35 cycles; 72 ℃ for 2min and 4 ℃ for 1h. The reaction system was checked by electrophoresis on a 1% agarose gel after completion of the reaction, with reference to Vazyme 2×Taq Master Mix.
If the target band is amplified by two pairs of primers PSPD-F1/F2 and PSPD-F2/nPSP-R2, the target band is a transgenic heterozygote; if the PSPD-F1/F2 positive band and the PSPD-F2/nPSP-R2 negative band are the wild type pigs; if PSPD-F1/F2 negative bands, PSPD-F2/nPSP-R2 positive bands are homozygotes.
Then mating the F0 generation transgenic cloned boar and sow of each family with the transgenic boar or sow of other families respectively, propagating the F1 generation grain-saving environment-friendly transgenic pig, see FIG. 37, according to Mendelian's genetic law, the transgenic pig homozygote (+/+) in the F1 generation cloned pig, the transgenic pig heterozygote (+/-) and the wild type (-/-), theoretical ratio 1:2:1, selecting a transgenic pig homozygote (+/+) boar and a sow, constructing a grain-saving environment-friendly pig breeding core group, and then breeding and expanding the group according to a group subculture breeding method. Because the Duroc pigs are terminal male parents in a pig hybridization breeding system, the breeding requirement of 150 ten thousand sow breeding can be met only by propagating 1 ten thousand transgenic homozygous boars, the slaughtering scale of 3000 ten thousand pork pigs of a large farm and herd group is met, and the feed cost is calculated by saving 25kg of feed per pig, 4 yuan/kg of feed cost can be saved, and 30 hundred million yuan of feed cost can be saved.
2. Experimental results
1. 6 family Duroc pig embryo fibroblast line establishment results
Selecting 6 or less than three generations of common ancestral special grade or 1-grade boar progenitors from a Winking S21-series Duroc core group, selecting 6 or less than three generations of common ancestral sow progenitors from a first-grade sow of the core group, avoiding the boars and sows with blood relationship in the three generations, performing one-to-one selection, taking a embryo establishment line 25-35 days after pregnancy, and identifying embryo gender as shown in figure 33.
2. Results of 6 Duroc family transgenic cell selection
The targeting vector PSPD-sgRNA11-Cas9 plasmid DNA was transfected with donor vector PSPD-npsp-BEXA plasmid DNA to isolate PEF cells as described above, resulting in transgenic cell lines of 6 families (D1 to D6 in FIG. 34).
3. Grain-saving environment-friendly transgenic pig breeding group construction
The transgenic cell line is constructed into cloned embryos to obtain transgenic cloned pigs of 6 families, then F1 generation transgenic pigs are propagated according to the mating method of fig. 35, and 80F 1 offspring are propagated in each family, wherein 20 transgenic pigs are homozygous and 40 transgenic heterozygotes are obtained (fig. 36-37). Selecting healthy transgenic homozygote with good development to construct a core group, and breeding according to a group subculture breeding method to enlarge the group scale so as to finish the construction of grain-saving environment-friendly pig breeding groups.
4. Transgenic pig genotype identification results
The transgenic pig genotype identification is carried out by using the transgenic genotype identification primer in the table 42, the wild type WT pig has no exogenous gene and is inserted at fixed points, the amplification result only has a single electrophoresis band 909bp, the transgenic heterozygote has two genotypes of WT and KI-TG, so that the banding of 909bp and 1278bp appears after the amplification, the transgenic homozygote only has the KI-TG genotype, the amplification only has a single 1278bp, and the F1 generation homozygote has exactly 6 heads/24 heads and has the ratio of 1/4 according to the amplification result shown in FIG. 36, thereby conforming to the Mendelian genetic rule.
Finally, it should be noted that, since each safety site currently found is easily generalized to other animals and cells, for example: h11 (PMID: 21092859, PMID:21464299, PMID:24304893, PMID:26381350, PMID: 30591434, PMID:34862376 and PMID: 31344144) and Rosa26 site (PMID: 18037879, DOI: 10.1007/978-1-60761-811-9_10, PMID:22564063, PMID:29991797, PMID:28596588 and PMID: 27063570). The foregoing embodiments are merely illustrative of the present invention and are not intended to limit the scope of the present invention, and other variations or modifications in light of the above teachings and concepts will be apparent to those skilled in the art and are not intended to be exhaustive or all embodiments of the invention. In addition to pigs, other animals and cells thereof, such as humans, monkeys, cows, sheep, rabbits, mice, chickens, ducks, etc., may be contemplated as falling within the spirit and principles of the invention, as any modifications, equivalent substitutions and improvements made thereto, are intended to be within the scope of the claims.
Sequence listing
<110> Wenshi food group Co., ltd
Guangdong middle core seed industry Co.Ltd
<120> a safe site for pig specific site-directed integration gene editing
<160> 159
<170> SIPOSequenceListing 1.0
<210> 1
<211> 350
<212> DNA
<213> Sus scrofa domestica
<400> 1
ctgcacattt gagtaatcag gctcctcttc cagactttac aggttcgctt tgacagagac 60
agttcttcac cagttggctc agtttagttt tatgggtgtg cctgctggtt atggccttgg 120
gtatatggga gctactatta tggccatttc tggagtaagg caactgtctg agctctgagg 180
atggggatca ggggagcatg cctttgtctg aaaacagttg gatagggctg ctggattggt 240
ttcctaccca ggtgaggctg taggatggac tttgtggttg cctgaattct ctggtcagtc 300
ttactagatg gttggggctg ggtactgtat tcagtacaag acgccttagg 350
<210> 2
<211> 3511
<212> DNA
<213> Sus scrofa domestica
<400> 2
gacaggaccc aggacctgta cagcttatca tttggaagcc tgaatgtgca ctgaattccc 60
tacttaggta aggccatcat tcccattctg caaacaagaa taagccatgg gctatgctca 120
cttcaagtgc cactacactt agggctgttg aaagggctct ggttagtctc tctagctgaa 180
caagatgaag gctgtattta acaatgagcg aagctattaa ttaggttctc tgcccaggca 240
cagtggtaga agcagctcag aagctggtaa agagttttgt ttcttgtctt aattcaagcc 300
aacccacacc ccaggtttcc tgggtgaaca gaaaactagc tttgctctgc aaactgtggg 360
ctctgcttgc ccttctccag gctccagctg ggcagcacag cctccaggag ttgtcactag 420
cccttctggt cagatgggat cagaagacaa tcttctcagt gggttgtgct gggactctac 480
tcctgacctg ggcctgggta aactgaactc cagaaccagc aaaactactt gtttgaggat 540
ctgaatcagg gagatttgct ccctgccaaa ttccctggtc agagagtgcg cccccaactt 600
ggttctgcag aagccactct ttgtgatgac tacttagcca ctgcaggtgt gaacttggtt 660
ggccaacatc catgtgttgg tttttgcaag cccctcccca gttctctgac acaatcagct 720
tcccaatggt ggagcccttc cttgcagatt cctaggcagt ccacatccca agaatagggg 780
ggagggggtc ccacaatgcc aacggggtcc tggttgttcc ttagggttct cttttcccat 840
tggaggaagt ggaggctcag gagagacctc tctgcacggt gttgcactgg cctgggggcg 900
ggggcagggc aacatggaga ttcttctttc ccctcttaat taactcagtc tgtcctggtc 960
tctgtggtgt ggaggatgct tcagcctcac ccttgcgtcc taggattctc tcagtggtgt 1020
tttgttctgc agtagttatt agctgttctt cttgtgaagg gagcaaatca ggaatgacct 1080
atatctccat ctaggtaaca tcactctctt actttttttc tttttctttc tttctatttt 1140
tttttttttt ttttttttta gggctgcacc tacagcatat ggaagtttcc aggctagggg 1200
tagaattaga gctcagctgc tggcctacac cacagacaca gcaacaccag atccaagcca 1260
catcttcgac ctacactgta gctcatagca atgctggatc cttaaaccac tgagcaaggc 1320
caggggctga acctgcatcc tcatggatac tagtcatgtt cttaacccgc tgagacacta 1380
tgggaactcc ctctcacttt tcttattcac ctctgccccc tgagcctgtg agctcctcca 1440
gggtaggcac ctccttcttc tcagcagccc cagtgcccag caaaggactc tgtgcaatac 1500
aagcactaat cagtgttgaa tgaaaggata gatggacgga tggatggatg gatggatgaa 1560
tgaatctctt cttgactgta cacagacact atcgctagga cagctaagtg ggtagtgaac 1620
aggaaaattc tagcaaccca cgactgtggg gtattggaga cccatcatta tttctggtga 1680
ccagggccag aggcctggcc cttgaggtca taagtaatgg actatggacc aggatagtcc 1740
aagggggtcc tggttgttcc ttagggttct cacagctctt agaggaatga agccagaaca 1800
tggagtgggt agggccaagg ccactcagct ccactggtga ctgatgtgac caatgaaggg 1860
ggaaaagcac aggtgaggaa gtgagggagt gttctgctac tgtgaccctg gcactcccag 1920
gaccagctct ggctctttgc ccacccagtg cagacctcaa agaggggtga gggcttcctg 1980
gagacagtga caaagaggag aatctgatga gagggtaagg gactatctgc cataagagaa 2040
catataggga gaaataagac aaaacctgag tcatccagaa actcaagtgc caaattccag 2100
tgcaagctct tttgaaggct taatccagtt atgtaaatgc cgaatctttc caatatttgc 2160
ataattccct caggataata tctcaggatc agaattaata actctatgtt tcagaacatt 2220
ttatggcccc aggaacacat cacaatgtta tttaccaaac cagatctttc tatttacact 2280
aaggtgagag agtatataca tttccccatg tatttgccag aaaatggaca cttttatttt 2340
aaatacttgc tcttaaaatg agggtagaat aaacttcatc acaaggaaga actaaggccc 2400
caaattagga aagtcaattg ttacatcaac tcaatagact attttctgaa tgttaaaaaa 2460
aaaaaaaaaa agcagtttcc cattgtggct cagcacatta agaacctgac attatgttca 2520
taaggagggg cttcaatccc tggccttgct cagtgggtca aggaacaggc attgccgcag 2580
ctatggtata ggtggaagat aacggcttgg attcagtgtg gctgtggctg tggtgtaggt 2640
tggcagctgc agctctgatt tgatccctag cctgggaacg tccatatgct gcaggaatgg 2700
ccttcaaaaa aagaagaaga aaaaaaaccc caaaacattg gaaaatactt gcgttttaca 2760
gctgagtgga aatatacaga tcttagaact gtgcttagct ggaatcatga cgacccccct 2820
gcccaggcag ggggtgagga agaggcaaca gagtgctgag aaaaagactc agaggagggt 2880
ctcgtgactc agcctggaga tgggaggagg agtcaaagca gacttcccag aaaaggtgct 2940
gatgcagtta aaactgaaag atgggagaga tgggagggga gggcatttac tgcaagcaaa 3000
agtgaccaaa agaatgaaga ctggggagtt cccgtcgtgg cgcagtggtt aacgaatccg 3060
actaggaacc atgaggttgc gggttcggtc cctgcccttg ctcagtgggt taatgatctg 3120
gcgttgccgt gagctgtggt gtaggttgca gacgcggctc ggatcccacg ttgctgtggc 3180
tctggcgtag gccagtagct acagctccga ttcgacccct agcctgggaa cctccatatg 3240
ccgcgggagc ggcccaagaa atagcaaaaa gacaaaaaaa aaaaaaaaaa aaaaaacaat 3300
gaaggctagg aactgagaat caacatggta gcacctggca cgtgtcgctg gcaatcaaac 3360
tccacagggt caagtggagg gcgaggcctc gaatgccaca ccaggctggt ttcaacgtgg 3420
tcctcttagc caggagttaa atttatttgt tcttatctca ctctcaaaaa ctgaacacct 3480
cgaagagttt tgtctgtatg gaccccatcc g 3511
<210> 3
<211> 979
<212> DNA
<213> Sus scrofa domestica
<400> 3
gttcttcacc agttggctca gtttagtttt atgggtgtgc ctgctggtta tggccttggg 60
tatatgggag ctactattat ggccatttct ggagtaaggc aactgtctga gctctgagga 120
tggggatcag gggagcatgc ctttgtctga aaacagttgg atagggctgc tggattggtt 180
tcctacccag gtgaggctgt aggatggact ttgtggttgc ctgaattctc tggtcagtct 240
tactagatgg ttggggctgg gtactgtatt cagtacaaga cgccttagga caggacccag 300
gacctgtaca gcttatcatt tggaagcctg aatgtgcact gaattcccta cttaggtaag 360
gccatcattc ccattctgca aacaagaata agccatgggc tatgctcact tcaagtgcca 420
ctacacttag ggctgttgaa agggctctgg ttagtctctc tagctgaaca agatgaaggc 480
tgtatttaac aatgagcgaa gctattaatt aggttctctg cccaggcaca gtggtagaag 540
cagctcagaa gctggtaaag agttttgttt cttgtcttaa ttcaagccaa cccacacccc 600
aggtttcctg ggtgaacaga aaactagctt tgctctgcaa actgtgggct ctgcttgccc 660
ttctccaggc tccagctggg cagcacagcc tccaggagtt gtcactagcc cttctggtca 720
gatgggatca gaagacaatc ttctcagtgg gttgtgctgg gactctactc ctgacctggg 780
cctgggtaaa ctgaactcca gaaccagcaa aactacttgt ttgaggatct gaatcaggga 840
gatttgctcc ctgccaaatt ccctggtcag agagtgcgcc cccaacttgg ttctgcagaa 900
gccactcttt gtgatgacta cttagccact gcaggtgtga acttggttgg ccaacatcca 960
tgtgttggtt tttgcaagc 979
<210> 4
<211> 1368
<212> DNA
<213> Sus scrofa domestica
<400> 4
tttttttttt agggctgcac ctacagcata tggaagtttc caggctaggg gtagaattag 60
agctcagctg ctggcctaca ccacagacac agcaacacca gatccaagcc acatcttcga 120
cctacactgt agctcatagc aatgctggat ccttaaacca ctgagcaagg ccaggggctg 180
aacctgcatc ctcatggata ctagtcatgt tcttaacccg ctgagacact atgggaactc 240
cctctcactt ttcttattca cctctgcccc ctgagcctgt gagctcctcc agggtaggca 300
cctccttctt ctcagcagcc ccagtgccca gcaaaggact ctgtgcaata caagcactaa 360
tcagtgttga atgaaaggat agatggacgg atggatggat ggatggatga atgaatctct 420
tcttgactgt acacagacac tatcgctagg acagctaagt gggtagtgaa caggaaaatt 480
ctagcaaccc acgactgtgg ggtattggag acccatcatt atttctggtg accagggcca 540
gaggcctggc ccttgaggtc ataagtaatg gactatggac caggatagtc caagggggtc 600
ctggttgttc cttagggttc tcacagctct tagaggaatg aagccagaac atggagtggg 660
tagggccaag gccactcagc tccactggtg actgatgtga ccaatgaagg gggaaaagca 720
caggtgagga agtgagggag tgttctgcta ctgtgaccct ggcactccca ggaccagctc 780
tggctctttg cccacccagt gcagacctca aagaggggtg agggcttcct ggagacagtg 840
acaaagagga gaatctgatg agagggtaag ggactatctg ccataagaga acatataggg 900
agaaataaga caaaacctga gtcatccaga aactcaagtg ccaaattcca gtgcaagctc 960
ttttgaaggc ttaatccagt tatgtaaatg ccgaatcttt ccaatatttg cataattccc 1020
tcaggataat atctcaggat cagaattaat aactctatgt ttcagaacat tttatggccc 1080
caggaacaca tcacaatgtt atttaccaaa ccagatcttt ctatttacac taaggtgaga 1140
gagtatatac atttccccat gtatttgcca gaaaatggac acttttattt taaatacttg 1200
ctcttaaaat gagggtagaa taaacttcat cacaaggaag aactaaggcc ccaaattagg 1260
aaagtcaatt gttacatcaa ctcaatagac tattttctga atgttaaaaa aaaaaaaaaa 1320
aagcagtttc ccattgtggc tcagcacatt aagaacctga cattatgt 1368
<210> 5
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 5
taagccttcg ggagtgaatc 20
<210> 6
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 6
aggccactgt cccgactgta 20
<210> 7
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 7
cattccagat tcactcccga 20
<210> 8
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 8
gtggagggta acgagtgtag 20
<210> 9
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 9
ggaacccaca agattatcgg 20
<210> 10
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 10
ctatcgctag gacagctaag 20
<210> 11
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 11
tgtacacaga cactatcgct 20
<210> 12
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 12
attctagcaa cccacgactg 20
<210> 13
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 13
ttctagcaac ccacgactgt 20
<210> 14
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 14
tattcagtac aagacgcctt 20
<210> 15
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 15
atgatggcct tacctaagta 20
<210> 16
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 16
ggggtcccac aatgccaacg 20
<210> 17
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 17
gacctgtaca gcttatcatt 20
<210> 18
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 18
gggggtccca caatgccaac 20
<210> 19
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 19
ctccaatacc ccacagtcgt 20
<210> 20
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 20
tctagcaacc cacgactgtg 20
<210> 21
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 21
gcccttgagg tcataagtaa 20
<210> 22
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 22
agtacaagac gccttaggac 20
<210> 23
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 23
ttcaagtgcc actacactta 20
<210> 24
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 24
cagagagtgc gcccccaact 20
<210> 25
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 25
tctccaatac cccacagtcg 20
<210> 26
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 26
cacggcaaat atcaacggat 20
<210> 27
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 27
gattgccagc gacacgtgcc 20
<210> 28
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 28
tcacggcaaa tatcaacgga 20
<210> 29
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 29
tagcacctgg cacgtgtcgc 20
<210> 30
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 30
acggcaaata tcaacggatg 20
<210> 31
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 31
aaccacctgg ctaggcgcta 20
<210> 32
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 32
tcaacgtggt cctcttagcc 20
<210> 33
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 33
gaaccacctg gctaggcgct 20
<210> 34
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 34
acaccaggct ggtttcaacg 20
<210> 35
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 35
tggtggagat ccgcacgaca 20
<210> 36
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 36
ggtggagatc cgcacgacaa 20
<210> 37
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 37
tctacccgga gcagacggaa 20
<210> 38
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 38
ggctagtgtc tagtagaatc 20
<210> 39
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 39
tcggggagct tagtcatcca 20
<210> 40
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 40
cggggagctt agtcatccaa 20
<210> 41
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 41
ttattgacac agccgtcaaa 20
<210> 42
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 42
gaaatcttac cccgaatgta 20
<210> 43
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 43
gaaggtgccc actagttgaa 20
<210> 44
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 44
tctcggcttc tcgccctgat 20
<210> 45
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 45
tacagtacgt gaatctagct 20
<210> 46
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 46
atacagtacg tgaatctagc 20
<210> 47
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 47
ttctggtgaa attgtgcggc 20
<210> 48
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 48
ccttatacct ttcaactagt 20
<210> 49
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 49
agggaggtct tatcctatca 20
<210> 50
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 50
acagtacgtg aatctagctg 20
<210> 51
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 51
ggtctaattg catcggggtt 20
<210> 52
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 52
gcagctatgt tgatcgttgg 20
<210> 53
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 53
ggagcagcta tgttgatcgt 20
<210> 54
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 54
aggtctaatt gcatcggggt 20
<210> 55
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 55
taatagagtg atgatccgag 20
<210> 56
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 56
gagccggtga gaatagctcg 20
<210> 57
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 57
aatagagtga tgatccgagc 20
<210> 58
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 58
accattaatc attacccgct 20
<210> 59
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 59
tgtttgttgg agctcgcccc 20
<210> 60
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 60
ttaaaggtct aattgcatcg 20
<210> 61
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 61
tccgagcggg taatgattaa 20
<210> 62
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 62
gtctaattgc atcggggttg 20
<210> 63
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 63
cttgcagatg atcgacttcc 20
<210> 64
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 64
acgttggatg tgttaggacc 20
<210> 65
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 65
atatcacagc agtcctcggg 20
<210> 66
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 66
ggtcattatc gaagatgagc 20
<210> 67
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 67
gatggtttcg tcggtccgca 20
<210> 68
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 68
ccgccagtaa ctcggttggt 20
<210> 69
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 69
aataccgcca gtaactcggt 20
<210> 70
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 70
taaccaacca accgagttac 20
<210> 71
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 71
atcactctaa gcgtagaagc 20
<210> 72
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 72
tgacaatacc gccagtaact 20
<210> 73
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 73
cttctacgct tagagtgatc 20
<210> 74
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 74
gagttttaaa cacgatagtc 20
<210> 75
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 75
gacctgcctt tgcgcaaagt 20
<210> 76
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 76
aagctgtaca tgggactcgc 20
<210> 77
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 77
ggaccgacga aaccatctcc 20
<210> 78
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 78
attcatctcc atttcgagcc 20
<210> 79
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 79
ggacctgcct ttgcgcaaag 20
<210> 80
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 80
atattcgatc agagctgaat 20
<210> 81
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 81
ggccgatacc tacttgcctt 20
<210> 82
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 82
gttaagaccc aggaccccgt 20
<210> 83
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 83
gccgatacct acttgccttt 20
<210> 84
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 84
agttaatcgt ggatgaccac 20
<210> 85
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 85
tggcagtttt ctaaaatcgc 20
<210> 86
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 86
accctggatt cagttaatcg 20
<210> 87
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 87
gaggacatta ccgaccccta 20
<210> 88
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 88
ggaggacatt accgacccct 20
<210> 89
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 89
aggacattac cgacccctag 20
<210> 90
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 90
actcagtccc taatccttac 20
<210> 91
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 91
atgcccagag cttcgtagat 20
<210> 92
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 92
gtgcccggtc atctaacatg 20
<210> 93
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 93
agaaccaatc tacgaagctc 20
<210> 94
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 94
catttggcaa ctatgcgact 20
<210> 95
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 95
ctaccaaccg gagcctgaat 20
<210> 96
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 96
actcccattc aggctccggt 20
<210> 97
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 97
ggtcaatggc tagtgcaagc 20
<210> 98
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 98
gaaccaatct acgaagctct 20
<210> 99
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 99
aaacctcatg ttagatgacc 20
<210> 100
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 100
gtcaatggct agtgcaagca 20
<210> 101
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 101
cagctcttaa gcgattcact 20
<210> 102
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 102
agtttagtga cccgcaagac 20
<210> 103
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 103
gctaaggttt ctcgcccagc 20
<210> 104
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 104
agctcttaag cgattcactt 20
<210> 105
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 105
gaagctcgag gcagttaatc 20
<210> 106
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 106
agtggtggct cggtatcagc 20
<210> 107
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 107
gtactgttgt ctgctatccg 20
<210> 108
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 108
gcataaagta tgccgtgaag 20
<210> 109
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 109
tgttatccag ggataggcgg 20
<210> 110
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 110
acaataacta gagaggccgt 20
<210> 111
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 111
catgtacatc ggtgatggcc 20
<210> 112
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 112
gtctccgtac tcaaagaagt 20
<210> 113
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 113
tagttcacgg tgctgattac 20
<210> 114
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 114
cctggaatgc acgattgcct 20
<210> 115
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 115
tgatcacggt ttccctagaa 20
<210> 116
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 116
ttttttttgt atgcgtcaac 20
<210> 117
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 117
gatgagtagg gcacgtatgt 20
<210> 118
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 118
gttagatagt caactctacc 20
<210> 119
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 119
ttaactgtgg gatccgcagc 20
<210> 120
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 120
ttggttctgt tcgttagtga 20
<210> 121
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 121
agatgagtag ggcacgtatg 20
<210> 122
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 122
ttaaagtatc tttagcgtgg 20
<210> 123
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 123
agagttgact atctaaccat 20
<210> 124
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 124
caacaacctg agcgtctgac 20
<210> 125
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 125
gcaatgccag tcagacgctc 20
<210> 126
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 126
aggagtgctg actagcctaa 20
<210> 127
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 127
gtgagggcaa ctcataactc 20
<210> 128
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 128
ccctactcat ctcaactatc 20
<210> 129
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 129
agctcccgca ggcattgaaa 20
<210> 130
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 130
ggggagcttg acggtaagcg 20
<210> 131
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 131
gtaagcgagg acctcgctgc 20
<210> 132
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 132
ggcatcgcac agggtttgtt 20
<210> 133
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 133
agtgtcatca caatcttgac 20
<210> 134
<211> 17
<212> DNA
<213> Sus scrofa domestica
<400> 134
actactgaca aaagtta 17
<210> 135
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 135
gaactttcgg tatgttgaat 20
<210> 136
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 136
ggaacccaca agattatcgg 20
<210> 137
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 137
aatctgcaca ggtctaccct 20
<210> 138
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 138
caaataggtt gaatctgcac 20
<210> 139
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 139
ggttatggcc ttgggtatat 20
<210> 140
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 140
atgatggcct tacctaagta 20
<210> 141
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 141
ggggtcccac aatgccaacg 20
<210> 142
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 142
cagagagtgc gcccccaact 20
<210> 143
<211> 20
<212> DNA
<213> Sus scrofa domestica
<400> 143
agtacaagac gccttaggac 20
<210> 144
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 144
tctccactgg actcttcagt tcag 24
<210> 145
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 145
tcgggtcaag ccagctgtca tttc 24
<210> 146
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 146
tacaagtaat ctagagggcc cg 22
<210> 147
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 147
tagccaggtg gttctcaaag tgtg 24
<210> 148
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 148
ctttcacagt ggtcacccag tttc 24
<210> 149
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 149
atggagttag gctgccagtc aaagg 25
<210> 150
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 150
tttttttgtg tgcgattctt gattc 25
<210> 151
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 151
ttgctggttc tggagttcag tttac 25
<210> 152
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 152
tgtctgaaaa cagttggata gggct 25
<210> 153
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 153
agccctggct gtcctagaac ctg 23
<210> 154
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 154
cgggacctga ccgactacct 20
<210> 155
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 155
gctcgttgcc gatggtgat 19
<210> 156
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 156
tgaacgcttt cattgtgtgg tc 22
<210> 157
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 157
gcctgtagcc tctgtgcctc ct 22
<210> 158
<211> 19760
<212> DNA
<213> Susscrofa domestica
<400> 158
tgcctccaac aaaggggtac tgttgcccac atagaaagat ctaaactaat taattaatcc 60
ctcacccgca aatctttcag tcactaagtt agcacgattg ttgaacaagt tctccaaagg 120
agagatacag atgagtgcgt atagggtgga cctggctgct gaggagacac ctgcatctga 180
ctaagaagag ccacggtgtt agttgaatgg tgtggagtag ggtggttctg tgggacagta 240
gaaaatcgag aggcatgtgc cgtttagtga actgatggaa gctaccccaa acgacagaga 300
ttgtcagtca ggccaatccg tttcgagttt gatgggcagc cggacagtga gacagacaca 360
cctactcagt tggaggaagg atgagaacaa tggccagcag ggattgagag accctgacag 420
gcgcaaggcc ctaacacaca cacctaccac ctcacttgac aaagctgcca aagaccaaag 480
acttgttctc cattagaaat gacagctggc ttgacccgac agcataataa gcagagtgta 540
ctctgattgg agaactttaa tgtgtttcat tcagtattat aaaaggacag tattacagat 600
tttgttgtac actgctgtta catgtggggc agtgtgtctt taagtagggt aaagtactct 660
ttaaaaatgg gtcctagata ttttttcctt taactcaagt ctcttactgt ttaaatgatt 720
tttattttgt ttaatatgga ggaaaaagaa gcgtaaatgg acaatatata tttagagaaa 780
gatggttagc tgtcagaaaa atatgcaaat caaaatcaca ccaagactgc agcacacccc 840
tgtcagatgg ctgtgatcaa gaaaataaat gacaatgagt ggtggtgaag atgtactaaa 900
gggaaacaca cacacacaca cacacacaca cacacacaca cactggagca accactgtgg 960
aaatcagtat gaatggtcct caaaaacctg aagatagagc ggggcgtggt ggcatacact 1020
tttattccca gcactgggga ggcagaggca ggtggatctc tgagttccag gccagcctgg 1080
tctatagcac aggttctagg acagccaggg ctacacagaa aaaccctgcc ttgattaaac 1140
caaaccaaac caaaccaaac caaaccaaac caaaccaaac caaaccaaac caaaccagac 1200
caaaccaaaa cactgaagat agaacttcag tattccattc ctagatatat acccaatgga 1260
gactaagtca gcaagacacc tgcacagcca tgttcactac tacactgttc accacagcca 1320
ggctgtggaa ccagcctgag tgtccatgat aaatgaatgg ataggtaact ttcaaggtaa 1380
atggactctg ctgtgtacat gcctcacatt ctgtttattc atttttcttt atgaggtgtc 1440
cattcaggag tcacatggta gttctatttt cagtcttctg aagatactac actggtcccc 1500
acagtttaca cttttatcag cagtgaataa gggttcctct atccttacca tcatttgttg 1560
taatttttct tgatgaccct ctttctgaca gggataggat gtaatatcag tgtgaggaag 1620
tacaacttgt tttctaagta tttattggcc ccttgcattt cttcttttga aaactgtcgg 1680
ttcctgacat ctgctcaggt attcattgga tgttgtttct ttggtgtttg agttcttatg 1740
aattctagat gttaaatccc tgcctgtggt tctctcccat tctgtaggct gcctcctcac 1800
cctggcaatt gttgtccttg ttttgcagaa acttttgact tcatggaatc tcatttgtca 1860
gttttccctc ctctgctata gcctgagcta atgcactggt ttttacagag ccctggtcta 1920
tgcctttatc ctcctctggc agcttcggag tttcatttct tacatttaga tctttgatcc 1980
actttgaaca agttttggag cagggtgaga gatacgaatc tagttccatt cttccatatg 2040
tgatcctagt ttacatagca tcgttggttg aagaggtttt attttatttt taaataatgt 2100
gtcataaaaa acgaggtggt tgtagcagtg tggatttgtt tctttgtcct ttgatctaca 2160
ggtcttgttt tgtgtcagtc tcatgatgtt ttattgctat ggctctgtca tacagtctga 2220
ggtcaggtat tgtgatatac cttcagtatt gctccctcag actcaggttt gctttggcca 2280
ggagtcatct tactcagtgc tcttagagct cccccagcat gtagctgcta ctattcttag 2340
ttgataaatc aggaaactgg ggctcagaga gattaactgt cttgaactac ttctggggag 2400
gtgaaacgtg gagacactaa actgtgttta ccctgtactg ctccagtagc tgtcgggtgc 2460
tgggctacag caaagcacct atactatata ttactcagga ggtggaaaaa ctcagcctcc 2520
cttggggttc ccaagctccc aggtgtccag tcactgctgg aaacctcatg gagtctgaaa 2580
ggaagggttg agggtacatg gggcagcgat gaggagcctg gggctgggat ctcccaaaca 2640
cctggatatc cagatgccac tgggtcaggg ggagttggga acagagttgg gatgtccatg 2700
gacctgtgac aaggccaggg ccagggggag gataactctg gctttactaa tttgcgaaag 2760
tccttagctt agcagcagtt gtctgggagc acagaggggc cttctgtaag aggctcaggc 2820
agtgccgctc tgtaggcgaa ggtcttctcc atgttcccca tggtggttct tgatgaaaga 2880
gacagtcctt ggctccaaac tggtttattg attgttcatt gtggaaaatg ggtgcacacc 2940
accttctcag ggtggaccag agatcaaata ccttttgcag ggaggaatat ctgggaaggg 3000
acgcttactg gctaaaccct cagggcctct agatacatca ttagcatgga gaactctgtt 3060
ctgggctaca tgaccacagg ccacatttcc acaagccaca tgtgggaagt gtggcacatg 3120
ttctaggcca ggaatctggt agggagcgtg gagccaccta ccatcccagg tgggtgcctg 3180
ggtgccaggg accctgaacc cgctcaacct taccaagttt cctggcaggg tccactgtcc 3240
tacacagaag ctggaggagg tgtgagggtt gtgtctttgt ggaatgtccc atgctgcttg 3300
gggctcagtt tctccacctg tacctcattg gtttgggtat aaaaagtggg gatactttat 3360
tattctctga ctcggtcctg aggaaaaagc atcgtggcag tccaggaacc acaccctgag 3420
gttcctgcac tgaagggact ccctaagtct ctggagtctc tccccttcac agagctgcca 3480
aagtctaggt tcttttgagg ataacagagc catgcttggt aagcagacaa cagcatttgt 3540
ttactcaacc ttcttttgtc agctccctct tcataaacaa gttgagacac catgctggct 3600
tgaggaagac ttctaaagcc agacaactgt gcaaggaaga agaagaaggg gcaagtggag 3660
ttagcctgga tgtagccctc aaagtctcca gagaccagcc atgaaggctc aagtggaggg 3720
caagacctgc agcagccaag catctggcag gagaggatcc tgggaacccc tctaccatga 3780
cacacattct tcctgcaggt cacacttaat aggccatttc ttatttggat ctatcatggt 3840
gttctgtgcg agattaatga ggtgttatgc tgcgaacaga aagttatata aaaacaagtc 3900
cccccccctt gtcactgctg ctaagaatgt agcagaaatt gtctcaagtg tctctctaat 3960
cagaaacaat aaaggtctcc ttggattcaa gccctccagt ttcctccttc cttgctgagc 4020
cttggacacc catacaaacc tcctggatgc tacagctctg ggcagagact ccaaggtggg 4080
gagagactga tggtacaaaa gcaaaatact tgtttggggg tacacccact cctctgcctg 4140
tgtggttcct gcagtcagtc ctgcagacag gccctcagtg ggtcttccat gggcaacacg 4200
cagagggagg caatggatgg gaatacccac accctggtta gtttaccccg gccatgctct 4260
ctgctcttca tccctcctct gccctctgcc acggctttct ctgcaggaat catatcttca 4320
tattggccca caggtgttct cctcacccta gctatgatgt ttactttaga gtgaccttag 4380
cagggctggt gggaatgagt tctagaaggc tcacggagat gctagggaag aaacgtcttc 4440
taactactga ggttactaag ttcctggtgg ttgtctctgc ctttcccttg ttaaagtcac 4500
cttgaagtta gtgcagaaga aatcagagcc cagtcacaga gtaaatatgg tcctgaagat 4560
ttcctttgag tgcccagaat ccatgacatt tcaagagccc tctttgtacc ttaagtcatt 4620
tggggttgta tcttctgctt gatgtatgtg tgtgtgttta tcaaagagtg agatggttac 4680
ataagaggtg ctctaaagga cagagaggat ttgcaattgt ggcatgtgac atcctcaggc 4740
cttgctctgg tgccaggagg aactgatgca gaaaagagta agaggtcatt tcctggaggc 4800
tgtcactata gaggagatct tacagtgcat tccctcctcc aggccctgcc tgaggataga 4860
catgtgctga ctgcaactga aacagaggct tgggatggag agttaggttc acagaaggga 4920
gggtgggaga tggatgcttg ctgggttctg ggtctcatca ccagctcctg accacccggt 4980
cagcccatgt gcttattcca tagctttctt ttgctatgtt tactcagtgt ggtgtttgtt 5040
gggacccagc agaagccagt cccaggctga cagctgtgga tacacagggc agcatgaggg 5100
tcctcagcct gaagcagtca ggctggcaga agagaaagac cagcacacat tccttcaacc 5160
aactatgtct tgaaaaacaa acatattata tcacatatat tgcatttatg agacagctaa 5220
aatgtactcg ggtagcatga ctccaggtgg ggatatctgc aagtgccatg agtggcagag 5280
ggacagccaa tgtgaggcaa gaaggaattc tggctcaaca cagcttagct ccctggtgtt 5340
ggttcaaact ttgagagttt gaccacaagc actttatttt tgacatattt aaacagagca 5400
caactttggg aaaaagtttt cttatgaaaa ttatcacaat aaagcttaag gcatgactac 5460
attaaaatgc ctttgcaaag tatatgtgcc ctcttccaca agaatggttc tattgactga 5520
gaaataatgt tcaggataaa gatccaggaa gaaaagatca gggataagta aaatactaaa 5580
ctcttttgca aagtacatag accctctttc ataacaatgg gttctattga ctgacaagca 5640
ctgctcagga gttgggaaag agtctagcat aagcacgata gcctggagac tctagtgagg 5700
tctagtctta cagacagcaa aaatcaccag gttacaaact acattcattt ccagttttct 5760
gatcaggcac aggtatgaat cccttctgtt gaagagaaaa gtccatgtgt ttaaaatatc 5820
tggtttctcc agtgctatta gcgagaagac ttgagcccta tacaactccc acctggagtg 5880
acatcctgtc ttcatggtat attacatacc tagacacgct catctcacag acttaggact 5940
ttgtcttctg atctccattt ctgatcccac ttccaccttt gccttgatag tgtcattttc 6000
ttcactgcct tggtgacaac catgttatcc tctgtgtatt tgagtgttac cattttcaga 6060
ttttacctgt atgcaagatc acacagtctt tgtctttctg tctggatgca tgctaatctc 6120
tacacaacaa cccttccccg tcactcagat cttcctccat taacacatac atggtgctga 6180
agaggctagg gagcttccct tcagtgggga gctagctggc tattgggcct ttttgactgt 6240
ccaggaaggc ccccaattgc tgagacaaga acttagattc ttcattattg actctaactc 6300
atgtatcaag cagaagctaa tgaatagtta tcaacaggat cagaggttcc agtgtaagac 6360
actttgacat gaaagaacgg aggaaggaca gatggatgca taaaagcagg accactgccc 6420
caggaaggtc ctggaaactg atgcagggca aaggacaggt tataaaccaa atcttaggga 6480
gtcaggaaga gcacagagga gctcaaccaa ctgaccactg cttaggggct accaacccaa 6540
tcctccctgt gggaacagct aagctatcag ccaagggtaa taaacaggca ggacctgtgg 6600
atgacatgga gagcataggg accctgggtc cagcctttag cacctgcact ctcaggatac 6660
tccaccattg tgtcttagag agcctaggga tactgggtcc agcctttggt accttcactc 6720
tcagggtacc ccatcactgt gtcttggaga gcctaggcac cctgggtcca gccttcagta 6780
cctgcgctct caggacaccc caccattgtc tcttgccccg tctcttcttc ctcttcctcc 6840
ctttcattgt ctcttctctg tttctttctt gactctcctt tcccctcaca ccctcactct 6900
agttctcccc ttccctctct gcatcaccct attctctctg tggtccctcc actttccttt 6960
atctctcatg cttctctcct ccctcaaata cttgtcaccc actatacttc aggggccagc 7020
tctagtgaca aagctgttaa tagcaagact ctcagatctc caacggctca gaggagccag 7080
acccaccaag aactctctcc aggtccaatt tcaggttcct tcgaaagctt tcagcaaatg 7140
ctcagggaac atgccactaa caagaagatg caaattccag ttgagagtgg gaaaggccct 7200
tgcgtaggtc ccatcttcca ggccaaggtc agaggggctc tgtgtaatcc ggattgacag 7260
ggctcagaac aatgttttgt ttttaaggtt tatttatttt aggtgttagt gtctttgctt 7320
gcatgacctt atgtgcatca tgtgtgtgca ggttcctgat gacagtagag gagggctttg 7380
aatccctggg gataggaagt tacaggaaat tataagctgc tttgtgggtc ttctagcttt 7440
cccaacagaa gtgaatgctc ttcaccactg agccatctct ctaggcccaa gagacattgc 7500
tttatggata taattgtgtg tgtgtgtcaa cattgaggaa agggaaataa aaaaaaaact 7560
tcagccgcta aggttgtaca gtttcactaa ttgctacttt tagttgtgat aaaatggcag 7620
gtgcttcaac atttatatat acaaaaactt ccctgctggt ggttcaactg tgagaactgg 7680
ggtaagtggg tgagttctct ttttctgtct ctgtctctgt ctctctcctt ccattctttc 7740
ttaaaggaaa taaacattgc agctgggtta tagctcatca atatggaagt tacagaagtg 7800
aaaaaaggca ttgccttggt gggtggtgtt accagctgat ttttggttgt cctgcaagga 7860
ggtctgggga ctggctgctc tgtctctgtc tgtatgagtg agggaagtct ggggagcaga 7920
ttccctaacc ttcagcctgg cctggttcct gagtgaaccc agcctctctg gtcctagtag 7980
ctttttccaa acaggaatct gagtggtgac agggaacaag taccagccca ttgcttaagt 8040
gccagggtta gtgagggcag gaagctgcca tagctgggat tagtagttgt attggatgta 8100
ggaagtccta tcctgggaca gctaatcctt aatgcttcac tggagatttt caatgagaaa 8160
tttatcccac ggcccatatg gccccatcct tttgtctcca acagccaagt attttccatt 8220
agaggagact tcctgtacac ttgatggatg ctcatttcaa ggtgacttgg ggcagtcagt 8280
acagacttgg gatgacctct gacagcctaa cctctcccca acaagggccc tctatgtttg 8340
ctatgtaatg taatgtcaga cattgtcagg agtgtccgca gcacagcctg cccagtgtga 8400
gggctctcat aggtttccca ctgtcttatc tacacaggga taacgaggag gtaagctgca 8460
gttcccagtc tcacttcaca gaggaagaga taaccccatc ccaggtcatg tagccagcag 8520
tggaaagaat gaggatttga actcaggtct tccaagtccc attgatagca tctcctcaca 8580
agtcccttgc caccctcacg atgccttaga cacttgcctg ccctttatac taaggagatg 8640
caggtacaag gggtttaccc atgtagcagc tgaggcagct ggggatagat accagcagca 8700
ggcctgatgt caccactcta actccagcat ccccagtctg tgttcctgga gtgtgaaaat 8760
ccctacttaa caagattgtg caacagtcct tggctctgtg acccatagct ggaaacagga 8820
ttctcattga tttgtggaac atggtggcag ccagccaaaa agagggtctg catacagaag 8880
acagctgtgg caaggccaca gcagactctg actaccttag cttacagaat tacaaggtca 8940
taatgtcctc tgctttggtc acctcatgtt aaggacaggc cctaatgaag atggggcaga 9000
agactgaagg aatggccaac caataactgg cccaacttga gacccatcct acaggcaagc 9060
atcaattcct gacactacta atgatactct gttatgcttg cagacagaag cctagcataa 9120
ctatcctccg agaggtccac ccagcaactg actgaaacag aaaaagatat ccacaggcaa 9180
acagtggatg gaggtcaggg actattatgg gagagctgtg ggaaggatta aaaaccctga 9240
aggggatagg aaccccacag gaagaccaac agagtcaact aagagacctg tgggagctct 9300
cagagactga gccaccaacc aaagagcata cacaggccgg tccgaggcac ctggcacgtg 9360
tgaagcagac atgcagctca gtctccatgt aggtcctcca ataagcggta gcctgactgc 9420
agtatccaat ccctaacagg gctgcacagt ctggcctcag tgggggaggg tgcccctaat 9480
cctgcagaga cttgatgagt ggagagctat ccagggggaa cccaccctct ctgagaaggg 9540
aatggggatg ggggagggac tctgtgaaga ggggacaagg acaaacaaga acctcaaata 9600
ggtcaggccc taaaggcttg ctaagtagca gtggcccagc tctgtcctgt tcctcagccc 9660
aaggctcagc tcccacctgt ttctgtgttt ttctggcttt tcatgggcct aggacttggt 9720
ggccagttca aacaatgggg cctgtggaag acacaatata caagactagg gacattcctg 9780
ttctgctgac tatccacagc ctgatgtagg tggaaggacc caatcactgg atttctaccc 9840
ttgcgcaacc ttgacagctg agggcctctc agaaacctat ttcttccact gaaaaatgag 9900
actctcaaat gaacgtcctg acaatcatca ggcttattaa agaggtgtat ctaacctgaa 9960
tggcaagcag acagcaggca aatgtctgta tcaacctcta ggaaggacaa gaactgctca 10020
ctgctgcccc ccaggaggcc atttgctgaa acagctgctc tcctgctggt gcacaggccc 10080
tgccttctca ttgcagctac agccccttcc tgtctgaacc tcctgtcagg tcactgggaa 10140
acagatcaag atggaacagg acagctcctg atggtaaata aaaaacagtg gtcatggcta 10200
ttcatagggg tttatgcttc ttcagtccac actgtgaaga gctgtgggca tgaaccacag 10260
tgttcgaggt agagttgggg ttctgaaatt cacagtgggg tgagctcagt aaatgtgagc 10320
tggaggtcac tcgtgagaca cacagtcctg ctgcttctgt tcccaatatc ctgaggagac 10380
gacacatcta ctttgttcag aggccacagt ctagttgacc tgagagttac cagtttctta 10440
tttgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg ttgttcgtgt gtgagtgcag 10500
gtgcacatat gatagcgtac acgttgaggt cagaggataa ctatcaggcg ttgtcccctc 10560
ctacttttcc tcggactctg gagaacaaac atgggtcctt attccagggg agcaagtgcc 10620
tgttggctga cacatcttgc tcacatacat tttacctaga caatggagcc tccatcagag 10680
tattacttta gctcctcacc gatggcaatg caccacctct ctacccacat aggagttggg 10740
tctccacaca cccccacacc cccttcacca aaacgttttc agttacttta tctggtaaag 10800
ttcatcagag aatgaagcca gtattaagaa catggaatca tttgggaacc tggatctagc 10860
aataccccac cctagatgga gttgctgagt tttcacctca gattataatt cccccctagc 10920
ttctatggtt tattctgaaa ccaggggaac tcgattcctc cctttggacc acagacatcc 10980
tggcttgtga attcacatgt catctactgc taatccattg gtagtatgtg gctcacagag 11040
acacactaca gtcatggcca atgtcaaggt aggacagatg tgaatcattc ccccagtcct 11100
gctgttttca tgactaaccc tcctcagcac agtgaccatg aacctacttt tcccctcctt 11160
ttatttttag aattgctgga attttctatt ttgagaaata atagccttgg ggcagcatta 11220
aacaaaatca tctagaaagc tggtttaaaa tacagatggt tgagtcagtg aaagagtgag 11280
gaatgtcatt attggcccct cacagaggct ggctcactcc agcagaggtg gttgaagctc 11340
ttggacacgg gtcaggtgca taggaagggt ggtctgggac acctgagaac cacaattgaa 11400
caaacagaag ctgctggctt tttttttttt aaatgagttc tcaaaaaatg actgggctag 11460
cttaggcaaa tacttcgagc caacccaaca gaacattctt ccattgattc attctggatc 11520
ttctttctag acaatactga actgacccct tgttggcagt ctcaagtttg acaacatagg 11580
gctttgaact tggcacaagg tccatcactg tcacccaagc atcctgggtg acctttgggt 11640
tggaatatct tggctaacct tagatatttt ctttggagta tctttagaac atccaggaaa 11700
tagggcttga ttctcatcct gggaccacaa tataagtcac cctagaatcc caggagatcg 11760
tgcagagaaa caaggatctc tctcgtgtgc atccttcttc aaagcagtga gtagtgactc 11820
cactaaactg agttcccatc tgagagtcca caggaggctt tggggcaaga agcagaggga 11880
aggcactgtt tgtgttggta aagttttgac tctaacaaat ttgaagacat agatgacatt 11940
gtgtcagact aacaacaacc tagactcatg tgggttctgt ttagggatca gattttattc 12000
atcaatgact tgtcttagtg tatagagaaa ggcttcctac tggagtgtag gctcaataat 12060
gacagaagag atagctattt cccctaggga ctgtgctgct ccaagtttgg tggagaaagg 12120
cagtggggaa cctagatgtg ctctctgggg agggggtctg aagctggctt catagaaggt 12180
gtgaagtttt gctgaaacat ctaaacagaa ttatagctta ggaaagtgag caggcaaggc 12240
agggaatgtg ttgcatatgt atatgtacat gaatatatta tgttatagat acacacacat 12300
ttgaacctca tttgcagatg acagaaaata ggttattttg cctctcttaa ctgctaagca 12360
caatgacttc cagttccatc catttcctga aatgccacaa tttcattttt cattgtggct 12420
gaataaaatt ccattgcaga ctgggcccta cttcatccac tcctgagggc aggcatatcc 12480
cctggctcca tttcttacct attgtgaaga gaagtgcaac tgtcttgttg aaaggcaagc 12540
gtgagagagg caggcactaa ttgtgggttt ttgtttcttc ttcctgctat gactctccat 12600
ttgtcagggc gcgccgccac catgtttcag ctctggaaac tcgtgttcct gtgtgggctc 12660
ctcattggca cctccgcttc tcagcagatt ggacgcatcc ccgaagtcca ccccaggctg 12720
accacacaga cctgcacaaa gcatggctgt accacacagc agacctctct ggtcctcgac 12780
gccctgtcac acccaatcga cgatattcat acaaatgaaa gctgcgagac ctccagcggc 12840
ggagtgaaca ccacaatctg tcctaccgtg gaagtctgcg ctgagaattg tgccctggag 12900
ggcgtgaact acgcctctca cggcgtctat acaaatggag actcagtgac cctgagacag 12960
tacctcaaca ttgatggaac cgaggaagag gtgtccccaa gggtctacct gctcgaccct 13020
agcgggagag attatgagat cctgaagctg ctcaaccagg aaatttcctt caccgtggac 13080
gtcagcaatc tcccatgcgg gatgaacggc gctctgtacc tcaccagcat ggatgcctct 13140
ggaggacgat cacagctgaa cccagctgga gccacatacg gaaccgggta ttgcgacgcc 13200
cagtgtaacg ctccagcctg gatcaacgga gtggctaatc tgaaaggcct cggagcctgc 13260
tgttctgaga tggatctgtg ggaagctaat tccgaggcca cacagctcac ccctcacgcc 13320
tgcaacgtca ccgggttcta cgagtgctct ggcgccgctt gtggatcaaa tggggtgtgc 13380
gacaaggatg ggtgtggctt caacccctac ggactggggg accattcttt ttatggccca 13440
tcagtgaccg acacaatcga tacaaagaaa cctttcacag tggtcaccca gtttctgacc 13500
tccgacggaa ccagcacagg aaccctctcc cagatccgac gactgtatct ccagaatggc 13560
aaagtgattc agaacgccaa ggtcgacttc gataactcta cactggactc aatcacccct 13620
gcttactgcg aggccacagc cgctaccttc gaagctgagg gagggtttcc ccagatggga 13680
aaagccctgg aaatggggat ggtgctcatc ttctccattt ggaatgaccc cagcgccttt 13740
atgaactggc tggattccgg cacagccgga ccctgcaata gcacccaggg caacccagct 13800
ctcatcgaag ccgagaatcc tggaacatcc gtgaccttca gcaacatcaa gtggggggac 13860
attggcacca catactctgg ctcaggatcc ggcggatgga accagtgtac taattatgct 13920
ctcttgaaat tggctggaga tgttgagagc aacccaggtc ccatgtttca gctctggaaa 13980
ctcgtgttcc tgtgtgggct cctcattggc acctccgctt ctatgcagac cggaggatcc 14040
ttctttgagc cattcaactc atacaattcc ggcctgtggc agaaagccaa cggctatagc 14100
aatggagaca tgtttaactg cacatggcga gccaacaatg tgtccatgac ctcttcagga 14160
gagatgcgac tggctctcac cagcccatct tacaataagt tcgactgtgg agagaaccgg 14220
tccgtccaga catacgggta tggcctgtat gaagtgagga tgaagccagc caaaaacacc 14280
ggcatcgtgt ccagcttctt tacatacacc ggacctacag acggaacccc atgggacgaa 14340
atcgatattg agttcctggg caaggatacc acaaaagtcc agtttaacta ctataccaat 14400
ggagctggga accacgagaa agtggccgac ctgggcttcg atgctgccaa cgcttaccat 14460
acctatgcct ttgactggca gcctaactcc atcaaatggt acgtggatgg gcagctgaag 14520
cacacagcca ccagccagat ccccaccaca ccaggcaaga ttatgatgaa cctgtggaat 14580
ggcattggag tcgacgattg gctgggatcc tacaacgggg tgaatcccct ctatgctcac 14640
tacgattggg tgcgatacac aaaaaaggag ggcagaggaa gtctgctaac atgcggtgac 14700
gtcgaggaga atcctggccc agccaccatg tttcaacttt ggaaacttgt tttcttgtgc 14760
ggtctgctca ttgggacctc agcatctagc acaccttcaa gcacaggcga aaacaatggg 14820
ttctattact ccttctggac cgacgggggc ggcgatgtca cctacacaaa cggagacgcc 14880
ggagcctaca ccgtggagtg gagcaacgtg gggaacttcg tgggaggaaa gggatggaac 14940
ccaggatccg cccaggatat cacctactcc ggcaccttta caccaagcgg caacggatac 15000
ctgtccgtgt acggatggac cacagaccct ctgatcgagt actacatcgt ggaaagctac 15060
ggcgattaca accccggatc cgggggcacc tacaaaggga ccgtgacatc cgacggcagc 15120
gtgtacgata tctacaccgc tacaaggacc aacgctgcca gcatccaggg cacagccacc 15180
ttcacacagt actggtccgt gcgccagaac aagcgggtgg gagggaccgt gaccacaagc 15240
aaccacttta acgcctgggc caaactggga atgaacctgg ggacacacaa ctaccagatt 15300
gtcgccaccg aaggctacca gtcctcaggc tcatcctcca ttacagtcca gggaagcgga 15360
gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacctatg 15420
ttccaactgt ggaagctggt cttcctgtgt ggtctgctga ttggcacctc tgcttcccag 15480
agcgaacccg aactgaaact ggaaagcgtc gtcatcgtct cccgccacgg agtccgcgcc 15540
cctacaaaag ccacccagct catgcaggac gtgacccccg atgcctggcc tacatggcca 15600
gtcaagctgg gatggctcac ccctagggga ggagagctga tcgcctacct cggacactat 15660
cagaggcaga gactggtggc tgacggactg ctcgctaaga aaggatgccc acagtccgga 15720
caggtggcta tcattgctga cgtggatgag cgcacccgga agacaggaga agccttcgcc 15780
gctggactgg ctccagattg cgctatcacc gtgcacacac aggccgacac cagctccccc 15840
gatcctctgt ttaaccccct caaaaccggc gtgtgccagc tggacaacgc caatgtcacc 15900
gatgctatcc tgtctagggc cggaggcagc attgctgact tcaccggcca tagacagaca 15960
gcctttcgcg agctggaacg ggtgctcaac ttccctcaga gcaatctgtg cctcaagcgc 16020
gagaaacagg acgaatcttg tagcctgacc caggccctcc catccgagct gaaggtgtct 16080
gctgataacg tcagcctgac cggagccgtg tccctcgctt ctatgctgac agagatcttc 16140
ctgctccagc aggctcaggg aatgccagaa ccaggatggg gccgcattac cgactcccac 16200
cagtggaaca cactgctctc tctgcataat gcccagtttt acctgctcca gaggacccca 16260
gaggtggcta ggtctagagc tacacccctg ctcgacctca tcaagaccgc cctgacacct 16320
cacccccctc agaaacaggc ttatggggtg accctgccaa caagcgtcct gttcattgcc 16380
ggacatgata ccaacctggc caatctcggg ggagctctgg aactcaactg gaccctgccc 16440
ggccagcctg acaatacacc acccggcggg gagctggtgt tcgaaaggtg gcgccggctg 16500
agcgataact cccagtggat ccaggtgagc ctggtctttc agaccctgca gcagatgaga 16560
gacaagaccc ccctgtccct caacacacct ccaggagagg tcaaactgac cctcgccggc 16620
tgcgaggaac gcaatgctca ggggatgtgc tctctcgccg gattcaccca gattgtcaac 16680
gaagcccgca ttccagcctg ctccctgtga ggcgcgccga tcaattctct agagctcgct 16740
gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc 16800
cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg 16860
catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca 16920
agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt 16980
ctgaggcgga aagaaccagc tggtttaaac tcgattataa cttcgtatag catacattat 17040
acgaagttat gatcgatatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg 17100
atgtacgggc cagatatacg cgttgacatt gattattgac tagcttaggg ttaggcgttt 17160
tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta 17220
ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac 17280
ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc 17340
aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt 17400
ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac 17460
gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac 17520
cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt 17580
gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc 17640
aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt 17700
tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg 17760
ggaggtctat ataagcagag ctctctggct aactagagaa cccactgctt actggcttat 17820
cgaaattaat acgactcact atagggagac ccaagctggc tagcgtttaa acttaagctt 17880
ggtaccgagc tcggatccac tagtccagtg tggtggaatt cgccaccatg ggatcggcca 17940
ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct 18000
atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc 18060
aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcagg 18120
acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg 18180
acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc 18240
tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc 18300
ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg 18360
agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc 18420
atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg 18480
atgatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc 18540
gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag 18600
cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg 18660
tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg 18720
agttcttcgc catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg 18780
tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg 18840
atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc 18900
cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg 18960
accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc 19020
gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg 19080
gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 19140
tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca 19200
agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg 19260
tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc 19320
ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg 19380
atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc 19440
tgtacaagta atctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta 19500
gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 19560
ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 19620
attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 19680
gcaggcatgc tggggatgcg gtgggctcta tggcttctga agatctataa cttcgtatag 19740
catacattat acgaagttat 19760
<210> 159
<211> 36690
<212> DNA
<213> Susscrofa domestica
<400> 159
cctcctgtgt ggcctgttgc cttccttcca tgggatttgg ggggaagtgc ggtttgtgca 60
actagagcag cagagacctg gatccagagc tgaaagggac atttggaatc atctggcctc 120
accttcttgt cttataaatg gggaatctga ggtccagaat gggcaagaaa ctgacccgag 180
gtcacccaga gacagcatca ctctgtttct gcaaacacca cccctgtctg ggagggctgc 240
cttttctggc acacagcctt gtccaggagc tttattcctt tgttttttct tctaggtgtt 300
tcaggtctta tctttaggtc tttaatctat tttttttttt aatttcccac tgtacagcaa 360
gggggtcagg ttatccttag atgtatacat tgcagttaca gttttttccc ccctaacttt 420
tgtcagtagt ggaagatatg ggtccagttt cattctttta catgtgaata tccaatcact 480
ccagcaccac tttgtgaggt attcactaaa cacaacatta ggttgatagc agaagcttgg 540
gtttatttct gggcccctga ttctgttcca tttgtctatt tgtctttgtt gatcccagta 600
tcaaactgga ttttgttttt gttttttctt cttatggctg cacccgccgc atatgaaagt 660
ttccaggcta ggattcaaat tggagctgca gctgctgacc tacgtcactg ccacagcaat 720
gctggatccc agccacatct gtgacctaca ctgcagcttg tggcaatgct ggatccttaa 780
cccactgagt gaggccaggg attgaatcca catcctcaca gacaatatgt caggttctta 840
acccactgag ccacaatggg aactccccca gtatcatact gttttgatga ctatggcttg 900
aattcagaaa atgtggtacc ttctgctttg ttcttcttcc tcaagatttc tttggctatt 960
cgtggtattt tgtggttcca tataaatttt aggagtgttt ttcctatttc ggttaaaaaa 1020
aaaaagtgtc atcacaatct tgacaggatt gcattgaatc tatagatggc ttttggtagt 1080
attggcattt taaccatgtt attaatcttt ccaatccatg aacttgggat acctgtccac 1140
ttatttgtat attcttcaat ttctttcatc agtgtcttgc acttttcaga gtaaagatca 1200
gctaccacct tagtcaaatg tactcctaag tattttattg ttgttaatac taaggtaaat 1260
gagaccaatt tttatttttt agagaaattc attgttaatg tataaaaatg ctattgattt 1320
tggtacatga attttgtacc ctgcatcctc actgaattca tgattagatt taacagattt 1380
ttttgtcttt aggatttcct atataaaatc atgttatttg caaatagggg cagttttact 1440
tattcctttc caattctgat cccttttatt tctttttctt gacttagtgt tctaactaga 1500
actttcggta tgttgaatag gaatagtcaa aatgggctcc tttgtcttat tgctgatata 1560
attttttttt ttcttttctg gctgtccctg caacatatgg aagttctggg gccagggatt 1620
gaatccaggc tggagctgca acctgcatca cagctgtagc aatgctagat ccttaaccca 1680
ctgcaccagg ctagggatca aaccagtgcc tccacagaga caagccagat cattaaccta 1740
ctgtgccaca gcaggaacct attctgatct tagaagaaac actttcaatt gtttaccttt 1800
gatggtgttg gatgtgggct tgtcctatac ggcttctctt atgttgagct atgttccttc 1860
tatgcctaat ttgttaagag gttttttaaa aatcatgaat ggatgtttga gttttttttt 1920
caaatgcttt ttctgcatct actgagatgc tcatataatt cttttctttc attcttttag 1980
tatgttgtat cacattgatc tgtgtatatg aactatcctt acattccagg gataaatccc 2040
acttgattat aatgactatc tttttaatac gcatctgaat ttgggttgct aatattttat 2100
tgagagtttt tacatctatg ttcatcagag atattggtct gaaattgtgc ttttgttgtt 2160
gttggtggtg gttttctagt agtgtcctct cctggttttg gaatcaaaat aattctggcc 2220
tcataaaatg agtttggtag tattctctcc cctcttcaat atttcggaat gttgaattct 2280
tttttaaaca tttggtaaaa ttcaccagtg aagccacctg gccctggact tttcaatgct 2340
aagagatttt tcattatgga ttcaatctcc ttactagtaa ttgttccatt catgtattct 2400
atttctcccc gattcagtct tggttagttt ccaagagttt ctccatttct tttaggctgt 2460
ccagcttgtt aacatgtagt agttcatagt agcctcttat gaccctttga atttctgtag 2520
tatcagttgt agtgtctcct tttttacttg tatttttgtt gattggggtc ctttctcttt 2580
tttccttggt tagtctggct aattgtttgt caatttcatt catcttttca aagtatctac 2640
tattaatttg gttaatcttt tctaatattt ttctgttctc tatttcattt acttctgctt 2700
tactctatac tatgtctttt tattctgcta actttgggct tagtttggtc tttgttatta 2760
ttctttcgtg tgtgtctctg ggtgtttgtg tgtgtgtgtg tgtgttttga tgtcttttgg 2820
tatcagtatg ttttgacttt ttattgtatt cttttatata attaccacag aattttccct 2880
tgtgcttacc aagaggctaa tataaaatat tttaaactta aaacaccctg ttttaaactg 2940
atcataactt aatttcagta gaattcataa actttacaat tgtacttatc ctcttcctca 3000
cattttagat tatcattaca atttacatgt tttatattgt gaaattaata acaaattatt 3060
agaattatag ttatctttct atgctttttt tttaactttt aaaccagaat cataagtgag 3120
ttatgcacca ctattatcat attgcagaat ctatgactat atatttacta tttccagtga 3180
aatttatgct actgttttat cttgttatga tgttaattag ccctatttta cttccactca 3240
aacaccttcc tttagcattt cttataaggc ctatctgatg gtgatgaact ctctcagctt 3300
ctgttcattt ggaaaagtct ttattcctcc tttgtttctg aaggaaaatt tttgattgac 3360
agttattttt tttcaatatt ttgactatgt catcccattc tctcctggtc agaatagttt 3420
ctgttgagaa agccgccgat aatcttgtgg gttcccctgt atacaacttt tcttttttct 3480
ctcttaaaat tctgtctttg tctttgactt ttgagaatat aattacaatg tgtcccaggg 3540
tagacctgtg cagattcaac ctatttgtgg ttttttggtc ctcatggatc tggatgtcca 3600
ttattttccc caagtttggt acattttgag ccattattgc tttacttttt gtccttttct 3660
ctttctcttc tcctggaatt cccataatgc aaatattatt tcttttcatt gtgttccata 3720
attcctatag gctttcttca ctcttttttc tttttgctcc tctgactggg taatttctaa 3780
tgtcctgtcc tccaggttgc tgattccctc ttctgcatgg tcaagtctac ttttgaaact 3840
ctccactgga ctcttcagtt cagttattgt attttttaat tctagggttt ctattttttt 3900
tttatggttg ctatttcctt gctgaactca ttttattcat gcattatttt cctaattttg 3960
ttggtgtttt cttgtagttc actaaacttt cttaagaaga ttatcctgaa ttctttgtct 4020
gacagtttag aaatgtccat ttctttaggg tcagttatcg gagttttact agtttccttt 4080
ggtggtgtca tagttaactg atttttttgt gtgcgattct tgattcctta cattggaatc 4140
tgcacatttg agtaatcagg ctcctcttcc agactttaca ggttcgcttt gacagagaca 4200
gttcttcacc agttggctca gtttagtttt atgggtgtgc ctgctggtta tggccttggg 4260
tatatgggag ctactattat ggccatttct ggagtaaggc aactgtctga gctctgagga 4320
tggggatcag gggagcatgc ctttgtctga aaacagttgg atagggctgc tggattggtt 4380
tcctacccag gtgaggctgt aggatggact ttgtggttgc ctgaattctc tggtcagtct 4440
tactagatgg ttggggctgg gtactgtatt cagtacaaga cgccttagga caggacccag 4500
gacctgtaca gcttatcatt tggaagcctg aatgtgcact gaattcccta cttaggtaag 4560
gccatcattc ccattctgca aacaagaata agccatgggc tatgctcact tcaagtgcca 4620
ctacacttag ggctgttgaa agggctctgg ttagtctctc tagctgaaca agatgaaggc 4680
tgtatttaac aatgagcgaa gctattaatt aggttctctg cccaggcaca gtggtagaag 4740
cagctcagaa gctggtaaag agttttgttt cttgtcttaa ttcaagccaa cccacacccc 4800
aggtttcctg ggtgaacaga aaactagctt tgctctgcaa actgtgggct ctgcttgccc 4860
ttctccaggc tccagctggg cagcacagcc tccaggagtt gtcactagcc cttctggtca 4920
gatgggatca gaagacaatc ttctcagtgg gttgtgctgg gactctactc ctgacctggg 4980
cctgggtaaa ctgaactcca gaaccagcaa aactacttgt ttgaggatct gaatcaggga 5040
gatttgctcc ctgccaaatt ccctggtcag agagtgcgcc cccaacttgg ttctgcagaa 5100
gccactcttt gtgatgacta cttagccact gcaggtgtga acttggttgg ccaacatcca 5160
tgtgttggtt tttgcaagcc cctccccagt tctctgacac aatcagcttc ccaatggtgg 5220
agcccttcct tgcagattcc taggcagtcc acatcccaag aatagggggg agggggtccc 5280
acaatgccaa cggggtcctg gttgttcctt agggttctct tttcccattg gaggaagtgg 5340
aggctcagga gagacctctc tgcacggtgt tgcactggcc tgggggcggg ggcagggcaa 5400
catggagatt cttctttccc ctcttaatta actcagtctg tcctggtctc tgtggtgtgg 5460
aggatgcttc agcctcaccc ttgcgtccta ggattctctc agtggtgttt tgttctgcag 5520
tagttattag ctgttcttct tgtgaaggga gcaaatcagg aatgacctat atctccatct 5580
aggtaacatc actctcttac tttttttctt tttctttctt tctatttttt tttttttttt 5640
tttttttagg gctgcaccta cagcatatgg aagtttccag gctaggggta gaattagagc 5700
tcagctgctg gcctacacca cagacacagc aacaccagat ccaagccaca tcttcgacct 5760
acactgtagc tcatagcaat gctggatcct taaaccactg agcaaggcca ggggctgaac 5820
ctgcatcctc atggatacta gtcatgttct taacccgctg agacactatg ggaactccct 5880
ctcacttttc ttattcacct ctgccccctg agcctgtgag ctcctccagg gtaggcacct 5940
ccttcttctc agcagcccca gtgcccagca aaggactctg tgcaatacaa gcactaatca 6000
gtgttgaatg aaaggataga tggacggatg gatggatgga tggatgaatg aatctcttct 6060
tgactgtaca cagacactat cgctaggaca gctaagtggg tagtgaacag gaaaattcta 6120
gcaacccacg actgtggggt attggagacc catcattatt tctggtgacc agggccagag 6180
gcctggccct tgaggtcata agtaatggac tatggaccag gatagtccaa gggggtcctg 6240
gttgttcctt agggttctca cagctcttag aggaatgaag ccagaacatg gagtgggtag 6300
ggccaaggcc actcagctcc actggtgact gatgtgacca atgaaggggg aaaagcacag 6360
gtgaggaagt gagggagtgt tctgctactg tgaccctggc actcccagga ccagctctgg 6420
ctctttgccc acccagtgca gacctcaaag aggggtgagg gcttcctgga gacagtgaca 6480
aagaggagaa tctgatgaga gggtaaggga ctatctgcca taagagaaca tatagggaga 6540
aataagacaa aacctgagtc atccagaaac tcaagtgcca aattccagtg caagctcttt 6600
tgaaggctta atccagttat gtaaatgccg aatctttcca atatttgcat aattccctca 6660
ggataatatc tcaggatcag aattaataac tctatgtttc agaacatttt atggccccag 6720
gaacacatca caatgttatt taccaaacca gatctttcta tttacactaa ggtgagagag 6780
tatatacatt tccccatgta tttgccagaa aatggacact tttattttaa atacttgctc 6840
ttaaaatgag ggtagaataa acttcatcac aaggaagaac taaggcccca aattaggaaa 6900
gtcaattgtt acatcaactc aatagactat tttctgaatg ttaaaaaaaa aaaaaaaaag 6960
cagtttccca ttgtggctca gcacattaag aacctgacat tatgttcata aggaggggct 7020
tcaatccctg gccttgctca gtgggtcaag gaacaggcat tgccgcagct atggtatagg 7080
tggaagataa cggcttggat tcagtgtggc tgtggctgtg gtgtaggttg gcagctgcag 7140
ctctgatttg atccctagcc tgggaacgtc catatgctgc aggaatggcc ttcaaaaaaa 7200
gaagaagaaa aaaaacccca aaacattgga aaatacttgc gttttacagc tgagtggaaa 7260
tatacagatc ttagaactgt gcttagctgg aatcatgacg acccccctgc ccaggcaggg 7320
ggtgaggaag aggcaacaga gtgctgagaa aaagactcag aggagggtct cgtgactcag 7380
cctggagatg ggaggaggag tcaaagcaga cttcccagaa aaggtgctga tgcagttaaa 7440
actgaaagat gggagagatg ggaggggagg gcatttactg caagcaaaag tgaccaaaag 7500
aatgaagact ggggagttcc cgtcgtggcg cagtggttaa cgaatccgac taggaaccat 7560
gaggttgcgg gttcggtccc tgcccttgct cagtgggtta atgatctggc gttgccgtga 7620
gctgtggtgt aggttgcaga cgcggctcgg atcccacgtt gctgtggctc tggcgtaggc 7680
cagtagctac agctccgatt cgacccctag cctgggaacc tccatatgcc gcgggagcgg 7740
cccaagaaat agcaaaaaga caaaaaaaaa aaaaaaaaaa aaaacaatga aggctaggaa 7800
ctgagaatca acatggtagc acctggcacg tgtcgctggc aatcaaactc cacagggtca 7860
agtggagggc gaggcctcga atgccacacc aggctggttt caacgtggtc ctcttagcca 7920
ggagttaaat ttatttgttc ttatctcact ctcaaaaact gaacacctcg aagagttttg 7980
tctgtatgga ccccatccgt tgatatttgc cgtgaaagaa actagccctg agaaaaattt 8040
aaaattcata attattgatt cctttagaaa tcacaataat aaactcattg cattttaaca 8100
aaatgacatg ttcttgtgaa aaataactca atgttccaaa aacatcatta gtaagaagag 8160
tggctctgtt ttacatcttt taaaatctcc agaaaaactg gcttaacaga ggacagctgg 8220
attctcacgt ctgctctgcc ttcaattggt ttcaatatca catgtcatgg aatgtctaga 8280
aaattctgct ccttgctcgt gagagaatga gagtaaattg aaaaataatg taatcaaatt 8340
aaattagaat ttgtttggac cttgcagatc ccctggaaag ggatttgggg acccccagac 8400
cacactttga gaaccacctg gctaggcgct agggagccat gaaaggtttc tgagcagaga 8460
agataccacc tcagtgttca gtgtaaggaa gtgtcttctg gaaggtgatg tgccaggtgg 8520
gttgaaagga gacaatgacg gtggctggtc caggctgatc cagggcagga gacagacttc 8580
gcttctaggg aagaggtgct caaggaaggg aaaggggcag ccagagaagg cctccctgca 8640
actcactcat gctgagcgtt tgccatcctc agtccccaga ctagagccca gggcagggct 8700
gatggctccg gggctgcgtt tctgccctcc ttgactttgg agggagacaa tgttgcagga 8760
aggtgcaaga cctcaacttt ggttccagaa gagggaacaa agtgattccc tttggctcct 8820
ctctaaggcc acaggcaaaa tcaacatttg tccctgttga cctgcaaaca ttggcccatc 8880
cagctgaccc tctgcaggcg tggccatcaa tgggaacagc tgggctgaca ttggtgcctg 8940
tgaatctaaa tgtcacactc tgatcaaaac ctcccaagtg tcttgccaca tgatccagca 9000
atcatgatct atggtatcca aaaaggttga aaacttaggt ccacactaaa cctgcccatg 9060
gatgtttata gctgttttat tcatgaatgc caagtcttgg aagcaaccaa gatgtcttcc 9120
agtaggtgaa tgaataaata aactatggta tattcagaca atggaatatt attcaaccct 9180
gaaaagaaat gggctatgga gtcatgaaaa gacatggagg aaatttaaat gcatgtgact 9240
acatgaaaga agcctaccaa aaggctacac gctgcatgat tccaactctg ggacattctg 9300
gaaaaggcaa agtatggaga cagtaaaaag atcagtggtt attaggggtt aggaaggagg 9360
gcggaatgaa cttgcagagc acagaggatt tttagtgcag tgaaactgtt ccatacatta 9420
tggtggcaaa gaaatgtcat cacacatttg acaaaagctg tgcagtgtac aacaccaaga 9480
aggagccttc atgtaaactc tgagtggtga tgatgtgtcg gtgtggttca ccgttataat 9540
aatgtcccac tctgtggagg atgttggtaa caggtgaggc tgtggggaca aaggggaagg 9600
agatgtataa gagctctctg cactttctgc tcagttttgc tgggaaggta aaattgctct 9660
taaaaactaa aatctatgga aaacacaacc tttcaggttg gaggctctaa gcctggcgtt 9720
gtgctttccc tagaacctga tctctgcctg ccccttcagc cacctttcct ggcccacgtt 9780
tttgtttgtt tgtttgtttg tttgtttttc tcttagggcc atacccatgg catatggaag 9840
ttcccaggct aggggtcaaa ttggagctac agctgctggc ctatatcaca tccatagcga 9900
ccctggatct gagctgcatc tgcaaccttc actgcagctc acagcaatgc cagaccctta 9960
aaccactgag agaggccagg gattgaacct gtatcatcat ggacactagt cgggttctta 10020
accccatgag ccacaaccag aactcccatt cattctttta acacagattt ctggaccatt 10080
tgctgtcttc caggcaggcc aggggaacag cttgagcaaa ggccctgagg caggaggggc 10140
aggacagcaa gtaaacagca gtggacttag ggtgcatttg gggatggttg gtggagatcc 10200
gcacgacaag ggccccacgg ctgggactcg gcaggattgt caaatcttca cccagagtag 10260
ttaagcgact taatgtagca gacaaggaaa tacatcacga agttgtccct ttaagaggac 10320
ctgccacaaa gagggtagtt ggctgatcat gtctctacat ctatgtctat cacttgtcac 10380
atggaacaaa gactagaggg aaatgaacgc aagtaatagt agggcttagt tctagataga 10440
gacactggtg tcttaacgcc tttctacatt ttaaaattat tcctggagtt tcctttgtta 10500
ctcagccttt aacgaacccg actagtatcg atgagcatgt ggattcgatc cttaggcctt 10560
gctcagtggg ttaaggatca ggcgttgctg tggctgtggt gcaggccagc agctacagct 10620
ctgatttgac ccttagcctg ggaacctcca tacgctgcgg gtgtggccct aaaaagtaaa 10680
aaaattaaaa aaaattttta aataaaatta tttctttcaa aacatggatt ctttgagtaa 10740
tctgaaatac actctttttt tttttttttt tttttgtctt ttttaggtcc gaacctgcgg 10800
catatggatg ttcccaggct aggggtccaa tcagcactgc agctgccagc ctacaccata 10860
gctcacagta atgctggatc cttaacccac tgagcaaggc cagggatcaa acccatgtca 10920
tcatggatat gagtcggatt cgttaccacc gagccacaat gggaactcct gaaatacaca 10980
ttttaagacc aatttgaaat taaggtttga agggtggtcc taacttttgg aacaaataat 11040
ggagaatgtc tgtccttggg gcctcgagga aaaagccttc tcagtgaaac agagaactgt 11100
cccttccacc ccaccaggag gcctcagctc tgctctaaaa ggaggactct gtgatgtggt 11160
gtcaagatca caagcttgga tatatggcag aaccagattc aaatcctgat tctactagac 11220
actagcccta tgaccttggg caaaatgttt cctctctcaa atcttagttc cttatctgta 11280
agtaacaaca gtcgttataa attaaggatg cacaatgagg atccgcaagc atttgtgaaa 11340
atggtccgag gcccaggagg gacacggggc tgaccagttt caaagagcaa ggaacaggcg 11400
gagccgggct agaacacaag gctccctggc ctgaggccag ggtaccttct ccctcacttc 11460
tacccggagc agacggaagg gcttcctcct cacagtcccc gggtccatgc cactcaagat 11520
gcagatgagg aaactggtgc agagcaggac aggatgtgga tgagtcagga acaagcctcc 11580
tggctctctt aatgccctcc aggttcccta cagctctgcc actggtgaca ttcatgaggt 11640
acagttccaa atatgttatt gacacagccg tcaaatggct taagtgactc cctacccctt 11700
aatcccccag ggccttctgt gcaggaaagt ttgctttgga ggtaagggat ggacgtggtg 11760
agattgaggg cttctcatca gattcccagc tgtccctcac tcggtgccac gtctccccca 11820
gtgaggggct cactcaccat ttataaagtc tgcccccctg cagagcccat gggggaccac 11880
ctgacgggga cccaggatat ctctacagca caccggagaa acccaggtga gcacgaggca 11940
atgccactct ccaacctgcc ttctcatcag atgccaaacc ggaagaaagc ttctgatata 12000
tgctactgtt gccaaaactt ggcctctgtc tgccgttgcc aaatagaaac atggagacga 12060
gtgttttcag tgaaggataa aaaattagct ttattgcttt gccagacaaa ggagggtcac 12120
agcaggctaa tgccctaaag attgtgcccc ccattggaaa gaactgtgag gagttttata 12180
gtgaaaagga gaaaaacggg tttccagaca ggaatcaggg tgagggcaga cacacattca 12240
tctttctttc ggggagctta gtcatccaag ggggcgtcag gagatctgag catgcgcatg 12300
atggtggtct tcggggttat tgcctagaaa aacagtactt gcaaaaaggg catattggga 12360
gttcccgtcg tggtgcagtg gttaacgaat ctgactagga accatgaggt tgcgggttcg 12420
atccctgccc ttgctcagtg ggttaaggat ctggcgttgc tgtgagctgt ggtgtaggtc 12480
gcagacacgg ctcggatccc gagttgctat ggctgtggcg tagaccggtg gctgcagctc 12540
tgattagacc cctagcctag gaacctccat gtgccacagg agcggcccta gaaaagtcaa 12600
aaagaccaaa aaagggggga ggcatattga tcagagatta gaacaaactg ggaaaattcc 12660
cgaaaaacat catgttctta taatctttaa cccacaggca actgtgctca gggtgcctaa 12720
tctttagctt acaggggatt gtgtttaggg tacaattaag ccagggagaa ggacaaggaa 12780
ggactctaag ctgttcaatt ttaaagtttt catttctaaa agaagcataa ggaatatagc 12840
ttttctctta ggtgtattaa gatgcctgta ttcttgagag ggaaccagga tgctgcccca 12900
aggctgtact actgtttctt ggctgttccc ccctcgtctt tgcatccgct tccttccctg 12960
atcagcaact gtctgaacct acccctcaga actcagggaa ggtcatggag gctgaatgag 13020
gcccatttcc taagaacaag aaatggggga cacagaaagg cttttgtgcc taggagcccc 13080
acaggcccct gcttggttac actaccagtt acaattgtgc ctggttctag cacagaacac 13140
aaaaaataat ggcttaaata atgatgagaa gtccaaaggt aggctgcttc actggcatgg 13200
gggaatcttg ccgactcttc cctctgctac cccctaggtt gtggctttca tcttcatgct 13260
aataaaatag ctgttgagct ccagccatca tgtctgatat ccaatcagca aaaagcagaa 13320
ttgggaaaac aaaagctgaa tcggccactt ttaaataatt ttttagaaga cccagcctaa 13380
aacttccatc catcatccct gattgcaaag aagtttgagg aagaaagttt ctttgggagt 13440
tcctgttgtg gctagactag aattcatgaa gacttgggtt tgattcctgg tcttgctcag 13500
tgggttaaag atcctgtatt gctgtggctg tggtgtaggc cagcagctat agctccaatt 13560
caacccctag cctgggaact tccatatgcc atgggcacag ccctaaaaag caaaaataaa 13620
aataaaaata aataaatgga ctgtttgggt gacctaaagg gacctaaagc agacttttct 13680
ctttcaccag aaatctatgg ggaaccagag ccttggtatc agcagagact ccttgggctc 13740
atctatctcc tcagagagtc cggatgaatt tgggtgagtc tatctcctgg ttctcagatg 13800
ctcatgagtt ttatttggca gtgtctgaca ggtacctggc tccccagttg aaagatactg 13860
ggggatgacc ccctaattga cagatactag gggagcccag tcaaaagaca ctgtggaggg 13920
agttcccatt gtggctcagc aggttatgaa accaactagt atccaggagg atgaggttca 13980
attcctggcc tctctcagtg gattaaggat ccggtattgc cctgagctgt agtgtaggtc 14040
acagacaggg cttggatccc gagttgctgt ggctgtggtg taggctggca gctatagctc 14100
caattcaatc cctagcctag aagcttccat tttccacagg tgcggcccca aaaagacaaa 14160
aagaaaaaaa gacacggggg ggaaaagtga cacccccccc atttaaaaga tattaggaga 14220
acccggttga aaagcactgg gagaaggtgc ccactagttg aaaggtataa ggggggccta 14280
tttgaaagat tctcggattt gctctgttaa aaagactctg aatatctgag ctgagaggtt 14340
agggaggagg ctgacttgac gtccttcctc agatgggtta ccttaagaat ttgtcaggat 14400
aaaagaaaaa atattacttg agacctttca gctccaaagc aaaggactct cctcctggct 14460
ggcaacagct ttctcaggca gctgagacac ttgcaagagc ggccagggac acaaataaga 14520
actggctgca aatacgaact catcatctga gcacccaggg aggtcttatc ctatcagggc 14580
gagaagccga gatgtgtgaa aggaactgaa atgaactcta gggtaaaaac ctagaggaat 14640
taagctcaca agcatcacat cggcccgccg cacaatttca ccagaaaatt ttatccccaa 14700
caaattcacc tttaaaatgg gaaaccgagt tctcttgtgg tgcagcagct taaggatcca 14760
gtattgtcac tgcagcagct tgggtcagtg ctgtccctgg cctgggtact tcctcatgcc 14820
acatgtgtgt ccaataaata taaataaaat gggaaacaga atctttcaaa cagagagaca 14880
aggggtgtca gaaagccaac ctctatctca taccccagct agattcacgt actgtatgtc 14940
cagagctcat tcttgcaagt acctacttta ttgaaaattc aaggaaatct taccccgaat 15000
gtaaggaaca ggagtctttt tctttcatat gcagttaagt tagaaaagct tgattaaaat 15060
atcttttcag aattcttctt cttttttttt ggctgtcccc aaggcatgtg gagttcccct 15120
ggccagggat caaatccaag ctacagctat gacctaatgt gtagctgcag taggccaggg 15180
attgaacctg tgtcccagtg ctcccaagag gccactgatc tgttgcacca cgatgggaac 15240
tcctcagaat tctgatgttt gtttgttggt tggtttttgt ctttttaggg ccccacctgc 15300
gacatatgga ggttcccagg ctaggggtca aatcagagtt gtaattgcct acaccacatc 15360
cacagcaatg ccagatccaa gctgtgtctg caacctacac cacagctcat ggcaacgcca 15420
gatccttacc ccactgactg aggccaggga tcgaatctga gtcctcatgg atactagtct 15480
cgtttccaat gagccatgtt gggaactccg gaattctgat cttaaacgaa agtcctcctg 15540
ggaggaactt gcatttctct tcctgtgcct ttgaaatata aatttcctac ctgatcttct 15600
tcaggctgtg tgcgtgtgtg tctgtgtctt ttgaaaactt ggtctctgcc atcaaaaggt 15660
acactcatag tggatatttc acagccaaac aaattgaaac caaaaacacc tctggctcga 15720
tcctagccag gacccagact ggggtttgaa cccatggttt tttaattaga atcactcacc 15780
tggtgtctgg acttactgaa attcaggtta tttttgtctc agtgcagaag gaattcaccc 15840
agagacaaag tgataggcaa gaaatagatt tattaagata ggacacttgt gagagatgca 15900
agcaggcagg caaggagact ccgccccgag gattaggtgg gctacatttt tatatggaaa 15960
gtggggaggg ggggaacact ttcttcctca ttttttgagt agacatcagg cttacagcat 16020
tagcgcctcc ttcgtatcca gtaagagaat atttgaccct atgaggtcaa actaggaatg 16080
tcatggtgct tattcaaatc agcagtagga tggtaacata tgctaacata tgttaaatca 16140
tctcaggttt ctgttatagt aagggtctcc ttctttggaa tgtcatcttt ccctttatta 16200
cctgtccttg agcctaagga tcattttctt gctgaacttg attgcaggca tcattttatc 16260
ttttgcttta atgacctgcg gcgtatttca tgcttttatt tgtgatttta tagttaagca 16320
agactgcttc gttccgcgac gttctgatag cttttttgaa agcgatcata aacttgcagt 16380
gagtggtctc ccaaagtgct ctaggttccc tcgctgtcta tggtccccca ctgggacttc 16440
tacaaccacc tgtgtaacta tcctactcta tctctatcac tcgggtccaa ttaacttgct 16500
agagtgactc acagatctca gagaaacatc ttacttacta aactgccagt ttcttataaa 16560
agtatataac tcagtaacag ccagatagaa gcaatgcata ggtcaaggga tgtgggacag 16620
gaccgccatt ctctctctgg gagcaccacc cagattcatc tccccgaatc tccatgttca 16680
cgcatccaga agctctatga accccatctt ttaggggttt catgaggact tcatgacata 16740
agtgtgttga tgaaatcatt agccattggt gattgaactc aacctccagc ctctctcccg 16800
tccctgaaaa ggggacattg gaggatgaaa ctccatcacc taggcggttc ccttggcaac 16860
cagcccccaa cctttggtgc tttccaagag tcacttcatc attggaacag aagacactgt 16920
cattgctctc ttcgcttagg aaattccaag gattttaaga gctaccggcc accaacaggg 16980
acaaagacca aatataaatt tcttcttatc aatcatagta tcacaagtct cacactgcac 17040
aggtgagcta aagagcagtc cagtgagttg aaggaaagct gtataccagt ttcaaggctg 17100
ccttcctctg aagccagcgc aagtcactgc caatgcctct ctgtaaagga agggtaccat 17160
gttttgcaca caatgtcaca tgatgaagcc acttgcccta catcccagga ttgtaagagg 17220
tagagagctt cagcagctgc tctcataact atgatgttat acagcctctg acaagggcag 17280
ccgcagcccc actggggaag gctgtgtccc tgtaggaaaa actgtatgtc cacatctctt 17340
gggcataaat atggtttgca tgccttaggc tggaccttcc attgcttgct ctatactatg 17400
aagaaagcat gtctcctctc tgagcttcag tggccccatc catagaatgg gtccatgtga 17460
gttctgcagc catcctggac ccaagctcca aggagaccct ctcagagaac aatccgtaac 17520
cagtcctctc tgccctgctt ccccacaggt acccgaccca agatgcggaa gctgtccagc 17580
ctcttcgttg tcctctgtgg gctactcgct ccatcctctg ctcaagaagt cctgtctcaa 17640
gtttctgaag gtgagacagc ccagccttgg gtcatgcctg gtcttgtcag acccataagc 17700
ctctcctaaa agtccaagcc attagccaat gttggttctt attagcatag tgcctgggac 17760
acagttaaca ccattaatca ttacccgctc ggatcatcac tctattacta tatttaatcc 17820
tcttctccaa gattcccctt tttcttgtct ccttgccttg cttcacctat caagatccac 17880
catcaaaccc ccacctgcct ctcctaccac ccacatgctg tccctcctgc cacagtagcc 17940
tcctcaggac attgacaccc caggatcttt gcacttgcta ttccccctgc ctggaacact 18000
cttcctccaa cgatcaacat agctgctccc ttccttcagg tctctgctaa aatttcgaac 18060
tttcatgagg ctgcaggttc catccctagc ctcgctcagt gggttaaaga tctggcattg 18120
ccatgagctg tggtgtaggt cacagacgca gctcagatcc ctcgttgcta cagccgcggt 18180
gtaggctggc agctatagct ctgattcaag tcctagcctg gaaacttcca tatgccatgg 18240
ggactgtcct aaaaagacaa aaaaaaaaaa aaaaaaatct gaagttttaa catctgacat 18300
agcccaggag ccccaacacc aggctacagc tatgcctgat gttctttctg actgccccgt 18360
gccaccccca gctggttgct aaatacagta aaactagtcc cttcccagca cttgtttgca 18420
cctccccacc cccatcccac cccgacccac ttgtctcttc attcacagct ctgtcctctc 18480
tctcctctca ccctcactgg aacacccgct ccacgagcta ttctcaccgg ctcagcccca 18540
tcccaggcag ggccccagca ctcagtaggc ccctataagt atttgctgaa tgttgtggcc 18600
atgagctgtg gtgtaggtcg cagatgcagc tcggatcgca tgtggctgtg gctgtggtgg 18660
agggtggcag cccctagcct gggaacctcc acatgccgca ggtgcggccc tgaaaagatt 18720
agaaaaaata tatctatttg ctgaatgact gtgtctgcaa cttggccccg cacagaggag 18780
gccagaaagg ctacaggaat gaaggggaaa tacctcatga gggacatgct cagaggtcaa 18840
gtttgcatct gagaatcttt agcatttaaa ggtctaattg catcggggtt gggggcaggt 18900
catgtttttc agctttgacc caagaactcc tcagcactgg ctttcttccc agcttgcaga 18960
tgatcgactt ccagggatca ctgaaaaatg tcttcagcca aattgcaggc accctggaca 19020
tcaacagcga cagcagtttc ttgtgagtgg tctctgatca ccagccccag gcccaggctg 19080
atctggtttg tctgtttgtt ggagctcgcc ccaggtttcc tgactctgca aagaccgttc 19140
cttctactaa cagcccttcc agcccctatg gcctgagacc cactggttgg aagaaattcc 19200
cacagtggtg gctgggggag gggcatgcag gagcttgggg gaggggtgcg ctacagacag 19260
aggttcagtc tctttgtctg ggaggaggat agcagtctct ccccctctct gagccccaag 19320
tcctgttcct gttaaatgac gaattagagc atctgccccc cccccgagga ctgctgtgat 19380
atcacaggaa gtaatagatg taaaatgctt gcatagctcc ccctcttagt aagactccaa 19440
tcagcagtaa caacaacctc tgcaataaca acgtgtaatc tattcggagt ccctgtcgtg 19500
gctcagcggg ttaagaacat gactggtatc catgagggcg tggggttgat ccctggcctt 19560
tctcagtggg tgaaggatcc agcattgcct tgagctgtgg tgttggtcaa agatgcggct 19620
tagattgcac attgctgtgg cagtggccta ggccagcagc tgcagctcca atttaacccc 19680
tagcctggga acttccatat gccatgggtg tggtccctaa aaagacaagt ggaaaacaac 19740
ataatattca atgtgatctg atcactagaa tgaagaagac cccagtccct ggtcctaaca 19800
catccaacgt cactgctgac tcccactctc agccccaccc tgcctgctcc tcccccttga 19860
cccagctcat cttcgataat gaccacatcc ccgccccacc ccatccagtt gctctgcccc 19920
aaaatcctga tcgtcctagg ctcctctctt tccgtcactc caacagcctg ttggcaaatc 19980
agccctacct tcaaaacaca cccaggcatc tgaccatttt gtccaccccc tctgatctga 20040
gccaccatcc tctccctggt gacttactgt tccagatccg tctgtttcca gtttaagccc 20100
ctcttctgca cttctattct attccactgg tatccagaag aatctaagag ataggtcagc 20160
ctctataact ctcctgccca aagcctccaa tgtcttctca ttgctttcag aaccaaaccc 20220
aaagtcctga tcattggccc acaaggctcc acgaaaccca gccctgactt cccctctccc 20280
ctcttctccg actgttctcc tctgacccac tgcccaccag ccacgccgag cctgtcactg 20340
ttcctcaaat aagccagaca agctgcagcc tcagggcctt tgcacttgct ctttgtctcg 20400
gtcctttatt ccctctggct gtccccatga cttgtaccct cacttcattc atgactctgg 20460
ccaaatgtca cctcccatga gaaggcttcc ctgactatcg tgtttaaaac tccactcttc 20520
ccaatattac cctctgtcac ctgatgcccc aaggcctttt cacagtacct atcacttggc 20580
atgttctata tgtcttcatt cactgggtta atgttttaca cacatacact ctttctaagc 20640
atgcactcta tatggctttc tatcttttcc ttactatttt gtcactcatg tattccagct 20700
ggcttgctac tgatcaaagg actaggcatg atcatcctga gctgcaataa gacaaaagct 20760
tctttctgct cagggctgga ttctcagagc tcataacaca tagtagctgt gcaataaatt 20820
ccatgattaa tgagcgaggg acaatctttg cctatcttca gcttctgctc atcaagggac 20880
ctgcctttgc gcaaagtggg gtccacctgc aggtggagcc acagctttcc cctgttgtgc 20940
tgtttcttta tgcaggttac cactgaggga cccttgactc catcagatct tcctccagaa 21000
ccccagcaat aaccaaccaa ccgagttact ggcggtattg tcattttctg tgcatgcaag 21060
gtaagcaaca cctgcgagtc ccatgtacag cttcatccgc ctttgcacag gtgtgcagtg 21120
ggggcgcgcg gggtgggggg cagcagccat aaataaaatg tgtccagcac catctcagaa 21180
agactgtgag ttgagacaaa acgcccactc cctctgggtc cagatcactc taagcgtaga 21240
agcaggaccc tgcagaacac ttgaatgctt ccatcctgtg gctcctctag agaaaggagg 21300
catgtttctt tcccctgctt catccaaaga cccccactct ttgagcccat tcagctctga 21360
tcgaatatga tttaagacga gaattcagga atctgctcta gaccccaaca ctccttggta 21420
ccaacaccta gagatgcctg gagatggttt cgtcggtccg catggtgtat ttttagagag 21480
gatgggcatt ttttcagttc atcacagctt tcacttctcc ctataaaacc aagccctggt 21540
ctgtttcatt catctccatt tcgagcctgg cccttgaggc agtgaatttt gagatctcta 21600
aaagagtact ttccaaggtc atagttgctc agttttgata ggatgccatg cttaaactca 21660
accatttcct ttctaactgt cccctaagcc aggggtgggg agtatcagtc acgtatcctg 21720
aatgcccctg gctggatgga tcacttcaag aaatctattc ctgtttccct gaatcagctc 21780
ttttgaaaaa tgaggaaagg accataatag agaaattccg agggtcctgt gcaaacatgg 21840
agaccctcat tctagaagta aatgggggcg tagaacgctc tgaaattctc tcctatcagg 21900
cttaattatt ccctcccacg gggtcctggg tcttaacctc ctcatatttc aagtgcagac 21960
aaatatcaaa gtccagcatc atttggagaa ggatataaat ggccgatacc tacttgcctt 22020
tgggaattgc atgctcagcc ctgagagtgt aaggatccaa cctggaagtt tgtaagttct 22080
ccatttttct ccttctggtt cctgttggtt atgacaaagc ctgtaatgag gcttcacctg 22140
aaagatgtcc ccggcaaaac agtaccttcc aaaatgagcc ttttgtgtac gtggggttgt 22200
gtattttata agtagaactt tatgttaata gagaatgcgt ttattacaaa aatagtattc 22260
cgttaacatg aaaagagatt caatgaacag cactgaggac aaagaaaacg gaatcatttg 22320
ataattggtt ggcatttagg ttatttctga actttttgct atcataagta atcctgagcc 22380
ttttgggaca gaaagatttt ccaaaaatag tgttataaat ggaatcattt ccttatggca 22440
gttttctaaa atcgctggaa aagtatcaac atttttatga cctttaaatt ttaagtcaca 22500
taatggcttt aagacattcc aagtcactta ccaaacactt ggccaataga actgggctaa 22560
gtgatgtgtt ctgagctggc attggtcggt ttcacttcat attcatcata ttcaatcaac 22620
gccagttata ccttctcaca atgtctctct ttagtaggta gaaaatatca tggatgccat 22680
tattttcttc ttctttttct ttgtaccaat agagcctgga cttcatcagt ctctcattag 22740
tctgtgatat tttctcttca gagtaaattt tttcagaggt ttgtcctttt attaactaaa 22800
attttagtgt ttccttctaa acttatagtc ttttacaatc ttccttggta agattttttt 22860
gaaaacttag gctgacatcc atgattaact gagtcctgtg gtcatccacg attaactgaa 22920
tccagggtat ctactctata ccctgtcttt gtcactcctg cattctttat tttgattcat 22980
ctcccaattt ctcactgttt cgaaaaagag ctgcatgtag aatcctttct gaacccctag 23040
gggtcggtaa tgtcctccag tcatgttcaa acataagtga gaatcaggcc agacatagcc 23100
cccttggctc agagccatct ccccccagga aggcttgaat aaggttcctg tgtcttttaa 23160
tgtgtcaaac aatcccattc tttgtgggaa gtcagagaca tcagactgca agtggtaaat 23220
taactgattt atataaaaag cttttataga ataaagacac aaacctgtcg ctgagaatta 23280
gacaggttta ccctcacctg taaggattag ggactgagtc aggaacacaa agatacctct 23340
tcactgttat ttcctgcttc cttctcatct ttctcaacta aacaatgatc ttgtaccgtc 23400
ccttccatga aggcatgacc atctggccaa ggagataagt tcaactgaag ttttgaataa 23460
tcagagaatt gggagagttg gccaaaggga acggagttgg ctagagagct ctgggaaggc 23520
tttctggggg aaaccagttg gctggacagt ccaggagaag aaaaacagaa tagacacaga 23580
ggagttcccc tgatggctca gcataaacga atctgactag tagccatgag gatgtgtgtt 23640
cgatccctgg ccttgctcag tgggttaagg atccagcatt gctgtgagct gtggtgcagg 23700
tcatagatgt ggctgggatc ctatgttgct gtggctgtgg cgtaggctgg aagctgcagc 23760
tccaattcaa cccctggccc aggaacttcc ataagctatg ggtgcggccc ttacaaacaa 23820
acaaaaaaca aaaatagaca cagagagaga aataaagggg aacaattata agggcaaggt 23880
gatttaagtg gaaagattaa ttttaagaag ccctaagtgg taggtgggag cctaagggca 23940
aggctgggtc acagagtgct atggaaacaa gggtagactt taagcaattt ggggtcagct 24000
gaacaaagaa atgaggtaaa gaaaggagag tttgggagaa aataatctgt cctactaact 24060
tctttcattc ttcatttaga acaagctgga tacaaacttt tcttctagga aatatagaga 24120
gaaccttgaa agacctgata attggtcatt tgggagaaaa agtaagtcta caaatccata 24180
ttacttcaga tttttggggt ttttttgtga gttttttttt gttttttttt tgtttttttt 24240
tttttgcctt ttcttgagct gctcctgcgg catatggagg ctcccaagct agaggtcgta 24300
tcagagctat agccaccagc ctacaccaga gccccagcaa cgtgggatcc aagctaagtc 24360
tgtgacccac accacagctc acagcaatgc cagatcctca acccactgag caaggccaag 24420
gatcgatctt gcaatctcat ggttcctagt tggattcatt aaccactgcg ccacaacggg 24480
aactcccata tttcttcagt ttttgagtcc caatgcccag agcttcgtag attggttctt 24540
tttttttttt ttctgtcttt ttgcctttcc tagggccgct cccgcagcat atggaggttc 24600
ccaggctagg ggtcgaatcg gagctgtaac caccggccta cgccagggcc acagccacgt 24660
gggatctgag ctgcgtctgt gacctacacc ccagctcatg gcaatgccgg atccttaacc 24720
cactgagcaa gggcagggat cgaacccaca agcttatggt tcctagtcgg attcgttaac 24780
cactgagcca cgacgggaac tcccttaagt tggttcttct taaactggaa ggaaatctcc 24840
cacggaggag aattggggag aaaggagttg gcatgtatta agttcccatt ctgtaaccag 24900
atatgggtga agtgctttac aaacatttaa ttctttgggt caaaaatcag catctgcgtg 24960
tctgcaggtg agttgataag cctttccaag acctggtttc cttatctgta aaatatgtag 25020
aagtggtcat aaaattgaca gctaatattc attaagctcc cagacactgt gcccggtcat 25080
ctaacatgag gtttcttatc ctttaaacaa tctcatactt taggtaacga tataatctcc 25140
agattccaga ttcaagaaca agtcccagag aagaatgact tacccaagtc gcatagttgc 25200
caaatggctc agctgacata tgatcccagg gcagtctgtt ccccaaaccc ctttacctgg 25260
ttgtccccta ggatgggttg gcttagaaga agaacttgcc ttgattactc ccattcaggc 25320
tccggttggt agagtcctcc tcctgactct tgcgtatttc ctcgtctttc aggtaagtcc 25380
cttcatgaat tcatggctct ataacctgag cccacaggtg tctagtgagc tgattagtaa 25440
gtgctggtag cgggaaggga cacgaaccct ctgggagaaa actccaactc ctccttcatc 25500
ttccagctcc tctgccctta tctcaccctg aacttctcct agaccaaaaa ccccttccct 25560
gctgagacag agtggtccct aataataaca atcctaatga actcagctgc tgtttattgc 25620
gagtcctctg cacctggccc agggataagc accctgcttg cactagccat tgaccctcta 25680
caactggctg agaatgtgct tgtttcaatt ctattctggt ccccaaagcc tagagaaact 25740
gaaatatgaa tggaggcttc gtgagaggag aaaagctgcc cgatacttct gttatcctcc 25800
tcctaaattt tattttattt tttacttttt gcttgttagg cctgcacctg tggcacatgg 25860
aagttcccag gctaggagtc aaattggaga tacagctggg gcctaagtca cagccacggc 25920
aattcaggat ctgagccaca tctgcaagcc acaccctagc tcacagtgat gccagattct 25980
taactcactg agtgaggcca gggattgaac tcgcatcctc atggatccta gttgggttca 26040
tttttgctga gccacagcag gaactcccac ctcctcctaa attttagatg ttaccattag 26100
gagagaaaat tggcctagtt tcagcccaga caaaaactgc ctcccttgga gtaatcttgt 26160
ggcatggcag gtcaaggatc tggcattgtc actgctgaag ctctggtctc tgctgtggtt 26220
caggttcaac ccctggccca ggaacattca catgctacag gcatggccaa aaacaaacaa 26280
acaaacaaac ctgcctcctt ccagtagtct ctagctccaa gcaccctaag tttcaacatc 26340
taccagaacc caaagcaaca aatgcaagat tgaatctcag caactgaggc cccagcggga 26400
atctgaaaac caatagaatt tactgggtca aaaggactct tccttctgtt gctgaaattt 26460
ggcctctgtc tgccggtgct gaatacaaac tcagagatag ggttttgggt gaaggagaaa 26520
aagacagctt catcgctttt cccggcaaag gaggccatag caggctagtg ccttaaagac 26580
tgtgccctct tggaatagat taggaggtgg ttttatagtt tggggagtgg agaatagggc 26640
caaagataag gatcaggttg tctttcaaag ctggagttta gtgacccgca agactggttc 26700
tggtggtcct tcttcccaga atgaagactg cttcatcaag tagttgttcc atttgttggg 26760
ggttttagtt ctacagaaag gctcaaagat attgttatgt atattccttg agcaggaacg 26820
gggaccctga ccccagagct acattattgt cttttgacgg ctcctccctg gtctctgtat 26880
cccctccctt ccctgattat actgtccttt ggaactcagg gaaggtcatg gaggccgagg 26940
cttagtccct aaaaacaaga aatggaggac acggaaaggc tcgtgtgccc aggagcccca 27000
cagggccctg cttggttaca tttccttcac tgtatacact acactagtcc taccaagtcc 27060
aaatgctaag gtttctcgcc cagcaggcaa ttttctggcc tctgtttgct gcttatgaga 27120
caggaagctc gaggcagtta atctggataa gacaaagctc ccaaagcttg gtccttaggt 27180
tccccaaagt aaatgcaaat agggttgctc aaacaacatc cacaccctgc tgataccgag 27240
ccaccacttt gaagaggtgc ccaggcaaac tggggactcc agctgctatc atgccaactt 27300
cctgcattgt ctcatttcaa tgcacagctc ttaagcgatt cacttgggaa ggatagttgt 27360
tcccaattgt ggatgaggaa aatgaggccc agcactgtca gtgaagtggt cagagacagt 27420
gggtgacaaa gagcctccag ccccagcctc ctatctccaa gcctttggct cactccctaa 27480
ccctaccctg ccttcaggta gaacagagga aactcttttg gcctcgctga tctgatttta 27540
ctccctgttt tgcattgtag gtttgctgct gcagtggggt gggtaccaaa ctgccatcta 27600
aatatcccca aagtgaagaa gtcacatttg tactaatcag gtaagacatc agagcacatt 27660
ctgtttctta gtgatggcac ttggccttta gggtcattat aaatgtgtca tttacttatt 27720
catttataaa atttcaattt aagcatctac tatgtgctag atactagaga tagagtcatg 27780
aacaatacaa agacccttct gccctgaggc ttgtattcta gttgggtgta ctaacaatga 27840
actaggaaat aaatacacag tcaactgaat tccagaggat ggtggatacc atgaagaaac 27900
cacagaagag gagagtgagt ggggtggagg cggggaggga ggcatcaaat taagagagca 27960
taggcaggca gaaggctctc tgaggaggag atctttcagc tgagctctga atgataaagc 28020
agcagttgtg tcagactttg ggagaagaac acaagcaaga ggatgagggt atgcagaagc 28080
ccagaggaag gaatgagctt tcctttttga aacaaagaaa ggagagcaaa gggaccgagg 28140
tgtggaaagg gctcagctat aggaagcatc tttacaatga tgatatttgg gagaaaagag 28200
gagattttct ataaaaagag atgccattgt tcatttatca tgcttttttt tttgcaacta 28260
ttctatgctc aataaacact ctgctcaagt gtcaccatct cgggtctctc aaaaaggctt 28320
atctgccact cccttcccta ctttattttt cttcataaca ttcattacta tctgacttcc 28380
tactaaatta cttgtttgtt ttttgtcccc ctactaggac ataaactgca taaaggcaag 28440
gaatttatta tttcattccc tgttttatcc tagttgtcaa ggttagaaag atcaatcaca 28500
catggcccat gacctcaaga gcccccagat tagtaaggaa taagacacat aaataggcaa 28560
ttttataggc agttatgtgc tctggttgat gttttgtgga aatagagagg caagatccag 28620
cagccctgtc atggaacttc caggaggttt cacagaggag gcaggattta aattgggcat 28680
ggaaaaatga ttacaagttt ctttgtggga aagacattca aagcagagga aacagcatgt 28740
gcagatacac aggagtaagg aagagtctgg tgctctggac catagggaga agatctgggt 28800
gcctggtcat aggagtcatg gggggtggag gtatcctcct ttaagggata aggataagtt 28860
tgtaatgaac tgtacacatg ctttctgggg aaactggtgc atgtgatgaa ttgagccatc 28920
aagaagttgt agcccatgat aagggacata agaacctgaa tgtcaacatc atattggatc 28980
tgcatccttg ggttgattag ctcagttatg caatattcta aacctatgct gttcaatgga 29040
aaaataacat aaaacatata taatttggag ttttctagta ataatatttt aaaggaacag 29100
caaatgggtt atattaattt cagcaataga tttttattta cctggtatat ctcaaatatc 29160
atgttgatac ataatcaata taaaaatatt aaaattcaat attctatttt gtactaagtc 29220
tttgatatct aatgtatatt ttacacttac agcacatttc aatttggact agctgtattt 29280
caagtgctaa tattcacatg tggttggtgg ctcctatatt ggacgatgca actctaattt 29340
gttgccagtg caaaattttc aaagcccaaa gcatcttgtt ttatcctaac cactatatca 29400
aggcatccat atatcataga agtcccaaac tatgtcccct ctattgtatt cagtccacag 29460
acatatttgg ttcaacctaa gaagtatttt ataaatatga atgagttgct aatattaaaa 29520
agtaagaaaa atgtcttttg atcaaccagg acatctggta atattttaca tctttgtggc 29580
ataaagtatg ccgtgaagag gaggatttat cttagttgat gagagtccag ggaacaatga 29640
gaaaatgtta tccagggata ggcggaggat ggatgtcggg acaagaatgt ggttcagcac 29700
cacggatagc agacaacagt accaggtgaa agcctgtgag ggagggggaa gggctctccc 29760
cactggctta cagcctcctt agaaagggta tgatgtcggc gaatatcttg attatctcct 29820
acagaatcag atcctgatgg ctgctgtcct ccctggaatg tcctacaagc tccaggccat 29880
caccgatgta catgttcatg ttgattccta tctggaattt cattacccac cccaaaccca 29940
aagatctcct acttctttga gtacggagac tatttccctg taaatcctca ccagaatcca 30000
taataaattt gttgcaactc tgaactgtgg actgcccagc ttccatggcg ccatgtgagc 30060
agggataaca ataactagag aggccgtggg cctggtcatg ataatgaaaa ttttcaaggt 30120
ggaggaagat gcttgggtta atttaggttt gacctcagaa atacctgaac actctggccc 30180
agagttttcc aaaagctaag gaacaagtct tcctgttttg tctgaacacc ttctgaactg 30240
attctgtgac cgtaggtagc tccctactct ggagcaccgt ctcctcactg tgaatttgag 30300
agtgttggtc taatcctcta aatgccctca ggctagatca gtctataatg ctttatggaa 30360
gaagtttgcc tttgccctgg taactccatt tttcctccct acaaacttcc aaactcctca 30420
aaagacatat catatggcta ctcactgtct ctatttcctt atcttcacct actgctcagt 30480
tttggataat atggcttctt cccccaactt ctcaaaatac ttgcccaaga attcctatgt 30540
cgctcagtgg aaatgaatcc gactagtatc ccttaggatg tgggttcaat ccctggcctc 30600
attcaagggg tgagggatcc gctgtcacca taagctgtgg tttaggtcac agatgtggct 30660
tggatccgga gttgctgtag ctgtgatgta ggctggcagc tgtagctcca attcgacccc 30720
agcctgggaa tttccatatg ctgcaggtgg ggtgtggccc taaaaagaca aaaaaaaaaa 30780
aaaaaaaaag aaagaaagaa agaaaaagaa gtttgcttcc aagttccata gggctccttt 30840
tcaaagatcc tcaaagctga agcttacccc aaccaccttc tcttgccact agacccataa 30900
ctaggttcag ttctcagcat gctccaggga aacaaaagta aaagataaaa taactcacat 30960
acttaaaata attacaattt tttgctgatt tatataagta gacgtggaga ctattgtagc 31020
atttgattat aaatatgtta gaccaaggaa gatagaatat aacattgagc acatttatga 31080
aaatgttttt aattaattga tgggtatatt tatgaaaggt tctggattta atactagatc 31140
acacaactaa aaatggcttt tatagttcac ggtgctgatt actggaaatc aaattccaca 31200
atgaccttca ttcagtgcgg ttgagatgct agaacttccc tggtttaata taaagtaagg 31260
cttcacttaa agtaagtcac ccaaaggctt agggaggtag gactattgga gtgggtttat 31320
cttttgtaaa catatgcgcc taccccacct cccacaccat tatatctcct gagggggcac 31380
agaggacaat atcttcacca aaaaattggg aaatacatta atgaggagag taacagtatc 31440
cttaaagagc tctgtgatgg ctttctgttg gaaatgctgc catgagatgg gctccctgat 31500
ttcaactggg atagtgaaac ataaaaataa cagagaggag gagttcatgc tgtggtgcag 31560
tggaaatgga tctgattagt atccatgagg atgcaagttg attcctggcc ttgctcagtg 31620
ggccacggat ccagcattgc catgagctgt ggtgcagatt gcagacacca ctcagatcct 31680
gtgttgctgt ggctgcggca taggctggcg actgtagctc taattcaacc tctagcctgg 31740
gaacttccat gcactgcagg tgcggcctta aaaatacaaa ggaaaaaaaa aaagtaacag 31800
aaaggaagtg gagagcttaa ctggcagaaa gaggatggca tgctcactgt aatgaatagc 31860
aaagatgaac tggccatcag aatatttggc aacttctaat tgatcacggt ttccctagaa 31920
aggaaataga tagtctatct actaaatgta agttgagaga gatcttcagg tttggaatcc 31980
agaaaactga cttgggttgc caaaatagag aatcagagcc tctcacacaa tgtccactta 32040
caagccagtt cagaatcagg aaagcattga ttgaaaggaa ggcaaagggt cctgaaatat 32100
tgccatagtg tatactgtaa atcttccttc aagtctctgc caaattgacc tgctgacatt 32160
tattagaatc atggtgtaca gtaagaaata cccaaaactt tggggaatat gagatactgg 32220
ctctgaattt atgctggttt ccaagaaccc caaatacctt catggtccat cagttaaagt 32280
gagtttatgg ttttcaagtg atagatgggg ttttggcaca agctggattc agagtgagcc 32340
tgatacttct ggggaaccac tctgcaatta tttccccagt taatgcatgt ataatttgaa 32400
tggacatgat tagcaaccgg cagaatcccc agttggctct ctgatccatg aagtaagagt 32460
tcttttagga gaaaatggca agcagaaact gctggacatt tctcttgtaa caggataata 32520
aaccaaaagc aaatgggaga attgaagaga caaaggccat catttagatc tgaggggaaa 32580
aaaagggtaa taatacttat gtgagtgaga gattgagaga aggagagaga gaatgaaaga 32640
aaaaggaact ggtttacatg attatagagg ttggttaagc aagataaaaa tccataaggc 32700
aagctatcag gaagggagaa tcaagggccg aggttgctat ccacaagaaa tattcttttt 32760
ctcctcttcc tactcctttt tcttcttttc tgcctctatc tctatccctc ccctcagcct 32820
tgcttgaaag gtcttctgat tgaattagcc ccacctaaat tatccaggat aatctcctct 32880
ccataaagcc atctcattat ggactttaat tatatctgaa aacttccttc agagtaacac 32940
ctagatttgc atctgattga atagggggac tatagcttag ccctgttgac gcatacaaaa 33000
aaaaaatcac agggagttcc cactgtggct caacggtaac aaattggact atatccacga 33060
ggatgcaggt ttgatctctg gtcttgctta gtgggttaag gatctggcac tgttgtgagc 33120
tgcagtgtag gttgcagaca cagctcagat ctggcattgc tttggctgtg gcataggcca 33180
gcagctgcag ctccaattcg tcccctaacc tggaaacttg catatgccac aggtgcagcc 33240
cttaaaagaa aaaaaaagaa aaaaaatcac agtaatgatc ccatccccac ctaacactgt 33300
taagtgttca cagtccacag gcctggcacc tggaatgcac gattgccttg gaaaacattt 33360
tttcgcccct ataccaagtt ataggaacca tcagaatcag cttgctttta cttggcagtg 33420
ctatcagcat accttcgtag ttttccttta ggctagtcag cactcctgct ctcagtttgc 33480
aaaaatcttg atcatcttga catcgcagaa cactatgtat gtccaatatg ttgttaacat 33540
tatgcctatt agattgatga gtgggaatta gcaagtattt aaatgcacta gcgaagtaca 33600
tgaaagcaag aaggtgagac aaaatcacat ggaaactctg gaccccaccg ctttgatgaa 33660
gtttctaaga ggatgatggt ttgaaacatg ttagggtatc ccttccaaga ttataacaac 33720
taattgttgg cccttacaat acatattctt aaaaaaacct caaggctcca tgggccattg 33780
tgatttttaa ggcaacatgc accataagta aaggtgctct tccgatacag ttactgaata 33840
actctcaagg catcagattt gagttggaat cagtaagaaa aatccctcca gcaagccatg 33900
caagtttctc tgccactttg tccttatgac ctagtcagat ccaattatgc ttttaaagta 33960
tctttagcgt gggggttctc tgtcatggct cagcaaaaca aatctgacta ggatccatga 34020
ggatgcaggt tccatccctg gccttgatca gtgggttaag gatctggtgt tgccgtgagc 34080
tctggtttag gtcacagagg tggcttgggt cccaagtttc tgtggctgtg gtgtaggccg 34140
gcaggtatag ctccaattcg acccctagcc tggggacctc catatgcggt gggtgcagtc 34200
ctaacaagac aaaaataaat aaataaataa ataaataaat aaataaataa ataaaaataa 34260
agtatcttta gcaaacaatg atgttgtctg gatgttctgg caaactctta agaagagaaa 34320
cagagcacag acttccagag ctttggagaa aagtactatc ctactgtgca gatgactctt 34380
gattttttaa caatagtttc tgtcttgcta ctgaatcctg gtagagttga ctatctaacc 34440
atgggacatt aaatgaccat gtgacctgag acgtcctcct gaactacacg tgatctgatc 34500
tatcaagttt taatactagg agtgtacagc aacaatcctg ctccaactga aaacataaaa 34560
gagactggtc ctgagctgta agtcacaagt aatttatagg agcaggggac tcagctatat 34620
ttgtaagaaa aaacacacta ttgttttcat ttatgttaca cacacaatac tctttatcac 34680
acactaactc tgacacttct gacaccaaat gtgtgtgttt ttcccacaca tcaagcaatc 34740
ccaggacacc agctgcggat cccacagtta aactcacttc taggagctcc ccttgtgcct 34800
cagcaggtta agaatcccac tagtatccat gaggatgtgg gttcaatccc tggcctggct 34860
cagtgggtta agtatctggt gtcaccgaaa gctgtggtga aggatgcaga tgtggctcag 34920
atctggtgtg gctgcggctg tggctatggc gcaggctggc agctgcagct ctgattcaac 34980
gccaagcctg gggatttcca catgccacag gcacagccct aaaaaaagga aaaacacaca 35040
ctaaaaaaac ccttaattct gacaccatct acctggagag agtgttaagg gcacaggtta 35100
tggttcagtc ctacaagaag accgcctcag ccttgatgca atgccagtca gacgctcagg 35160
ttgttgcttg tacttctgac ccgccggcta tagatcagag gttcccaaga cctgctcctt 35220
tggttctgtt cgttagtgag ggcaactcat aactcaggaa aatacttgac ttgctggatt 35280
accagtttat tataaaaaga agagccagat agttgagatg agtagggcac gtatgtggga 35340
aggtctgcag cattccatga gtctctgagc ccaccactct ccctgcacct gcccatgttc 35400
accaaaccca gaacctccaa accctctcct ttgggttttt atgagggctc cactacacag 35460
gcatggctgg ttgaattgtt ggccattgat ggctgattca atgtccagca tctttcccgt 35520
cctggaggtc agcaagatgg gacagaaagt tccagcaccc taatcacttg gttggttccc 35580
ctggcaacca gcccccatcc ttaggggatt tccaaaatcc acctcattaa cataaactca 35640
gttgtggttg aaaggggttt gttataacaa gacacccttt tcatctttat ggctctgaaa 35700
cttctttgaa aacgaatggc aaaagaccaa atattataac aaaagatgtg cctatgctgt 35760
cacctctctc attctccttc ttattcatat tggtctattg tgtcttctcg ccccctttcc 35820
tcatctccat aactagaatt ttttcagttt tattaatctg agagacaact ttttttcttt 35880
gttttcttag agctgcaccc ccagcataag gaagttccca ggctagaggt ccaatcagag 35940
ctgcagccac cagcctacac cacggccaca gcaacgcagg atcgagttgc gtctgcaacc 36000
taccccacag ctcacagcaa cgctggatcc tgagcgaggc caggaattga acccgagtcc 36060
tcatgaatac tagttgggat ctttactgct gagccatgac gggaactccc tgagagagac 36120
aacttttgat tttgttgatt ttctctatta tttttctgtg tctattttat tagtttctac 36180
tctgatcttt attacttcct tgcttctgct tgctttgggt ttacctgact ctagtttcca 36240
aagtctgatg tgtgcggtaa agagaaggac tcgagcgcat tccctccttt caactggctg 36300
acatttaagg ctgacattgt ggctctgaat gcctgctcat tgtggatgac ctgacttctt 36360
cctttcccct gagaacccct ctgccaggca ggaccctggg ctgcaagggc agatcctgcc 36420
tgcttgctgt ctagagcatt catggttatg gtgagacggg cagtcttcac tccccctggt 36480
acattgactc cagctcattg aaaacctttg atatgtgcta tgtgtgtccc ttggagactt 36540
tagtggcctc tttagaacat gagagggaca gggcaggctc aggagcccaa gaccaggggc 36600
aaaggctgtg agtgaattta tccagacaca cagtagagcc tcctagagcc aggcagcctg 36660
ggtttgaatc ctggctccac acttaacaac 36690
Claims (6)
1. Located on chromosome 17 of pig 36,699,989 ～ 36,736,679, its nucleotide sequence is shown in SEQ ID NO:159, and genome data pig genome sequencing alliance Sscofa 11.1, NCBI project 13421, GCF_000003025.6.
2. The use according to claim 1, wherein the safety site gene fragment is as set forth in SEQ ID NO: 1-2, SEQ ID NO:9 to 124, SEQ ID NO: 129-139, SEQ ID NO: 3-4, or a combination of one or more of the sequences shown in figures.
3. The construction method of the grain-saving environment-friendly pig is characterized by utilizing a nucleotide sequence shown as SEQ ID NO:1 and 2, or a recombinant cell, a crjspr/Cas 9 expression vector, or a recombinant cell, wherein the polycistronic BEXA has a nucleotide sequence as shown in SEQ ID NO: as shown at 158, the number of the cells,
wherein,
the homologous recombination vector comprises: the upstream homology arm, the insertion fragment and the downstream homology arm have nucleotide sequences shown in SEQ ID NO:1, the nucleotide sequence of the downstream homology arm is shown as SEQ ID NO:2, wherein the insert is the polycistronic BEXA;
the nucleotide sequence of the sgRNA is shown as SEQ ID NO:5 to SEQ ID NO: 143;
the CRISPR/Cas9 expression vector comprises the sgRNA;
the recombinant cell is a recombinant cell of a pig genome integrated exogenous gene, and is transformed or transfected with the CRISPR/Cas9 expression vector and/or the homologous recombinant vector.
4. The construction method according to claim 3, wherein the CRISPR/Cas9 expression vector and the homologous recombination vector are co-transfected and resuscitated porcine embryo fibroblasts, the transfected cells are subjected to limiting dilution and plating, positive clone cell clusters are screened after 12 to 15 days of culture, cells after screening marker genes are removed as nuclear transfer donor cells, a transgenic embryo is constructed by utilizing a somatic cell cloning technology, the reconstructed embryo is subjected to operation transfer into a surrogate sow body, the transgenic cloned pig is prepared, namely a grain-saving environment-friendly pig, the insertion fragment of the homologous recombination vector is the polycistronic BEXA, and the nucleotide sequence of the polycistronic BEXA is shown as SEQ ID NO: shown at 158.
5. A method for constructing a grain-saving environment-friendly pig breeding group, which is characterized in that the safe site gene fragment and the nucleotide sequence of the safe site gene fragment are shown in SEQ ID NO:1 and 2, a homologous recombination vector, an sgRNA, a CRISPR/Cas9 expression vector, or a recombinant cell, or one or more of them
Wherein,
the homologous recombination vector comprises: the upstream homology arm, the insertion fragment and the downstream homology arm have nucleotide sequences shown in SEQ ID NO:1, the nucleotide sequence of the downstream homology arm is shown as SEQ ID NO:2, wherein the insert is the polycistronic BEXA;
the nucleotide sequence of the sgRNA is shown as SEQ ID NO:5 to SEQ ID NO: 143;
the CRISPR/Cas9 expression vector comprises the sgRNA;
the recombinant cell is a recombinant cell of a pig genome integrated exogenous gene, and is transformed or transfected with the CRISPR/Cas9 expression vector and/or the homologous recombinant vector.
6. The method for constructing a grain-saving and environment-friendly pig breeding group according to claim 5, comprising the following steps:
s1, selecting at least 6 boars without common ancestor within three generations as boar progenitors; selecting sows with the same number of first three or less generations and without common ancestor from sows as sow progenitors, and taking the selected sow progenitors as mating objects of the above boar progenitors;
S2, selecting the sow progenitors and the boar progenitors one by one, wherein no common ancestor exists in 3 generations between the sow progenitors and the boar progenitors to be bred, and taking out embryos when the embryos are pregnant for 25 to 35 days old;
s3, after three times of washing by DMEM, digesting the tissue to be fluffy, adding serum for plating overnight, adding the tissue to a high-sugar DMEM culture solution containing 10 to 14% (V/V) FBS for maintenance culture for 2 to 3 days, obtaining embryo fibroblasts of offspring of each pair of sow progenitors and boar progenitors, and identifying the sex;
s4, constructing grain-saving environment-friendly pigs by embryo fibroblasts of offspring of each pair of sow progenitors and boar progenitors according to the construction method of claim 3 or 4, and producing transgenic cloned boars and sows with the number of families not less than 6 as F0 transgenic pig groups;
s5, mating the F0 transgenic cloned boars and sows of each family with F0 transgenic boars or sows of other families respectively, propagating to obtain F1 transgenic grain-saving environment-friendly transgenic pigs, selecting transgenic pig homozygotes boars and sows, and constructing to obtain grain-saving environment-friendly pig breeding core groups.
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