Expression elements

Abstract

genetic elements capable of improving the levels of expression of operably-linked transcription units. In particular, said genetic elements are derived from the 5' untranslated regions of ribosomal protein genes and may comprise a CpG island. Also provided are vectors and host cells comprising said genetic elements and methods of use to obtain high levels of recombinant gene expression.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]This application claims priority from GB Application # GB 0509965.0, filed May 17, 2005 and U.S. Provisional Patent Application No. 60/682,277, filed May 18, 2005, the contents which are hereby incorporated by reference in full.

BACKGROUND

[0002]The present invention relates to polynucleotides comprising elements conferring improved expression on operably-linked transcription units. These elements are naturally associated with the promoter regions of ribosomal protein genes and, in recombinant DNA constructs, confer high and reproducible levels of gene expression. The present invention also relates to vectors comprising such polynucleotide sequences, host cells comprising such vectors and use of such polynucleotides, vectors or host cells in therapy, for production of recombinant proteins in cell culture and other biotechnological applications.

[0003]The current model of chromatin structure in higher eukaryotes postulates that genes are organised in "domains" (Dillon, N. & Grosveld, F. Chromatin domains as potential units of eukaryotic gene function. Curr. Opin. Genet. Dev. 4, 260-264 (1994); Higgs, D. R. Do LCRs open chromatin domains? Cell 95, 299-302 (1998)) Chromatin domains are envisaged to exist in either a condensed, "closed", transcriptionally silent state, or in a de-condensed, "open" and transcriptionally competent configuration. The establishment of an open chromatin structure characterised by increased DNasel sensitivity, DNA hypomethylation and histone hyperacetylation, is considered a pre-requisite to the commencement of gene expression.

[0004]The open and closed nature of chromatin regions is reflected in the behaviour of transgenes that are randomly integrated into the host cell genome. Identical constructs give different patterns of tissue-specific and development stage-specific expression when integrated at different locations in the mouse genome (Palmiter, R. D. & Brinster, R. L. Ann. Ref. Genet. 20, 465-499 (1986); Allen, N. D. et al. Nature 333, 852-855 (1988); Bonnerot, C., Grimber, G., Briand, P. & Nicolas, J. F. Proc. Natl. Acad. Sci. USA 87:6331-6335 (1990)).

[0005]The chromatin domain model of gene organisation suggests that genetic control elements that are able to establish and maintain a transcriptionally competent open chromatin structure should be associated with active regions of the genome.

[0006]Locus Control Regions (LCRs) are a class of transcriptional regulatory elements with long-range chromatin remodelling capability. LCRs are functionally defined in transgenic mice by their ability to confer site-of-integration independent, transgene copy number-dependent, physiological levels of expression on a gene linked in cis, especially single copy transgenes Fraser, P. & Grosveld, F. Curr. Opin. Cell Biol. 10, 361-365 (1998); Li, Q., Harju, S. & Peterson, K. R. Trends Genet. 15: 403-408 (1999). Crucially, such expression is tissue-specific. LCRs are able to obstruct the spread of heterochromatin, prevent PEV (Kioussis, D. & Festenstein, R. Curr. Opin. Genet. Dev. 7, 614-619 (1997)) and consist of a series of DNase I hypersensitive (HS) sites which can be located either 5' or 3 of the genes that they regulate (Li, Q., Harju, S. & Peterson, K. R. Trends Genet. 15: 403-408 (1999)).

[0007]The generation of cultured mammalian cell lines producing high levels of a therapeutic protein product is a major developing industry. Chromatin position effects make it a difficult, time consuming and expensive process. The most commonly used approach to the production of such mammalian "cell factories" relies on gene amplification induced by a combination of a drug resistance gene (e.g., DHFR, glutamine synthetase (Kaufman R J. Methods Enzymol 185, 537-566 (1990)) and the maintenance of stringent selective pressure. The use of vectors containing LCRs from highly expressed gene domains, using cells derived from the appropriate tissue, greatly simplifies the procedure, giving a large proportion of clonal cell lines showing stable high levels of expression (Needham M, Gooding C, Hudson K, Antoniou M, Grosveld F and Hollis M. Nucleic Acids Res 20, 997-1003 (1992); Needham M, Egerton M, Millest A, Evans S, Popplewell M, Cerillo G, McPheat J, Monk A, Jack A, Johnstone D and Hollis M. Protein Expr Purif 6, 124-131 (1995).

[0008]However, the tissue-specificity of LCRs, although useful in some circumstances, is also a major limitation for many applications, for instance where no LCR is known for the tissue in which expression is required, or where expression in many, or all, tissues is required.

[0009]U.S. Pat. No. 6,689,606 and, co-pending patent application WO 00/0539, incorporated by reference herein, describe elements that are responsible, in their natural chromosomal context, for establishing an open chromatin structure across a locus that consists exclusively of ubiquitously expressed, housekeeping genes. These elements are not derived from an LCR and comprise extended methylation-free CpG islands.

[0010]In mammalian DNA, the dinucleotide CpG is recognised by a DNA methyltransferase-enzyme that methylates cytosine to 5-methylcytosin. However, 5-methylcytosine is unstable and is converted to thymine. As a result, CpG dinucleotides occur far less frequently than one would expect by chance. Some sections of genomic DNA nevertheless do have a frequency of CpG that is closer to that expected, and these sequences are known as "CpG islands". As used herein a "CpG island" is defined as a sequence of DNA, of at least 200 bp, that has a GC content of at least 50% and an observed/expected CpG content ratio of at least 0.6 (i.e. a CpG dinucleotide content of at least 60% of that which would be expected by chance) (Gardiner-Green M and Frommer M. J Mol Biol 196, 261-282 (1987); Rice P, Longden I and Bleasby A Trends Genet 16, 276-277 (2000).

[0011]Methylation-free CpG islands are well-known in the art (Bird et al (1985) Cell 40: 91-99, Tazi and Bird (1990) Cell 60: 909-920) and may be defined as CpG islands where a substantial proportion of the cytosine residues are not methylated and which usually extend over the 5' ends of two closely spaced (0.1-3 kb) divergently transcribed genes. These regions of DNA are reported to remain hypomethylated in all tissues throughout development (Wise and Pravtcheva (1999) Genomics 60: 258-271). They are often associated with the 5 ends of ubiquitously expressed genes, as well as an estimated 40% of genes showing a tissue-restricted expression profile (Antequera, F. & Bird, A. Proc. Natl. Acad. Sci. USA 90, 1195-11999 (1993); Cross, S. H. & Bird, A. P. Curr. Opin, Genet. Dev. 5, 309-314 (1995) and are known to be localised regions of active chromatin (Tazi, J. & Bird, A. Cell 60, 909-920 (1990).

[0012]An `extended` methylation-free CpG island is a methylation-free CpG island that extends across a region encompassing more than one transcriptional start site and/or extends for more than 300 bp and preferably more than 500 bp. The borders of the extended methylation-free CpG island are functionally defined through the use of PCR over the region in combination with restriction endonuclease enzymes whose ability to digest (cut) DNA at their recognition sequence is sensitive to the methylation status of any CpG residues that are present. One such enzyme is HpaII, which recognises and digests at the site CCGG, which is commonly found within CpG islands, but only if the central CG residues are not methylated. Therefore, PCR conducted with HpaII-digested DNA and over a region harbouring HpaII sites, does not give an amplification product due to HpaII digestion if the DNA is unmethylated. The PCR will only give an amplified product if the DNA is methylated. Therefore, beyond the methylation-free region HpaII will not digest the DNA a PCR amplified product will be observed thereby defining the boundaries of the "extended methylation-free CpG island".

[0013]It has been shown (WO 00/05393) that regions spanning methylation-free CpG islands encompassing dual, divergently transcribed promoters from the human TATA binding protein (TBP)/proteosome component-B1 (PSMBI) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2)/heterochromatin protein 1 Hsγ (HP1^Hsγ) gene loci give reproducible, physiological levels of gene expression and that they are able to prevent a variegated expression pattern and silencing that normally occurs with transgene integration within centromeric heterochromatin.

[0014]It is known that methylation-free CpG islands associated with actively transcribing promoters possess the ability to remodel chromatin and are thus thought to be a prime determinant in establishing and maintaining an open domain at housekeeping gene loci (WO 00/05393) and that such elements confer an increased proportion of productive gene delivery events with improvements in the level and stability of transgene expression.

[0015]Ribosomes are large RNA and protein complexes responsible for the translation of mRNA into polypeptides. Each ribosome is comprised of 4 ribosomal RNA (rRNA) molecules and large number of ribosomal proteins (currently thought to be 79 in mammalian cells). Ribosomal proteins have functions including facilitation of rRNA folding, protection from cellular ribonucleases, and co-ordinating protein synthesis. Some ribosomal proteins have additional extraribosomal functions (Wool, 1996, TIBS 21: 164-165). Given the structural and functional similarities of ribosomes across species, it is unsurprising that the amino acid sequence conservation of ribosomal proteins is high, and among mammals the sequences of most ribosomal proteins are almost identical (Wool et al, 1995, Biochem Cell Biol 73: 933-947.

[0016]Two ribosomal proteins appear atypical in that they are expressed in the form of propeptides (carboxy-extension proteins) fused to ubiquitin. Ubiquitin is a highly conserved 76-residue polypeptide involved in a variety of cellular functions, including the regulation of intracellular protein breakdown, cell cycle regulation and stress response (Hershko & Ciechanover, 1992, Annu Rev Biochem 61: 761-807; Coux et a/, 1996, Annu Rev Biochem 65: 801-847).

[0017]Ubiquitin is encoded by two distinct classes of gene. One is a poly-ubiquitin gene encoding a linear polymer of ubiquitin repeats. The other comprises genes encoding natural fusion proteins in which a single ubiquitin molecule is linked to the ribosomal protein rps27A or rpL40 (Finley et al, 1989, Nature 338: 394-401; Chan et al, 1995, Biochem Biophys Res Commun 215: 682-690; Redman & Burris, 1996, Biochem J 315: 315-321).

[0018]The common structural features of ribosomal protein promoters are discussed by Perry (2005, BMC Evolutionary Biology 5: 15). The promoters may be classified according to the nature of the TATA box motifs, number and type of transcription factor binding sites and location of AUG start codons. However, such classification does not appear to predict promoter strength and evidence suggests that several such promoters tested have equivalent transcriptional activity as measured by expression of a linked reporter gene (Hariharan et al, 1989, Genes Dev 3: 1789-800).

[0019]U.S. Pat. No. 6,063,598 discloses the hamster-ubiquitin/S27a promoter its use to drive high level production of recombinant proteins. However, there is no suggestion of its use to enhance the expression of a gene primarily transcribed from a further promoter (i.e one other than hamster-ubiquitin/S27a promoter).

[0020]US application US 2004/0148647 discloses a reporter assay using an expression vector comprising a hamster ubiquitin/S27A promoter functionally linked to a gene for a product of interest and a fluorescent protein reporter. Again, the application only discloses constructs in which transcription of gene of interest is from the hamster-ubiquitin/S27a promoter itself.

[0021]It remains an objective in the field of recombinant gene expression to obtain higher and more reliable levels of expression, particularly for in vivo and ex vivo therapeutic applications and for in vitro recombinant protein production.

SUMMARY OF THE DISCLOSURE

[0022]Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

[0023]Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0024]Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0025]As used herein, a promoter region is defined as being a genomic nucleotide sequence consisting of a promoter and transcriptional start site together with 5 kb of 5' sequence upstream of the transcriptional start site and 500 bp 3' sequence downstream of the distal end of the first exon.

[0026]A 5' untranslated region means a region 5' of the translational start encoded in the genomic or cDNA sequence. It is taken to include all upstream regulatory elements. A 5' upstream sequence is used to mean sequence 5' to the transcriptional start encoded in the genomic sequence.

[0027]As used herein, `transcribable nucleic acid` means a nucleic acid which, when operably linked to a functional promoter and other regulatory sequences, is capable of being transcribed to give a functional RNA molecule, such as mRNA. Such sequences may comprise open reading frames, which encode translatable polypeptide sequences. Alternatively, the functional RNA may have another function, such as ribosomal RNA, ribozymes or antisense RNA.

[0028]`Gene` is commonly taken to means the combination of the coding region of the transcribable nucleic acid, together with the promoter from which it is transcribed and other regulatory sequences such as enhancers and 3' polyadenylation signals. In genomic DNA genes also contain introns. `Transcription unit` is sometimes used to describe a functional combination of at least a promoter and minimal regulatory sequences with a transcribable nucleic acid, often derived from cDNA from which the introns have been spliced out. `Cistron` is defined as a nucleic acid encoding a single polypeptide together with functional initiation and termination signals. `Transgene` implies a gene that has been transferred from one genome to another, although the term may be more loosely applied to any gene or even transcribable nucleic acid comprised in a recombinant DNA construct such as a vector.

[0029]Promoter and enhancer are terms well known in the art and include the following features which are provided by example only, and not by way of limitation. Promoters are 5', cis-acting regulatory sequences directly linked to the initiation of transcription. Promoter elements include so-called TATA box and RNA polymerase initiation selection (RIS) sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.

[0030]In simple terms, promoters are directional elements that act to initiate transcription of sequences situated less than 100 (and usually less than 50) nucleotide base pairs (bp) downstream. They contain a number of short consensus nucleotide sequences that act as binding sites for various proteins that participate in the initiation of transcription and the assembly of a multi-subunit complex known as the pre-initiation complex (McKnight and Tjian, 1987, Cell 46: 795-805). In most genes, this occurs at a very widely conserved sequence known as the TATA box (TATAAA) to which the TATA box-binding protein (TBP, a subunit of the general transcription factor TFIID) binds. There follows an ordered assembly of more than ten further transcription factors to finally form the PoI II holoenzyme complex. RNA transcription actually starts at an initiator site about 25-30 bases downstream (Breathnach and Chambon, 1981, Annu Rev Biochem 50: 349-393) to which TBP also binds.

[0031]Most functional promoters contain further upstream promoter elements (UPEs), of which the most highly conserved are the CAAT box (CCAAT, the binding site for the transcription factors CBF, C/EBP and NF-1), about 70-200 bp upstream, and the GC box (GGGCGG, binding site for the general transcription factor Sp-1) a similar distance upstream. Although basal levels of transcription occur from the TATA box alone, for most promoters at least the CAAT and GC boxes are required for optimal levels of transcription.

[0032]Enhancers are sequences that act non-directionally to increase transcription from promoters situated locally but not necessarily immediately adjacent (up to several kilobases away (Kadonaga (2004) Cell 116: 247-257). Enhancers contain short (8-12 bp) consensus sequences representing the binding sites for a wide range of transcriptional activator proteins (Ondek et al, 1988, Science 236: 1237-1244) including some, such as NF-1 and SP-1 that are also associated with promoter elements. These sequences are often duplicated in tandem or inverted repeats.

[0033]In some natural transcription units, including the very active immediate/early gene transcription units of many DNA viruses such as cytomegalovirus, enhancer and promoter elements may be functionally combined into what is effectively one extended upstream element.

[0034]Promoters may be regulated, being responsive to cell type, temperature, metal ions or other factors; or constitutive, giving transcription that is unresponsive to such factors. For many purposes a strong, constitutive promoter giving consistent, high, levels of transcription in many, if not all, cell types is highly advantageous. For many years the enhancer/promoter element driving immediate/early gene expression in human cytomegalovirus has been very widely used for driving such expression of heterologous genes in eukaryotic expression vectors (Foecking & Hoffstetter, 1986, Gene 45: 101-105).

[0035]It was hypothesised that promoter regions of ribosomal protein genes might have useful activity in boosting and stabilising expression of linked transgenes and that the regulatory regions from highly expressed genes might be more likely to contain elements that are very effective at maintaining chromatin in a transcriptionally active conformation. The linking of such elements to a heterologous promoter might then generate a more open chromatin environment surrounding that promoter, resulting in increased expression. It will be understood by one of skill in the art that promoters derived from ribosomal protein genes are to be distinguished from ribosomal promoters, which are RNA polymerase Type I dependent promoters from which rRNA is transcribed.

[0036]To test the hypothesis, RNA was obtained from exponentially growing CHO-K1 and NS0 cell lines and a microarray analysis against 13,443 murine genes was performed. We limited our analysis to elements having a high CpG island content and a likelihood of bi-directional promoters. Using criteria based on the minimal effective sequence from the hnRNPA2 regulatory region, approximately 3 kb of DNA from a selection of these genes was amplified by PCR from NS0 genomic DNA. These sequences were then cloned into EGFP expression vectors and transfected into CHO-K1 along with hnRNPA2 control versions of the same vector.

[0037]It was found that sequences derived from the promoter regions of two ribosomal proteins gave consistently high levels of expression of the heterologous reporter sequence in the assay used. In each case, the promoter region comprised a GC-rich sequence extending from the 5' region upstream of the actual promoter elements well into the first exon and, indeed into the first intron. This GC-rich sequence fulfilled the criteria of being an extended CpG island, as defined herein as extending over 300 bp.

[0038]Accordingly, the invention provides an isolated polynucleotide comprising

[0039]a) an element comprising at least 500 contiguous nucleotides from the promoter region of a ribosomal protein gene

[0040]b) a heterologous promoter

[0041]c) a transcribable nucleic acid sequence adjacent said heterologous promoter

wherein the transcribable nucleic acid sequence is transcribed from said heterologous promoter and the level of said transcription is enhanced by said element.an element. Preferably said element comprises more than 1 kb and most preferably more than 3 kb 5' untranslated sequence from a ribosomal protein gene.

[0042]The contiguous nucleotides are selected from the promoter area, which extends from a point 5 kb upstream (5' relative to the sense strand) of the transcriptional start site to a point 500 bp downstream (3' relative to the sense strand) of the distal (3') end of the first exon.

[0043]Preferably said ribosomal protein gene is selected from the list consisting of RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13; RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL28, RPL29, RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL41, RPLP0, RPLP1 and RPLP2 and their orthologues. More preferably it is RPS3 or RPS11.

[0044]In one preferred embodiment the element comprises a CpG island, preferably an extended CpG island of at least 300 bp and more preferably 500 bp. Preferably the CpG island is unmethylated. It is also preferred that said element comprises the promoter from a ribosomal protein gene and from which transcription of the ribosomal protein gene is naturally initiated. Such a promoter is often referred to as the endogenous promoter. In a preferred embodiment, the element further comprises or more exons of said ribosomal protein gene.

[0045]It is preferred that the ribosomal protein gene is a mammalian gene, although such genes and their promoters and 5' upstream sequences are highly conserved across species and might alternatively be an insect, nematode or yeast gene. Preferably, however, it is a human or rodent gene and most preferably it is a mouse gene.

[0046]In a highly preferred embodiment, the isolated polynucleotide of the invention comprises nucleotides 38 to 3154 of the mouse rps3 nucleotide sequence depicted in SEQ ID NO:1. Alternatively it comprises nucleotides 12 to 3032 of the mouse rps11 nucleotide sequence as listed in SEQ ID NO:2.

[0047]In one aspect, in addition to the element described, the polynucleotide further comprises a promoter not naturally associated with said element from a ribosomal protein gene. In this embodiment a heterologous promoter (distinct from the endogenous promoter which may or may not be present in the first element) is situated in an adjacent and operably-linked position downstream from the element containing ribosomal protein gene-derived 5' sequence. In this arrangement, expression directed by this heterologous promoter is enhanced by the effect of the ribosomal protein gene element.

[0048]In one embodiment, said promoter is a constitutive promoter, more preferably selected from the list consisting of the cytomegalovirus early/immediate promoter, SV40, EF-quadrature, Rous sarcoma virus (RSV) LTR, or HIV2 LTR or combinations of sequences derived therefrom. More preferably the promoter is a CMV immediate/early promoter. Most preferably it is the mouse or guinea pig CMV immediate/early promoter.

[0049]Alternatively, said promoter may be a tissue-specific promoter, which directs expression in a limited range of tissues. Such promoters are well-known in the art and include those from quadrature-globin, the quadrature and quadrature immunoglobulin light chains, immunoglobulin heavy chain, desmin, tyrosinase, CD2, IL-3, myosin light chain, human melanoma inhibitory activity gene promoter and keratins. In a particularly preferred embodiment the promoter is a tumour-selective promoter, which directs-expression preferentially one or more tumour types. Examples of such promoters include those from carcino-embryonic antigen (CEA), prostate-specific antigen (PSA), cyclooxygenase-2 (COX-2), alpha-fetoprotein (AFP), tyrosinase, and T-cell Factors 14 (TCF)-based promoters.

[0050]The transcribable nucleic acid may encode any useful polypeptide for in vitro expression and is preferably selected from the list consisting of an antibody, a functional epitope-binding fragment of an antibody, growth factor, cytokine, protein kinase, soluble receptor, membrane-bound receptor, or blood clotting factor. Alternatively, the transcribable nucleic acid may encode a therapeutic gene of use for in vivo or ex vivo gene therapy. Such a therapeutic nucleic acid may act by replacing or supplementing the function of a defective gene causing a disease such as cystic fibrosis, thalassaemia, sickle anaemia, Fanconi's anaemia, haemophilia, severe combined immunodeficiency (SCID), phenylketonuria (PKU), alpha-1 antitrypsin deficiency, Duchenne muscular dystrophy, ornithine transcarbamylase deficiency or osteogenesis imperfecta. Alternatively, it may encode a cytotoxic agent or prodrug-converting enzyme selectively expressed in a target cell, such as a malignant cancer cell, in order to kill it. Such applications, and many others, are well-known to those of skill in the art and the relevance of the current invention in enhancing the expression of therapeutic nucleic acids will be clear to such skilled practitioners.

[0051]In another aspect, the invention provides a vector comprising the polynucleotide of invention as disclosed-above. Preferably said vector is an expression vector adapted for eukaryotic gene expression.

[0052]Typically said adaptation includes, by example and not by way of limitation, the provision of transcription control sequences (promoter sequences) which mediate cell/tissue specific expression. Adaptations also include the provision of selectable markers and autonomous replication sequences which both facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host. Vectors which are maintained autonomously are referred to as episomal vectors. Episomal vectors are desirable since they are self-replicating and so persist without the need for integration. Episomal vectors of this type are described in WO98/07876.

[0053]Adaptations which facilitate the expression of vector encoded genes include the provision of transcription termination/polyadenylation sequences. This also includes the provision of internal ribosome entry sites (IRES) which function to maximise expression of vector encoded genes arranged in bicistronic or multi-cistronic expression cassettes.

[0054]These adaptations are well-known in the art. There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y. and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach Vol III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0055]The vector may be an episomal vector or an integrating vector. Preferably, the vector is a plasmid. Alternatively, the vector may be a virus, such as an adenovirus, adeno-associated virus, a herpesvirus, vaccinia virus, lentivirus or other retrovirus.

[0056]Alternatively, such a vector may comprise

[0057]a) an element comprising at least 500 contiguous nucleotides from the promoter region of a ribosomal protein gene

[0058]b) a heterologous promoter

[0059]c) a multiple cloning site

wherein a transcribable nucleic acid sequence inserted into said multiple cloning site is capable of being transcribed from said heterologous promoter and the level of said transcription is enhanced by said element.

[0060]In a further aspect the invention provides a host cell comprising the isolated polynucleotide or vector as herein described. Preferably said host cell is a mammalian cell, more preferably selected from the list consisting of CHO, NS0, BHK, HeLa, HepG2.

[0061]Also provided by the invention is a method of expressing a polypeptide comprising inserting expression vector comprising the polynucleotide of the invention into an appropriate host cell as herein described and culturing said host cell in suitable conditions to allow expression. Preferably said polypeptide is a therapeutically useful polypeptide.

[0062]In a further aspect the invention provides a pharmaceutical preparation comprising the polynucleotide, vector or host cell as herein described and a pharmaceutically acceptable carrier, excipient, buffer or medium.

BRIEF DESCRIPTION OF THE DRAWINGS

[0063]FIG. 1 shows a plasmid map of vector rps3-1005-EGFP (see Example 1).

[0064]FIG. 2 shows a plasmid map of vector rps11-1005-EGFP (see Example 2).

[0065]FIG. 3 shows the expression of the EGFP reporter as expressed by various rps3 constructs in CHO-K1 cells analysed by FACS analysis, 8 days post transfection. Figure A shows mean fluorescence, and Figure B a indicates the percentage of cells expressing the reporter gene to a detectable level (% positive cells). See Example 1.

[0066]FIG. 4 shows reporter gene expression of rps11 constructs in CHO-K1 cells 7 days post-transfection (analysed by FACS). A and C are total counts, B to E are results based on just the expressing cells within the population. Figure A shows the mean fluorescence of cells in the stably selected pool, and Figure B shows percentage (%) positive cells. See Example 2.

[0067]FIG. 5 shows the expression level of a reporter gene in stably transfected NS0 cells when it is driven by the hCMV promoter with no additional elements present or when either an 8 kb hnRNPA2 or a 3 kb RPS3 element are placed immediately 5' to the hCMV promoter. Figure A shows the mean fluorescence intensity of the stable pools at 28 days and Figure B shows the percentage (%) positive cells.

[0068]FIG. 6 shows similar data to FIG. 5 for rps11 constructs. Figure A shows the mean fluorescence intensity values for stable pools generated with either hCMV driven constructs or identical constructs with a 8 kb hnRNPA2 or 3 kb RPS3 elements immediately 5' to the promoter. Figure B shows the percentage of cells within the pool expressing the reporter gene (percentage (%) positive cells).

DETAILED DESCRIPTION

Materials and Methods

[0069]Microarray analysis.

[0070]Total RNA was extracted from ˜80% confluent CHO-K1 cells using RNeasy RNA extraction kit (Qiagen, Crawley, UK) according to the manufacturers protocol. Total RNA (2 quadratureg/quadraturel) was subjected to microarray expression analysis using the mouse 70-mer oligonucleotide library (Operon V.1) representing 13,443 known transcripts. The University of Cincinnati, Genomics and Microarray Laboratory undertook Microarray analysis according to referenced protocols (http://microarray.uc.edu).

[0071]Gene transcript sequences were ranked according to increasing fluorescence. Since our previous study detailed the HNRPA2B1/CBX3 loci as a chromatin-remodelling element and conferring benefit to hCMV the HNRPA2 transcript was identified as the baseline expression level. However, using the available microarray analysis the HNRPA2 transcript was barely detectable. Since the expression level of the HNRPA2 transcript was minimal, using HNRPA2 as our reference would have identified 3829 sequences for potential analysis. Therefore, 7 sequences from the top 2% (76 sequences) of the ordered, expressed transcripts were identified according to the criteria of containing a CpG island and one or more putative/known transcriptional start sites (see Table 1). CpG islands position, size and GC: CG ratios were verified using GrailEXP (http://compbio.ornl.gov). Putative/known transcriptional starts sites were identified from NIX blast analysis (http://www.hgmp.mrnc.ac.uk) and Ensembl databases (http://www.ensembl.org).

PCR Amplification of CpG Island-Containing Fragments.

[0072]PCR oligonucleotides we redesigned to amplify approximately 3 kb fragments encompassing the complete CpG island embedded promoter region whilst including approximately 500 bp of coding sequence according to known or predicted coding sequence structure (see Table 2).

[0073]PCR reactions contained oligonucleotide sets specific each genomic fragment (2 pmol of each primer; Table 2). PCR amplification was achieved using the Failsafequadrature

[0074]PCR premixes A-F (Cambio, UK), 1 unit Taq DNA polymerase (Promega, UK) and 200 ng of template DNA. Initial denaturation was 96° C. for 2 min, whilst PCR amplification was carried out for 35 cycles (94° C. for 1 min, 55-60° C. for 1 min, 72° C. for 5 min). A final extension step (72° C. for 10 min) was included.

[0075]PCR products were gel purified, using GFX DNA purification columns (Amersham, UK) according to the manufacturers protocol, and subjected to TOPO TA cloningquadratureaccording to the manufacturers protocol (TOPO; Invitrogen, UK). Sense and anti-sense orientations were obtained for each CpG island-containing fragment cloned into TOPO vectors (Invitrogen, UK).

Expression Vector Construction.

[0076]A control expression vector (designated CET1005EGFP, SEQ ID NO:20) was constructed by the insertion of an hCMV/EGFP/sv40 pA (Nhe//Age/deleted multiple cloning site) from pEGFP-N1 into CET 900 followed by the insertion of the Ascl cassette from this vector into the Ascl site of CET 1005.

[0077]All CpG island fragments were removed from TOPO2.1 (Invitrogen, UK) unless otherwise stated. Terf2ip Acc65llEcoRV fragment was inserted into Acc65llSwal of 1005. GAPDH SpellSnaBl was inserted into PmellXbal of 1005. RPS3 XballSpel fragment was inserted into Xbal of 1005. RPS11 and TUBA1 EcoRl blunt fragments was removed from TOPO4.0 and TOPO2.1 respectively (Invitrogen, UK) and inserted into Pmel of 1005. Finally, A430106P18Rik (EcoRV) and 2510006D16Rik (BstXl) fragments were also inserted into Pmel of 1005. All CpG island containing fragments were inserted in both sense and anti-sense orientations immediately upstream of the hCMV promoter.

Cell Lines and Transfections.

[0078]CHO-K1 cells were grown in HAMS F12 (Invitrogen, Paisley, UK) plus 4500 mg/l L-ananyl-L-glutamine, 10 quadratureg/ml each of penicillin and streptomycin, and 10% (v/v) heat inactivated foetal calf serum (FCS; Invitrogen, Paisley, UK). Transfection was carried out by electroporation using approximately 10⁷ cells from 80% confluent cultures and a BioRad Gene Pulser II® set to deliver a single pulse of 975quadratureF at 250V. Transfections used 2 quadratureg of linearised CET1005EGFP plasmid and equivalent molar quantities for expression vectors of different size. Stably transfected cells were selected and maintained in growth medium containing 12.5 quadratureg/ml puromycin sulphate (Sigma, UK).

Quantification of Transgene Expression

[0079]Analysis of cells transfected with EGFP reporter constructs was with a Becton-Dickinson FACScan using the parental CHO-K1 cell line as a background, autofluorescence control.

TABLE-US-00001 TABLE 1 Sequences analysed CpG island^c % GC/ Locus Acc. #^a Description^b bp CG CG Terf2ip AB041557 Telomeric repeat 968 64.56 0.90 binding factor 2 interacting protein 1 (TRF2-interacting telomeric protein Rap1). Gapd^d M32599 Glyceraldehyde-3- 1187 60.50 0.84 phosphate dehydrogenase RPS3 NM012052 RPS3 - 40S ribosomal 419 60.39 0.87 protein S3. TUBA1 M13445 Tubulin alpha-1 chain 850 66.30 0.91 (Alpha-tubulin 1). RPS11 AK011207 RPS11 - 40S 957 59.71 0.95 ribosomal protein S11. A430106P18 AK020778 Expressed sequence 982 63.62 0.93 Rik tag 2510006D16 AK010915 Expressed sequence 679 67.69 0.75 Rik tag ^aGenbank Accession ^bEnseml description (http://www.ensembl.org/) ^cGrailexp (http://compbio.ornl.gov/grailexp) ^dGapd - derived from human sequence

TABLE-US-00002 TABLE 2 PCR oligonucleotides and amplicon sizes Locus Sense Antisense Amplicon Terf2ip gtagtttctgacttggaaatgt aactgacctgccatgccattc 2995 bp (SEQ ID NO: 3) (SEQ ID NO: 4) Gapd gagcagtccggtgtcacta gcagagaagcagacagttatg 3096 bp (SEQ ID NO: 5) (SEQ ID NO: 6) RPS3 cagagcatcaagtacctgtga taaccactaagccatctctcc 3056 bp (SEQ ID NO: 7) (SEQ ID NO: 8) TUBA1 caagaacaaggaagctggcc taaaacccacagcactgtaggg 3049 bp (SEQ ID NO: 9) (SEQ ID NO: 10) RPS11 aagactgtttgcctcatgcc ggatgacaatggtcctctgc 3020 bp (SEQ ID NO: 11) (SEQ ID NO: 12) A430106P18Rik atggttgtaggttcacgtcc atccctcacattgccaagcc 3128 bp (SEQ ID NO: 13) (SEQ ID NO: 14) 2510006D16Rik acttaagacctgatgcctcc gctagcttacataggcagcc 2997 bp (SEQ ID NO: 15) (SEQ ID NO: 16)

Example 1

Rps3 Element Driven Expression

[0080]SEQ ID NO:1 shows the RPS3 cloned sequence (Nucleotides 38 to 3154); SEQ ID NO:17 shows the complete plasmid sequence of pRPS3-1005-EGFP; SEQ ID NO:18 shows the complete plasmid sequence of pCET1015-EGFP.

[0081]EGFP expression levels, 8 days post-transfection, were investigated, within CHO-K1 pools containing hCMV alone (control construct; plasmid pCET1005-EGFP, linearised with Pmel prior to transfection), constructs containing an 8 kb RNPA2 fragment (plasmid pCET1015-EGFP, linearised with Pmel prior to transfection) and Rps3 (plasmid pRPS3-1005-EGFP; linearised with Pmel prior to transfection).

[0082]Pools generated with Rps3 containing constructs show a significant increase in EGFP expression levels compared to control constructs. Addition of the Rps3 sequence upstream of the hCMV promoter resulted in a 5.5- or 1.5-fold increase in mean fluorescence intensity relative to the control or hnRNPA2 element containing constructs respectively (FIG. 3A).

[0083]The activity of the constructs was investigated in NS0 cells. The increase in mean fluorescence intensity in stable pools when RPS3 element or hnRNPA2 elements are included in the constructs, compared to the hCMV promoter alone, was 28-fold or 18-fold respectively (FIG. 5A).

[0084]In both CHO-K1 and NS0 cells, the percentage positive cells was significantly increased with the hnRNPA2 element but this increase was greater with the RPS3 element (FIGS. 3B and 5B)

Example 2

Rps11 Element-Driven Expression

[0085]SEQ ID NO:2 shows the RPS11 cloned sequence (nucleotides 12 to 3032); SEQ ID NO:19 shows the complete sequence of pRPS11-1005-EGFP.

[0086]Rps11 containing- and control vectors (Pmel linearised) were transfected into CHO-K1 and NS0 cell lines and stable pools were generated by puromycin selection. Mean EGFP expression levels were assessed by FACscan analysis.

[0087]The addition of the Rps11 element upstream of the hCMV resulted in a 1.2-fold increase in mean EGFP expression levels, in CHO-K1 pools, compared to a construct containing the previously described RNPA2 fragment (FIG. 4A).

[0088]NS0 cell lines stably transfected with Rps11 containing constructs demonstrated a 1.8 and 1.5 fold, (respectively) increase in mean EGFP expression levels compared to hCMV and RNPA2 constructs (FIG. 6A).

[0089]An increase in percentage positive cells was observed for CHO-K1 cell lines transfected with Rps11 constructs compared to RNPA2 constructs (FIG. 4B). Furthermore, an increase in percentage positive cells was observed in NS0 pools transfected with Rps11 constructs compared to both hCMV and RNPA2 (FIG. 6B)

[0090]While the present invention has been particularly shown and described with reference to the foregoing preferred and alternative embodiments, it should be understood by those skilled in the art that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention without departing from the spirit and scope of the invention as defined in the following claims. It is intended that the following claims define the scope of the invention and that the method and apparatus within the scope of these claims and their equivalents be covered thereby. This description of the invention should be understood to include all novel and non-obvious combinations of elements described herein, and claims may be presented in this or a later application to any novel and non-obvious combination of these elements. The foregoing embodiments are illustrative, and no single feature or element is essential to all possible combinations that may be claimed in this or a later application. Where the claims recite "a" or "a first" element of the equivalent thereof, such claims should be understood to include incorporation of one or more such elements, neither requiring nor excluding two or more such elements.

Sequence CWU 1

2013145DNAMus musculus rps3 1ctagtaacgg ccgccagtgt gctggaattc gcccttataa ccactgagcc atctctccag 60ccctgagtca tgattttagt gtgagaggca tcattgaatt ttctgagcac ggccatcagg 120gtagctggca caggtcttca gatacaagga gatagttata agaaggcagc catggctgtg 180gtgcactaga aatggagaaa cagcttcatc aggtgacaga ccagtctgac tctgtcccat 240gattagaagc catcttgtta caaggtcaaa ataagttcat tcctgttttc tgtaacactt 300gggtttgatc ctgtcgtcaa cccattttct ggaatttgac atgttccata ctccattata 360ccctgacttc caccctgata agatgttctg ccaagttcct gtgtagccaa cattcccctg 420gaaatctctc ttcccttgga aaccacctag tcttagaaat tttgagttat ataaattcca 480cttctatgtt tgatgctatt ctttaaaact ccactttagg gagatagccc tgtctgatag 540aaaataaaac ttgcttaatt tgtctaaaag agtttaagta atagttttta cttttgttcc 600gtgggattaa tacagggtga aacagactcc cgtgtttcca gtgtgaagtg agccacacac 660tgcagtacaa gttatatcag caggttctgc ctctgggcaa tgaacttttg cttgtgtgga 720catcagggtc tgtgtgaagg gaaggtccta tggcctagat ttatactatt caacagtctg 780tccccgaagc cctggtgctt tattattttg acaagcccct gctgctggta ttccaccctg 840ctgcgagtca aaaaagttcc tgtctcggaa aaacaaaaca aaacaaaaca accaaaaaat 900aaaatttttt tttcccacag gttctagtgg aggtgctcac taccagaaat cctacaaata 960agcccatctc atggatcagg gtttaccttt gtaataatat taaatctgtg tgcatgtgcg 1020cacgcatgtg ttttatgctt gcatatatgt atacgcagcc atggttttct actgtcccac 1080tcactctgta acttactgag ccatccagct ggtcctctaa atacatttca atgaaagttt 1140tcattagcgt gaacgtgaag gtggtaaaat ctgttagtgt gtgcttatgc ctgtggtttg 1200cacctctagt ctgaaggttg ctcttttcaa attttttatt tatttacgtt tttacttttg 1260agtcagaaac tcataaaggc caagctggcc tcgaattcgc tatgtagtca atgatgacct 1320taaacttgtg accctctact tcgttagtgc tggaacccca agcttgctga gtacagagca 1380ctttcagacc ggaactagat gtctacttcc tgttccgcct acattacagg ttgctaggtt 1440acaccccccc tacgccgttt tagacgcaaa acttcatttc ccatgcaaaa cttcatttcc 1500catgaacact tgcaagggtc gccgcgctgc gcggcgtcat tgctcccgcc ctatatacct 1560acttccgccc gcgagccact tcctttcctt tcagcggcgc gcggctgcaa gatggcggtg 1620cagatttcca agaagaggaa ggtaagcgtc tgggcccggt tcgggagtcc gccgcgggtt 1680ctacaagtgc cagggaggcc tgtggctccg tgatcagtcc tgtggagcgt ctggggccgc 1740ctgccgtctc ttcgagcctc ggatggccgt agattgtgta ttgggccgga gccgggcgag 1800tgctgtgtgc ctgggcaagg gagggacaaa ctcctcgagt tctggaccga ctcgaacacc 1860gggcgcctcc agttccggac tagacacctt tgagcgtttc ttggtctcca taatagtaat 1920cctgtggcac agttagaggg cgtgtgccat cagatctagt ccagtttctt tagtaagtga 1980agtttagcag tcccttctct tagtcgcgtg atcctgcaag tggccatagt tgaaagccta 2040cttactgact gctgccgtgt tcactcggga cccggagctg cagcgtccct gtggttatca 2100tttcatgggg gaaaagtgtg caggttgcca ggtttagaaa tagatggtct gtcgtttgtg 2160cttatgcaca cagatgataa acctgttttg agtcaggatt cctctcctat ccgaggtaca 2220acttacagtc ccagctgtac atgtgctact tggagacaga tttttctttg tctcttgggt 2280gtagattatg ccgtagagcc cttcgatgaa gaggtgatga cgagtctgag taggaagtgt 2340tgtctttgtc caagatgcct cactatgctg cgttctgtgg cacagctgaa agcactgtgg 2400tcaaaagaaa cttcctaaag atgaccaaga ggcatttgtc tgagaagggt tgctgctttt 2460ctgtagggcc attgggcttg ctctgactaa ccctgtcttc acctcagagg taacttgttt 2520cctttggttc agtttgtagc tgatggcatc ttcaaagctg agctgaatga atttctcact 2580cgggagctgg ctgaagatgg ctactctgga gttgaagtcc gagttacacc aaccaggaca 2640gaaatcatta ttttagccac caggtagaaa taccattgat tgtcacctgt aaatactgtg 2700tgtactgaga tgctgtgtaa acttgggcca accaagcagt aaatctggcc tcagtgggtg 2760taactgcttt gttagaactg catttgggaa gaacttacct tccatttaac gtgtgtgctg 2820gcgttgtggt gggcggcagg tgggatcttg agtaaatggt tgcgcttccc ctctacagga 2880cacagaatgt tcttggggag aagggtcgtc ggatcagaga gttgaccgca gttgtccaga 2940agcgctttgg cttccctgaa ggcagcgtag aggtgagttc ctctgcttta tctcccgggg 3000gttttagact gagttgggat gtggcttctg ctatagaatt gtacttctga aaacctgaca 3060tggccagtga cagtcacagg tacttgatgc tctgagggcg aattctgcag atatccatca 3120cactggcggc cgctcgagca tgcat 314523039DNAMus musculus rps11 2aattcgccct taagactgtt tgcctcatgc ctgcctggcc tgcccttcct ccgccgccaa 60ctagggaagt ggggaccaaa ggttccttag gcactgctcc tgtgggtaga ggggacatta 120gagagctgac agcgcaccac ctgcatgagt ttttattaaa gtgcaaacca tgggatgaat 180cagttgagct tcagtgttga aaatgagtag cagggctgcc ccacccacct gaccaagtac 240cctattctgc agctatgaaa atgagatctg cacatgagct ggggttcaca agtgcacact 300tggagcactg ccttgctcct tcccagcaga ccacaaagca gtatttttct ggaggatttt 360atgtgctaat aaattatttg acttaagtgt gtacgatgtg tgctgtgcag agaggggcag 420agggcaccag caggtcatct gcatgggggg cccctttggg tgaatccttg ctcacgggat 480aggctttgtt gctcaaaagt tgcagatata catcttgggt cctgtcctag atggtgttac 540tgtaagtcag caccaagata caagagctgg tacctggact gtaggaggtc aggccatgac 600acaaaggctg ggactaaagg catttaccac gcctgagtct tctggttctt taaacatcaa 660atccttccgg gggctggcga gatggctcag tggttaagag cacagactgc tcttacgaag 720gatccgagtt caaatcccag caaccaaatg gtgcctaaca actatccata atgaaatctg 780atgccctctt ctggagtatc tgagaacagc tacagtgtac ttacatataa tcttaaaaat 840gcttcccatg ttaaccacca ctagagtttt tattacagct agctgacctg gaagccaagt 900ccttatgcct ccgtgagtgc tggggttaaa aagatccagc accactcaaa atgtcaatct 960attttgaaaa tatgctttat actgttctag cccatctgtg cagggctaga acggtgaata 1020cgagaaactg acacaagctt ttgccacctg gctaaatggt tcctctatta cctggggtgg 1080tcacctaagg ttagacactc atccacgagt agtcaggaca taaacccatc aaagtgtggg 1140tagacgcgca gcctgagata ctgtcaacaa aggacatgcg accttggtga cgtcggcctt 1200taataaaagg aagaaaggtt gactattcgg tcgacgctgg ctgctcctga catcgtatgg 1260cagatactct gctgtaaagc ggttcacccc tttcttgaga cccgctctgc acggccgctt 1320ctctctggaa actgaatccc agcacgtgtt tcccaacccg tacggcacgc cttctccgcc 1380ctaagcctcg ccgtaccaca tgatgcacgt ttcctccaca tcgtgctcct gaaatctcgc 1440gagatgatag gatcttcccg ccccttagtc ctcccccgtc atggcggcgt acggacagtc 1500ccaggaacgc gggctctcgc cggaagtacc tcccacctcc gtgaggataa ccccgcgtca 1560cttccgcccc gacctcgcgt ggtgaataag gaagccggga gcggccctgc ctctcccttt 1620ctccggcggc cgggaagatg gcggacattc aggttcgagc gtttagttgc tttcccccga 1680cgcttcggtg tggagcgtat cccttggcgt cctcgttgtc ttacgcatta gctgaagcga 1740ggatgcctgc gaatgccttc gtctcaggcg gctcggaaat ccgggctcta cgcagtaatg 1800gggtccctgg cgcttcggga gttggttctt aaagctcaga gcttaacggg tgagggattg 1860tggcgggagg agggcatcct gcggcgcggg agtcctgcgg cggcagagcc ggggacactg 1920ggtaaagcag gttttttccc cttgatggag actgaggccc ggacctcgtg cgctctacgg 1980cagggctgcg gtcccgacct cgctgtagtt ttcagtgtga gcgcagctct ggcctcgatg 2040agcttaggct tgtcttaaac ttgccatcct gcctcaacct caaccgggat gacagatccg 2100gcccaccagg ctcggctacg tggacataag cttgaatccc gaatgagtgg atttgtatgt 2160tttggaggtc cagtctggct gaaaagctct ttttgatctc agccgtgagt tctgcaggct 2220gtggaggtgt tagatgggac gcagtgtgtg agctaaacta gacttggggt ggttggagag 2280ccctgaccag ccggttttgg cgattggggc aaataaggtt gaaggtagga aggaagaaat 2340attgtctctg atttccttga actttacctg caacctcacc aaattctcat ccctacagac 2400ggagcgtgct taccaaaagc agcctacgat ctttcaaaac aagaagcggg ttctgctggg 2460agaaaccggc aaggaaaaac tccctcggta ctacaagaat atcggtctag gcttcaagac 2520gcctaaagag gtacaggacc ctccagcaga tgagatccct gctgccctgc acgtgtggga 2580gcacagccac cccgccccct tcacagtggc ttcccatggg cccctgggaa ttgtagtatg 2640ggccctgagg cgtcatcctt ggttctgttt aggaagtggt aatctaaacc ccactttctt 2700aactttgcag gctattgagg gtacctacat agacaagaaa tgccccttca ctggtaacgt 2760ctccatccga ggtcggatcc tgtctggtga gtgggatgtt ggaagggtgg ttctaggttc 2820ctgcgtccag gggcgctggc aagtgatgtc tgttctcacg atggtcttca gatgtcctct 2880agggcactgc tgagacagcc agttgacaaa gctgatgcca taaatggagc ttcttgggag 2940ccccgttcaa ctgactccta cctgctaaca cctttctgtt actctcccag gtgtcgtgac 3000gaagatgaag atgcagagga ccattgtcat ccaagggcg 3039322DNAArtificialSynthetic PCR oligonucleotide 3gtagtttctg acttggaaat gt 22421DNAArtificialSynthetic PCR oligonucleotide 4aactgacctg ccatgccatt c 21519DNAArtificialSynthetic PCR oligonucleotide 5gagcagtccg gtgtcacta 19621DNAArtificialSynthetic PCR oligonucleotide 6gcagagaagc agacagttat g 21721DNAArtificialSynthetic PCR oligonucleotide 7cagagcatca agtacctgtg a 21821DNAArtificialSynthetic PCR oligonucleotide 8taaccactaa gccatctctc c 21920DNAArtificialSynthetic PCR oligonucleotide 9caagaacaag gaagctggcc 201022DNAArtificialSynthetic PCR oligonucleotide 10taaaacccac agcactgtag gg 221120DNAArtificialSynthetic PCR oligonucleotide 11aagactgttt gcctcatgcc 201220DNAArtificialSynthetic PCR oligonucleotide 12ggatgacaat ggtcctctgc 201320DNAArtificialSynthetic PCR oligonucleotide 13gtggttgtag gttcacgtcc 201420DNAArtificialSynthetic PCR oligonucleotide 14atccctcaca ttgccaagcc 201520DNAArtificialSynthetic PCR oligonucleotide 15acttaagacc tgatgcctcc 201620DNAArtificialSynthetic PCR oligonucleotide 16gctagcttac ataggcagcc 20178691DNAArtificialVector pRPS3 1005 EGFP 17cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga attgggtacc 60gggccccccc tcgaagttta aacatttaaa tctagtaacg gccgccagtg tgctggaatt 120cgcccttata accactgagc catctctcca gccctgagtc atgattttag tgtgagaggc 180atcattgaat tttctgagca cggccatcag ggtagctggc acaggtcttc agatacaagg 240agatagttat aagaaggcag ccatggctgt ggtgcactag aaatggagaa acagcttcat 300caggtgacag accagtctga ctctgtccca tgattagaag ccatcttgtt acaaggtcaa 360aataagttca ttcctgtttt ctgtaacact tgggtttgat cctgtcgtca acccattttc 420tggaatttga catgttccat actccattat accctgactt ccaccctgat aagatgttct 480gccaagttcc tgtgtagcca acattcccct ggaaatctct cttcccttgg aaaccaccta 540gtcttagaaa ttttgagtta tataaattcc acttctatgt ttgatgctat tctttaaaac 600tccactttag ggagatagcc ctgtctgata gaaaataaaa cttgcttaat ttgtctaaaa 660gagtttaagt aatagttttt acttttgttc cgtgggatta atacagggtg aaacagactc 720ccgtgtttcc agtgtgaagt gagccacaca ctgcagtaca agttatatca gcaggttctg 780cctctgggca atgaactttt gcttgtgtgg acatcagggt ctgtgtgaag ggaaggtcct 840atggcctaga tttatactat tcaacagtct gtccccgaag ccctggtgct ttattatttt 900gacaagcccc tgctgctggt attccaccct gctgcgagtc aaaaaagttc ctgtctcgga 960aaaacaaaac aaaacaaaac aaccaaaaaa taaaattttt ttttcccaca ggttctagtg 1020gaggtgctca ctaccagaaa tcctacaaat aagcccatct catggatcag ggtttacctt 1080tgtaataata ttaaatctgt gtgcatgtgc gcacgcatgt gttttatgct tgcatatatg 1140tatacgcagc catggttttc tactgtccca ctcactctgt aacttactga gccatccagc 1200tggtcctcta aatacatttc aatgaaagtt ttcattagcg tgaacgtgaa ggtggtaaaa 1260tctgttagtg tgtgcttatg cctgtggttt gcacctctag tctgaaggtt gctcttttca 1320aattttttat ttatttacgt ttttactttt gagtcagaaa ctcataaagg ccaagctggc 1380ctcgaattcg ctatgtagtc aatgatgacc ttaaacttgt gaccctctac ttcgttagtg 1440ctggaacccc aagcttgctg agtacagagc actttcagac cggaactaga tgtctacttc 1500ctgttccgcc tacattacag gttgctaggt tacacccccc ctacgccgtt ttagacgcaa 1560aacttcattt cccatgcaaa acttcatttc ccatgaacac ttgcaagggt cgccgcgctg 1620cgcggcgtca ttgctcccgc cctatatacc tacttccgcc cgcgagccac ttcctttcct 1680ttcagcggcg cgcggctgca agatggcggt gcagatttcc aagaagagga aggtaagcgt 1740ctgggcccgg ttcgggagtc cgccgcgggt tctacaagtg ccagggaggc ctgtggctcc 1800gtgatcagtc ctgtggagcg tctggggccg cctgccgtct cttcgagcct cggatggccg 1860tagattgtgt attgggccgg agccgggcga gtgctgtgtg cctgggcaag ggagggacaa 1920actcctcgag ttctggaccg actcgaacac cgggcgcctc cagttccgga ctagacacct 1980ttgagcgttt cttggtctcc ataatagtaa tcctgtggca cagttagagg gcgtgtgcca 2040tcagatctag tccagtttct ttagtaagtg aagtttagca gtcccttctc ttagtcgcgt 2100gatcctgcaa gtggccatag ttgaaagcct acttactgac tgctgccgtg ttcactcggg 2160acccggagct gcagcgtccc tgtggttatc atttcatggg ggaaaagtgt gcaggttgcc 2220aggtttagaa atagatggtc tgtcgtttgt gcttatgcac acagatgata aacctgtttt 2280gagtcaggat tcctctccta tccgaggtac aacttacagt cccagctgta catgtgctac 2340ttggagacag atttttcttt gtctcttggg tgtagattat gccgtagagc ccttcgatga 2400agaggtgatg acgagtctga gtaggaagtg ttgtctttgt ccaagatgcc tcactatgct 2460gcgttctgtg gcacagctga aagcactgtg gtcaaaagaa acttcctaaa gatgaccaag 2520aggcatttgt ctgagaaggg ttgctgcttt tctgtagggc cattgggctt gctctgacta 2580accctgtctt cacctcagag gtaacttgtt tcctttggtt cagtttgtag ctgatggcat 2640cttcaaagct gagctgaatg aatttctcac tcgggagctg gctgaagatg gctactctgg 2700agttgaagtc cgagttacac caaccaggac agaaatcatt attttagcca ccaggtagaa 2760ataccattga ttgtcacctg taaatactgt gtgtactgag atgctgtgta aacttgggcc 2820aaccaagcag taaatctggc ctcagtgggt gtaactgctt tgttagaact gcatttggga 2880agaacttacc ttccatttaa cgtgtgtgct ggcgttgtgg tgggcggcag gtgggatctt 2940gagtaaatgg ttgcgcttcc cctctacagg acacagaatg ttcttgggga gaagggtcgt 3000cggatcagag agttgaccgc agttgtccag aagcgctttg gcttccctga aggcagcgta 3060gaggtgagtt cctctgcttt atctcccggg ggttttagac tgagttggga tgtggcttct 3120gctatagaat tgtacttctg aaaacctgac atggccagtg acagtcacag gtacttgatg 3180ctctgagggc gaattctgca gatatccatc acactggcgg ccgctcgagc atgcatctag 3240aagcttatcg ataccggtgg cgcgccaatt gaattaagat ctggcccaat gggccgtacg 3300aattcgagct cggtacccgg ggatcctgat ctaatagtaa tcaattacgg ggtcattagt 3360tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 3420accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 3480aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc 3540agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 3600gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 3660ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg 3720tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag 3780tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt 3840gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt 3900gaaccgtcag atccgtcgcc accatggtga gcaagggcga ggagctgttc accggggtgg 3960tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg 4020agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca 4080agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca 4140gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 4200acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg 4260tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 4320aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata 4380tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg 4440aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 4500ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca 4560acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg 4620gcatggacga gctgtacaag taaagcggcc gcgactctag atcataatca gccataccac 4680atttgtagag gttttacttg ctttaaaaaa cctcccacac ctccccctga acctgaaaca 4740taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata 4800aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 4860tttgtccaaa ctcatcaatg tatcttaact agagtcgacc tgcaggcatg caagcttacc 4920ggtggcgcgc gcgccaattg ttaattaaga tctggcccaa tgggccgtac gaattcctta 4980ggctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc tttagcagcc 5040ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc cacatccacc 5100ggccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac cttctactcc 5160tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga cgtgacaaat 5220ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc aatggaagcg 5280ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct gggctcagag 5340gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg gggcgggcgc 5400ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt ctgccgcgct 5460gttctcctct tcctcatctc cgggcctttc gaccagctta ccatgaccga gtacaagccc 5520acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg 5580ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc 5640accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc 5700gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg 5760gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag 5820caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc 5880accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc 5940ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc 6000aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa 6060ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg 6120cagcgcccga ccgaaaggag cgcacgaccc catgcatcgt agacgaaatg accgaccaag 6180cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg 6240gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc 6300tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag acaataccgg 6360aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt gttgggtcgt 6420ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac 6480cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg 6540gtgaaggccc agggctcgca gccaacgtcg gggcggcagg cccccagctt ttgttccctt 6600tagtgagggt taatttcgag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 6660tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 6720ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 6780tcgggaaacc tgtcgtgcca gcatcgcgag cacttttcgg ggaaatgtgc gcggaacccc 6840tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 6900ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 6960ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 7020gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 7080caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 7140ttttaaagtt ctgctatgtg

gcgcggtatt atcccgtatt gacgccgggc aagagcaact 7200cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 7260gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 7320taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 7380tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 7440agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 7500caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 7560ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 7620tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 7680agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 7740tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 7800agactcgcga cactgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 7860gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 7920gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 7980ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 8040ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 8100cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 8160ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 8220tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 8280gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 8340tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 8400gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 8460tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag 8520ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 8580agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 8640gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc a 86911813827DNAArtificialVector pCET 1015 EGFP 18cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga attgggtacc 60gggccccccc tcgaagttta aacatttaaa tctagaagct tcaatgtttt tagcaccctc 120tgtgtggagg aaaataatgc agattattct aattagtgta atatctaacc acattaaaat 180atattacata gtaaactaca ctccataatt ttataaattt gactccccag ggtaataaac 240tagtctctag tctgctcacc ttcaactgta caataaagtc ttggttcttt tgaaatagac 300ctcaaatgag acacctaaaa ttcaaagtgt ctttacattt aaagacacct acaggaaagc 360aggtaaaaga gccaggttaa aaacaaattc taaaaccact tagctgcagt taaacatata 420gtaaagatgc actaaagttt cttactctgt aaatcccttc cacttcagga aatattccac 480tttcccattc actacacgtc gatctagtac tttttccacg acaaattctt caggctctgc 540ctcttcaact tttttactct ttccattctg tttttttccc attttttgct aaaataaaac 600aaaagagaaa ttaagaaata ttcctcttga attttgagca cattttcaag gctcaattgc 660ttatattatt atcacattcg acataaattt ttacttctat atcccagggc agacaccttc 720tggaaagatt aaaagtcaac agacaataaa ataaaagaat gctttatctt gttcatttag 780ttcaaactta caacccacca ccaaaataat acaataaaaa aacactatct ggaaacagtt 840atttttttcc agtctttttt tttgagacag ggtctcacac tcttgtcgcc caggctggag 900tgcagtggcg tgatctcagc tcactgcaac ctccgcctcc ccaggttcaa gcagttctca 960tgcctcagcc tccagagtag ctgggattat aggcggatgc caccatgccg ggctaatttt 1020ttttgtgttt ttattagaaa cagggtttca ccatgttgac caggctggtc tcaaactcct 1080gacctgaagt gattcaccag cctgggcctc ccaaagtgct ggcattacag gcgtgagcca 1140ctgcgcccgg ccctgtagtc ttaaaagacc aagtttacta attttcactc attttaacaa 1200cactgcaaca aacaactatg caggaagtac ctaaagggtg atccagagaa gcaagtagta 1260gtgacaggtc ttaggtgaac ctatgacaga ccttgtatcc acccccagat ggtaaaagcc 1320ccagccccct tctcaattca aatattaatg tcaaaagcat caatgataca gagaaaagat 1380aaatgcagaa tgaaaacatg gttcaaaatc ctgataccaa ctgcagggtc aactatagag 1440accactagga ggttcaatta aaggacaaga ttatttttcc ataatctctg tagataatat 1500ttcctaccac ttagaacaaa actataaagc tatcacttca agagaccaac attacaaatt 1560tattttaatt ccctaaggtg aaaaaaatcc ttccttcctg gtttctcaag agaaagtcta 1620tactggtaac caaattcact ttaaacaggc attttctttg gtatgacact atttaagaga 1680agcaggaaac caacgtgaac cagctctttc caatggctca agatttccta tgagaggact 1740aaaaatgggg aaaattttta tgagaggatt aaaaatgggg gaaaaaaaac cctgaaatgg 1800ttaatcagaa gatcctatgg gctgagaagg aatccatctt aacatttcat cttaaagcaa 1860atgctattgc cgggggcagt ggctcatgcc tgtaatccca gcactttggg aggccgaggt 1920gggcagatca tctgaggtca ggagtttgag accagcctga ccaacatgga gaaaccccgt 1980ttctactaaa aatacaaaat tagccaggca tagtggtgca tgcctgtaat cccagctact 2040tgggaggctg aggcaggaga actgcttgaa cccaggaggc ttaagttgcg gtgagccaag 2100atcacgccat tgcactctag cctggacaac aagagaaaaa ctctgtctca aaaaaacaca 2160aaaacaaaaa acccaaatac tatttaaaaa agataaacct taattgctca atcattaaag 2220ccatcccaca agtaaagcag caagcagaaa aaagttaaga acacctcaag gctacagaag 2280gacatttcaa gctatgcagg catatgaagt gtgcagacag atatgtaaga aaggcctcaa 2340gactgcaaaa gggcatttca agctatgcaa gcatataggt aacacataca cacacacaaa 2400ataaaatccc ctgaaataca aaaacatgca gcaaacacct gacgtttttg gataccattt 2460ctaagtcagg tgttatgatt ctcattagtc aagatacttg agtactgggc ccaaacagct 2520ttctgccact gtacagtaca agaaggtagg aataatggtg ggaggagcaa agacaaactg 2580taatagacag aagtgtatca gatacctata ctacatgaaa aacaaaacag ctactgccac 2640aaagggagaa ggctaacaaa ataaagtcaa caataaatac agaaaatgaa aaggatacac 2700actaaggttt acaaaaaaaa aaaggcagac aaaatgccat acagtattca ttcactacta 2760tggcattcat aagctagttt caaatgctca ctattttctt ttatagtata tatttgcctt 2820aacccagcac ttttttccaa aagtggatga gtcaaaataa atttcccatt atttaagtga 2880aattaacagc acacatatct cacaacacta atgaattttt aaaatggaaa gttaagaact 2940tttaaagtgg ccaacctgtg atccttcaca aaataaacta aatacaataa cagaccccaa 3000aggctatcaa ttgcgtgcaa aaacaacttc tgttttccag ggtaaacaga atctaatgca 3060gaatctaatg cagggtaaac agacttaatg cagaatctaa tgatggcaca aattaaaaat 3120cactaacgtg ccctttttag tgtgaaaccc agagagagca catacaagcc aaaaacaaat 3180gctttatttt acctaggaga cattaacatt cacctttacg tgtttaagat taatgcaatg 3240ttaaatattg tgaaaactgt aactttgaat ttcatgattt ttatgtgaat attccagggt 3300ttaaaaaaac ttgtaacatg acatggctga ataagataaa aaaaaaatct agccttttct 3360cccttctggc tcatatttgc gatttcgatc attttgttta aaaaacaaaa cactgcaatg 3420aattaaactt aatattcttc tatgttttag agtaagttaa aacaagataa agtgaccaaa 3480gtaatttgaa agattcaatg acttttgctc caacctaggt gcacaaggta ccttgttctt 3540taaattgggc tttaatgaaa atacttctcc agaattctgg ggatttaaga aaaattatgc 3600caaccaacaa gggctttacc attttatgta acatttttca acgctgcaaa aatgtgtgta 3660tttctatttg aagataaaaa tcctcagcaa aatccacatt gcactgtcct tcaaagatta 3720gccttctttg aactagttaa gacactatta agccaagcca gtatctccct gtaatgaatt 3780cgtttttctc ttaattttcc cctgtaattt acactgggag agctgggaaa tatgtggatg 3840taaatttctc agccacagag atgcaaagtt atactgtggg gaaaaaaaac ttgagttaaa 3900tccttacata ttttaggttt tcattaactt accaatgtag ttttgttgga ggccattttt 3960tttattgcag acttgaagag ctattactag aaaaatgcat gacagttaag gtaagtttgc 4020atgacacaaa aaaggtaact aaatacaaat tctgtttgga ttccaacccc caagtagaga 4080gcgcacactt tcaaacgtga atacaaatcc agagtagatc tgcgctccta cctacattgc 4140ttatgatgta cttaagtacg tgtcctaacc atgtgagtct agaaagactt tactggggat 4200cctggtacct aaaacagctt cacatggctt aaaatagggg accaatgtct tttccaatct 4260aagtcccatt tataataaag tccatgttcc atttttaaag gacaatcctt tcggtttaaa 4320accaggcacg attacccaaa caactcacaa cggtaaagca ctgtgaatct tctctgttct 4380gcaatcccaa cttggtttct gctcagaaac cctccctctt tccaatcggt aattaaataa 4440caaaaggaaa aaacttaaga tgcttcaacc ccgtttcgtg acactttgaa aaaagaatca 4500cctcttgcaa acacccgctc ccgacccccg ccgctgaagc ccggcgtcca gaggcctaag 4560cgcgggtgcc cgcccccacc cgggagcgcg ggcctcgtgg tcagcgcatc cgcggggaga 4620aacaaaggcc gcggcacggg ggctcaaggg cactgcgcca caccgcacgc gcctaccccc 4680gcgcggccac gttaactggc ggtcgccgca gcctcgggac agccggccgc gcgccgccag 4740gctcgcggac gcgggaccac gcgccgccct ccgggaggcc caagtctcga cccagccccg 4800cgtggcgctg ggggaggggg cgcctccgcc ggaacgcggg tgggggaggg gagggggaaa 4860tgcgctttgt ctcgaaatgg ggcaaccgtc gccacagctc cctaccccct cgagggcaga 4920gcagtccccc cactaactac cgggctggcc gcgcgccagg ccagccgcga ggccaccgcc 4980cgaccctcca ctccttcccg cagctcccgg cgcggggtcc ggcgagaagg ggaggggagg 5040ggagcggaga accgggcccc cgggacgcgt gtggcatctg aagcaccacc agcgagcgag 5100agctagagag aaggaaagcc accgacttca ccgcctccga gctgctccgg gtcgcgggtc 5160tgcagcgtct ccggccctcc gcgcctacag ctcaagccac atccgaaggg ggagggagcc 5220gggagctgcg cgcggggccg ccggggggag gggtggcacc gcccacgccg ggcggccacg 5280aagggcgggg cagcgggcgc gcgcgcggcg gggggagggg ccggcgccgc gcccgctggg 5340aattggggcc ctagggggag ggcggaggcg ccgacgaccg cggcacttac cgttcgcggc 5400gtggcgcccg gtggtcccca aggggaggga agggggaggc ggggcgagga cagtgaccgg 5460agtctcctca gcggtggctt ttctgcttgg cagcctcagc ggctggcgcc aaaaccggac 5520tccgcccact tcctcgcccg ccggtgcgag ggtgtggaat cctccagacg ctgggggagg 5580gggagttggg agcttaaaaa ctagtacccc tttgggacca ctttcagcag cgaactctcc 5640tgtacaccag gggtcagttc cacagacgcg ggccaggggt gggtcattgc ggcgtgaaca 5700ataatttgac tagaagttga ttcgggtgtt tccggaaggg gccgagtcaa tccgccgagt 5760tggggcacgg aaaacaaaaa gggaaggcta ctaagatttt tctggcgggg gttatcattg 5820gcgtaactgc agggaccacc tcccgggttg agggggctgg atctccaggc tgcggattaa 5880gcccctcccg tcggcgttaa tttcaaactg cgcgacgttt ctcacctgcc ttcgccaagg 5940caggggccgg gaccctattc caagaggtag taactagcag gactctagcc ttccgcaatt 6000cattgagcgc atttacggaa gtaacgtcgg gtactgtctc tggccgcaag ggtgggagga 6060gtacgcattt ggcgtaaggt ggggcgtaga gccttcccgc cattggcggc ggatagggcg 6120tttacgcgac ggcctgacgt agcggaagac gcgttagtgg gggggaaggt tctagaaaag 6180cggcggcagc ggctctagcg gcagtagcag cagcgccggg tcccgtgcgg aggtgctcct 6240cgcagagttg tttctcgagc agcggcagtt ctcactacag cgccaggacg agtccggttc 6300gtgttcgtcc gcggagatct ctctcatctc gctcggctgc gggaaatcgg gctgaagcga 6360ctgagtccgc gatggaggta acgggtttga aatcaatgag ttattgaaaa gggcatggcg 6420aggccgttgg cgcctcagtg gaagtcggcc agccgcctcc gtgggagaga ggcaggaaat 6480cggaccaatt cagtagcagt ggggcttaag gtttatgaac ggggtcttga gcggaggcct 6540gagcgtacaa acagcttccc caccctcagc ctcccggcgc catttccctt cactgggggt 6600gggggatggg gagctttcac atggcggacg ctgccccgct ggggtgaaag tggggcgcgg 6660aggcgggaat tcttattccc tttctaaagc acgctgcttc gggggccacg gcgtctcctc 6720ggcgagcgtt tcggcgggca gcaggtcctc gtgagcgagg ctgcggagct tcccctcccc 6780ctctctcccg ggaaccgatt tggcggccgc cattttcatg gctcgccttc ctctcagcgt 6840tttccttata actcttttat tttcttagtg tgctttctct atcaagaagt agaagtggtt 6900aactattttt tttttcttct cgggctgttt tcatatcgtt tcgaggtgga tttggagtgt 6960tttgtgagct tggatcttta gagtcctgcg cacctcatta aaggcgctca gccttcccct 7020cgatgaaatg gcgccattgc gttcggaagc cacaccgaag agcggggagg gggggtgctc 7080cgggtttgcg ggcccggttt cagagaagat atcaccaccc agggcgtcgg gccgggttca 7140atgcgagccg taggacaaag aaaccatttt atgtttttcc tgtctttttt ttcctttgag 7200taacggtttt atctgggtct gcagtcagta aaacgacaga tgaaccgcgg caaaataaac 7260ataaattgga agccatcggc cacgaggggc agggacgaag gtggttttct gggcggggga 7320gggatattcg cgtcagaatc ctttactgtt cttaaggatt ccgtttaagt tgtagagctg 7380actcatttta agtaatgttg ttactgagaa gtttaaccct tacgggacag atccatggac 7440ctttatagat gattacgagg aaagtgaaat aacgattttg tccttagtta tacttcgatt 7500aaaacatggc ttcagaggct ccttcctgta atgcgtatgg attgatgtgc aaaactgttt 7560tgggcctggg ccgctctgta tttgaacttt gttacttttc tcattttgtt tgcaatcttg 7620gttgaacatt acattgataa gcataaggtc tcaagcgaag ggggtctacc tggttatttt 7680tctttgaccc taagcacgtt tataaaataa cattgtttaa aatcgatagt ggacatcggg 7740taagtttgga taaattgtga ggtaagtaat gagtttttgc tttttgttag tgatttgtaa 7800aacttgttat aaatgtacat tatccgtaat ttcagtttag agataaccta tgtgctgacg 7860acaattaaga ataaaaacta gctgaaaaaa tgaaaataac tatcgtgaca agtaaccatt 7920tcaaaagact gctttgtgtc tcataggagc tagtttgatc atttcagtta attttttctt 7980taatttttac gagtcatgaa aactacagga aaaaaaatct gaactgggtt ttaccactac 8040tttttaggag ttgggagcat gcgaatggag ggagagctcc gtagaactgg gatgagagca 8100gcaattaatg ctgcttgcta ggaacaaaaa ataattgatt gaaaattacg tgtgactttt 8160tagtttgcat tatgcgtttg tagcagttgg tcctggatat cactttctct cgtttgaggt 8220tttttaacct agttaacttt taagacaggt ttccttaaca ttcataagtg cccagaatac 8280agctgtgtag tacagcatat aaagatttca gctctgaggt ttttcctatt gacttggaaa 8340attgttttgt gcctgtcgct tgccacatgg ccaatcaagt aagcttatcg ataccggtgg 8400cgcgccaatt gaattaagat ctggcccaat gggccgtacg aattcgagct cggtacccgg 8460ggatcctgat ctaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt 8520tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc 8580cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac 8640gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 8700tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc 8760agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 8820ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac 8880ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc 8940aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc 9000gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgtcgcc 9060accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 9120gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 9180tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc 9240accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg 9300aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 9360ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 9420ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg 9480cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag 9540aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc 9600gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac 9660cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg 9720gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag 9780taaagcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg 9840ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg 9900ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 9960tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 10020tatcttaact agagtcgacc tgcaggcatg caagcttacc ggtggcgcgc caattgttaa 10080ttaagatctg gcccaatggg ccgtacgaat tccttaggct accgggtagg ggaggcgctt 10140ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg gcgctacaca 10200agtggcctct ggcctcgcac acattccaca tccaccggcc ggtaggcgcc aaccggctcc 10260gttctttggt ggccccttcg cgccaccttc tactcctccc ctagtcagga agttcccccc 10320cgccccgcag ctcgcgtcgt gcaggacgtg acaaatggaa gtagcacgtc tcactagtct 10380cgtgcagatg gacagcaccg ctgagcaatg gaagcgggta ggcctttggg gcagcggcca 10440atagcagctt tgctccttcg ctttctgggc tcagaggctg ggaaggggtg ggtccggggg 10500cgggctcagg ggcgggctca ggggcggggc gggcgcccga aggtcctccg gaggcccggc 10560attctgcacg cttcaaaagc gcacgtctgc cgcgctgttc tcctcttcct catctccggg 10620cctttcgacc agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac 10680gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc 10740cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc 10800acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg 10860gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc 10920atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg 10980ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac 11040cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc 11100ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc 11160ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc 11220cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca 11280cgaccccatg catcgtagac gaaatgaccg accaagcgac gcccaacctg ccatcacgag 11340atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg 11400ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg 11460ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa 11520taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt 11580cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg 11640tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca 11700acgtcggggc ggcaggcccc cagcttttgt tccctttagt gagggttaat ttcgagcttg 11760gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac 11820aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc 11880acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcat 11940cgcgagcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 12000ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 12060aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 12120ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 12180gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag 12240ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 12300ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 12360gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 12420aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 12480gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 12540aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 12600caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact 12660tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 12720acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 12780gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 12840agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 12900gataggtgcc tcactgatta agcattggta actgtcagac tcgcgacact gcattaatga 12960atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 13020actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 13080gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 13140cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 13200ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 13260ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 13320ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 13380agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 13440cacgaacccc ccgttcagcc

cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 13500aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 13560gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 13620agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 13680ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 13740cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 13800tctgacgctc agtggaacga aaactca 13827198585DNAArtificialVector pRPS11 1005 EGFP 19cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga attgggtacc 60gggccccccc tcgaagttta attcgccctt aagactgttt gcctcatgcc tgcctggcct 120gcccttcctc cgccgccaac tagggaagtg gggaccaaag gttccttagg cactgctcct 180gtgggtagag gggacattag agagctgaca gcgcaccacc tgcatgagtt tttattaaag 240tgcaaaccat gggatgaatc agttgagctt cagtgttgaa aatgagtagc agggctgccc 300cacccacctg accaagtacc ctattctgca gctatgaaaa tgagatctgc acatgagctg 360gggttcacaa gtgcacactt ggagcactgc cttgctcctt cccagcagac cacaaagcag 420tatttttctg gaggatttta tgtgctaata aattatttga cttaagtgtg tacgatgtgt 480gctgtgcaga gaggggcaga gggcaccagc aggtcatctg catggggggc ccctttgggt 540gaatccttgc tcacgggata ggctttgttg ctcaaaagtt gcagatatac atcttgggtc 600ctgtcctaga tggtgttact gtaagtcagc accaagatac aagagctggt acctggactg 660taggaggtca ggccatgaca caaaggctgg gactaaaggc atttaccacg cctgagtctt 720ctggttcttt aaacatcaaa tccttccggg ggctggcgag atggctcagt ggttaagagc 780acagactgct cttacgaagg atccgagttc aaatcccagc aaccaaatgg tgcctaacaa 840ctatccataa tgaaatctga tgccctcttc tggagtatct gagaacagct acagtgtact 900tacatataat cttaaaaatg cttcccatgt taaccaccac tagagttttt attacagcta 960gctgacctgg aagccaagtc cttatgcctc cgtgagtgct ggggttaaaa agatccagca 1020ccactcaaaa tgtcaatcta ttttgaaaat atgctttata ctgttctagc ccatctgtgc 1080agggctagaa cggtgaatac gagaaactga cacaagcttt tgccacctgg ctaaatggtt 1140cctctattac ctggggtggt cacctaaggt tagacactca tccacgagta gtcaggacat 1200aaacccatca aagtgtgggt agacgcgcag cctgagatac tgtcaacaaa ggacatgcga 1260ccttggtgac gtcggccttt aataaaagga agaaaggttg actattcggt cgacgctggc 1320tgctcctgac atcgtatggc agatactctg ctgtaaagcg gttcacccct ttcttgagac 1380ccgctctgca cggccgcttc tctctggaaa ctgaatccca gcacgtgttt cccaacccgt 1440acggcacgcc ttctccgccc taagcctcgc cgtaccacat gatgcacgtt tcctccacat 1500cgtgctcctg aaatctcgcg agatgatagg atcttcccgc cccttagtcc tcccccgtca 1560tggcggcgta cggacagtcc caggaacgcg ggctctcgcc ggaagtacct cccacctccg 1620tgaggataac cccgcgtcac ttccgccccg acctcgcgtg gtgaataagg aagccgggag 1680cggccctgcc tctccctttc tccggcggcc gggaagatgg cggacattca ggttcgagcg 1740tttagttgct ttcccccgac gcttcggtgt ggagcgtatc ccttggcgtc ctcgttgtct 1800tacgcattag ctgaagcgag gatgcctgcg aatgccttcg tctcaggcgg ctcggaaatc 1860cgggctctac gcagtaatgg ggtccctggc gcttcgggag ttggttctta aagctcagag 1920cttaacgggt gagggattgt ggcgggagga gggcatcctg cggcgcggga gtcctgcggc 1980ggcagagccg gggacactgg gtaaagcagg ttttttcccc ttgatggaga ctgaggcccg 2040gacctcgtgc gctctacggc agggctgcgg tcccgacctc gctgtagttt tcagtgtgag 2100cgcagctctg gcctcgatga gcttaggctt gtcttaaact tgccatcctg cctcaacctc 2160aaccgggatg acagatccgg cccaccaggc tcggctacgt ggacataagc ttgaatcccg 2220aatgagtgga tttgtatgtt ttggaggtcc agtctggctg aaaagctctt tttgatctca 2280gccgtgagtt ctgcaggctg tggaggtgtt agatgggacg cagtgtgtga gctaaactag 2340acttggggtg gttggagagc cctgaccagc cggttttggc gattggggca aataaggttg 2400aaggtaggaa ggaagaaata ttgtctctga tttccttgaa ctttacctgc aacctcacca 2460aattctcatc cctacagacg gagcgtgctt accaaaagca gcctacgatc tttcaaaaca 2520agaagcgggt tctgctggga gaaaccggca aggaaaaact ccctcggtac tacaagaata 2580tcggtctagg cttcaagacg cctaaagagg tacaggaccc tccagcagat gagatccctg 2640ctgccctgca cgtgtgggag cacagccacc ccgccccctt cacagtggct tcccatgggc 2700ccctgggaat tgtagtatgg gccctgaggc gtcatccttg gttctgttta ggaagtggta 2760atctaaaccc cactttctta actttgcagg ctattgaggg tacctacata gacaagaaat 2820gccccttcac tggtaacgtc tccatccgag gtcggatcct gtctggtgag tgggatgttg 2880gaagggtggt tctaggttcc tgcgtccagg ggcgctggca agtgatgtct gttctcacga 2940tggtcttcag atgtcctcta gggcactgct gagacagcca gttgacaaag ctgatgccat 3000aaatggagct tcttgggagc cccgttcaac tgactcctac ctgctaacac ctttctgtta 3060ctctcccagg tgtcgtgacg aagatgaaga tgcagaggac cattgtcatc caagggcgaa 3120acatttaaat ctagaagctt atcgataccg gtggcgcgcc aattgaatta agatctggcc 3180caatgggccg tacgaattcg agctcggtac ccggggatcc tgatctaata gtaatcaatt 3240acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 3300ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 3360cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 3420actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 3480aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 3540acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag 3600tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 3660gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 3720aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 3780agagctggtt tagtgaaccg tcagatccgt cgccaccatg gtgagcaagg gcgaggagct 3840gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 3900cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 3960ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg 4020cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc 4080catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 4140gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 4200catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 4260ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 4320ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 4380catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct 4440gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 4500cgggatcact ctcggcatgg acgagctgta caagtaaagc ggccgcgact ctagatcata 4560atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc 4620ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat 4680aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg 4740cattctagtt gtggtttgtc caaactcatc aatgtatctt aactagagtc gacctgcagg 4800catgcaagct taccggtggc gcgcgcgcca attgttaatt aagatctggc ccaatgggcc 4860gtacgaattc cttaggctac cgggtagggg aggcgctttt cccaaggcag tctggagcat 4920gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac 4980attccacatc caccggccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg 5040ccaccttcta ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc 5100aggacgtgac aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct 5160gagcaatgga agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct 5220ttctgggctc agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg 5280ggcggggcgg gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc 5340acgtctgccg cgctgttctc ctcttcctca tctccgggcc tttcgaccag cttaccatga 5400ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca 5460ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc 5520acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg 5580gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg 5640tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc 5700ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg 5760cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg 5820ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga 5880cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg 5940tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgacgcc 6000cgccccacga cccgcagcgc ccgaccgaaa ggagcgcacg accccatgca tcgtagacga 6060aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt 6120ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg 6180cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa 6240ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca 6300cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga 6360taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca 6420ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccccca 6480gcttttgttc cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt 6540ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 6600agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 6660tgcccgcttt ccagtcggga aacctgtcgt gccagcatcg cgagcacttt tcggggaaat 6720gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 6780agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 6840catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 6900ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 6960atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 7020ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 7080gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 7140ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 7200ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 7260gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 7320ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 7380gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 7440ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 7500gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 7560gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 7620caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 7680cattggtaac tgtcagactc gcgacactgc attaatgaat cggccaacgc gcggggagag 7740gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 7800ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 7860caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 7920aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 7980atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 8040cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 8100ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 8160gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 8220accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 8280cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 8340cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct 8400gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 8460aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 8520aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 8580actca 8585205546DNAArtificialVector pCET 1005 EGFP 20cgttgtaaaa cgacggccag tgaattgtaa tacgactcac tatagggcga attgggtacc 60gggccccccc tcgaagttta aacatttaaa tctagaagct tatcgatacc ggtggcgcgc 120caattgaatt aagatctggc ccaatgggcc gtacgaattc gagctcggta cccggggatc 180ctgatctaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc 240gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg 300acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa 360tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca 420agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac 480atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc 540atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga 600tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg 660gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta 720cggtgggagg tctatataag cagagctggt ttagtgaacc gtcagatccg tcgccaccat 780ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg 840cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg 900caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct 960cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca 1020gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt 1080caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt 1140gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa 1200gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg 1260catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga 1320ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta 1380cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct 1440gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag 1500cggccgcgac tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta 1560aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt 1620aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 1680aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 1740taactagagt cgacctgcag gcatgcaagc ttaccggtgg cgcgcgcgcc aattgttaat 1800taagatctgg cccaatgggc cgtacgaatt ccttaggcta ccgggtaggg gaggcgcttt 1860tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa 1920gtggcctctg gcctcgcaca cattccacat ccaccggccg gtaggcgcca accggctccg 1980ttctttggtg gccccttcgc gccaccttct actcctcccc tagtcaggaa gttccccccc 2040gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct cactagtctc 2100gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg cagcggccaa 2160tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg gtccgggggc 2220gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg aggcccggca 2280ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc atctccgggc 2340ctttcgacca gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg 2400acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc 2460acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca 2520cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg 2580tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca 2640tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc 2700cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc 2760agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg 2820gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg 2880gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc 2940gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac 3000gaccccatgc atcgtagacg aaatgaccga ccaagcgacg cccaacctgc catcacgaga 3060tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc 3120cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg 3180gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat 3240aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc 3300ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt 3360ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa 3420cgtcggggcg gcaggccccc agcttttgtt ccctttagtg agggttaatt tcgagcttgg 3480cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 3540acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 3600cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcatc 3660gcgagcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 3720tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 3780aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 3840tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 3900ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 3960tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 4020gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 4080aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 4140agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 4200acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 4260actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 4320accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 4380actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 4440cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 4500cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 4560gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 4620ataggtgcct cactgattaa gcattggtaa ctgtcagact cgcgacactg cattaatgaa 4680tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 4740ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4800taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4860agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4920cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4980tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 5040tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 5100gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 5160acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5220acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5280cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5340gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5400gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 5460agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5520ctgacgctca gtggaacgaa aactca 5546

Claims

1. An isolated polynucleotide comprising:a) a methylation-free GC-rich element comprising at least 500 contiguous nucleotides from the promoter region of a ribosomal protein gene;b) a heterologous promoter; andc) a transcribable nucleic acid sequence adjacent said heterologous promoter wherein the transcribable nucleic acid sequence is transcribed from said heterologous promoter and the level of said transcription is enhanced by said element.

2. The polynucleotide according to claim 1, wherein said ribosomal protein gene is selected from the group consisting of RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37ARPL38, RPL39, RPL41, RPLP0, RPLP1, RPLP2.

3. The polynucleotide according to claim 1, wherein said ribosomal protein gene is RPS3 or RPS11.

4. The polynucleotide according to claims 1, wherein said element comprises at least 1000 contiguous nucleotides from the promoter region of a ribosomal protein gene.

5. The polynucleotide according to claim 1, wherein said element comprises more than 3 kb 5' untranslated sequence from said ribosomal protein gene.

6. (canceled)

7. The polynucleotide according to claim 1, wherein said methylation-free GC-rich element is an extended methylation-free CpG island.

8. (canceled)

9. The polynucleotide according to claim 1, wherein said ribosomal protein gene is a mammalian gene.

10. The polynucleotide according to claim 9, wherein said ribosomal protein gene is a murine gene.

11. The polynucleotide according to claim 10, wherein said methylation-free GC-rich element comprises the nucleotide sequence of SEQ ID NO:1.

12. The polynucleotide according to claim 10, wherein said methylation-free GC-rich element comprises the nucleotide sequence of SEQ ID NO:2.

13. The polynucleotide according to claim 9, wherein said ribosomal protein gene is a human gene.

14. The polynucleotide according to claim 1, wherein said heterologous promoter is a constitutive promoter.

15. The polynucleotide according to claim 14, wherein said constitutive promoter is selected from the group consisting of cytomegalovirus early/immediate promoter, SV40, EF-1.alpha., Rous sarcoma virus (RSV) LTR and HIV2 LTR

16. The polynucleotide according to claim 1, wherein said heterologous promoter is a tissue-specific promoter.

17. The polynucleotide according to claim 16, wherein said heterologous promoter is a tumour-selective promoter.

18. The polynucleotide according to claim 17, wherein said promoter is selected from the group consisting of carcino-embryonic antigen (CEA), prostate-specific antigen (PSA), cyclooxygenase-2 (COX-2), alpha-fetoprotein (AFP), tyrosinase, and T-cell Factors 1-4 (TCF)-based promoters.

19. The polynucleotide according to claim 1, wherein said transcribable nucleic acid encodes a polypeptide selected from the group consisting of an antibody, a functional epitope-binding fragment of an antibody, a growth factor, cytokine, a protein kinase, a soluble receptor, a membrane-bound receptor, and a blood clotting factor.

20. A vector comprising the polynucleotide of claim 1.

21. The vector according to claim 20, wherein the vector is a eukaryotic expression vector.

22. An expression vector comprising:a) a methylation-free GC-rich element comprising at least 500 contiguous nucleotides from the promoter region of a ribosomal protein gene;b) a heterologous promoter; andc) a multiple cloning site;wherein a transcribable nucleic acid sequence inserted into said multiple cloning site and expression of said transcribable nucleic acid sequence from said heterologous promoter is enhanced by said element.

23. A host cell comprising the polynucleotide according to claim 1.

24. The host cell according to claim 23, wherein said cell is selected from the group consisting of CHO, NS0, BHK, HeLa, and HepG2.

25. A method of increasing expression of a polypeptide encoded by a transcribable nucleic acid comprising inserting the expression vector according to claim 22 into an appropriate host cell and culturing said host cell under suitable conditions, thereby to increase expression of said polypeptide.

26. The method according to claim 25, wherein said polypeptide is a therapeutically useful polypeptide.

27. (canceled)

28. The polynucleotide according to claim 1, wherein the heterologous promoter is a guinea pig CMV immediate/early promoter.

29. The polypeptide of claim 25, wherein the polypeptide is an antibody or a functional epitope-binding fragment thereof.

30. A method of increasing the expression of a transcribable nucleic acid in a host cell, the method comprising the steps of:a) transfecting the host cell with a vector comprising the transcribable nucleic acid operably linked to a heterologous promoter which is operably linked to a methylation-free GC-rich element derived from an rps3 gene;b) culturing the host cell under suitable conditions, thereby to allow for the expression of the transcribable nucleic acid,wherein the expression of the transcribable nucleic acid is increased in the presence of the methylation-free GC-rich element relative to expression obtained in the presence of the heterologous promoter alone.

31. The method of claim 30, wherein the transcribable nucleic acid encodes an antibody.

32. The method of claim 30, wherein the rps3 gene is a murine gene.

33. The method of claim 32, wherein the murine gene is a mouse gene.

34. The method of claim 32, wherein the host cell is a CHO cell.

35. The method of claim 32, wherein the heterologous promoter is a guinea pig CMV promoter.

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