MXPA99002661A - High level expression of proteins - Google Patents

Abstract

The invention features a synthetic gene encoding a protein normally expressed in a mammalian cell wherein atleast one non-preferred or less preferred codon in the natural gene encoding the protein has been replaced by a preferred codon encoding the same amino acid.

Description

SXHIESION OF HIGH LEVEL PROTEINS Field of the Invention The invention concerns genes and methods for expressing eukaryotic and viral proteins at high levels in eukaryotic cells. BACKGROUND OF THE INVENTION The expression of eukaryotic gene products in prokaryotes is sometimes limited by the presence of codons that are used infrequently in E. coli. The expression of these genes can be increased by systematically replacing the endogenous codons with codons on represented in highly expressed prokaryotic genes (Robinson et al., Nucleic Acids Res. 12: 6663, 1984). It is commonly assumed that rare codons cause the pausing of the ribosome, leading to a failure to complete the nascent polypeptide chain and a decoupling of transcription and translation. It is thought that pausing the ribosome leads to exposure of the 3 'end of the mRNA to cellular ribonucleases. SUMMARY OF THE INVENTION The invention provides a synthetic gene that encodes a protein normally expressed in a mammalian cell or other eukaryotic cell wherein at least one non-preferred or less preferred codon in the natural gene encoding the protein has been replaced by a preferred codon encoding the same amino acid. The preferred codons are: Ala (gcc); Arg (cgc); Asn (aac); Asp (gac); Cys (tgc); Gln (cag); Gly (ggc); His (cac), lie (ate); Leu (ctg); Lys (aag); Pro (ecc); Phe (ttc); Ser (age); Thr (acc); Tyr (tac); and Val (gtg). The less preferred codons are: Gly (ggg); lie (att); Leu (ctc); Ser (tec); Val (gtc); and Arg (agg). All codons that do not satisfy the description of preferred codons or less preferred codons are non-preferred codons. In general, the degree of preference of a particular codon is indicated by the codon prevalence in highly expressed human genes as indicated in Table 1 under the heading "High". For example, "ate" represents 77 percent of the lie codons in genes of highly expressed mammals and is the preferred lie codon, "att" represents 18 percent of the lie codons in genes of highly expressed mammals and is the codon lie less preferred. The "ata" sequence represents only 5 percent of the lie codons in highly expressed human genes since it is a non-preferred lie codon. The replacement of a codon with another codon that is more prevalent in highly expressed human genes will generally increase the expression of the gene in mammalian cells. In accordance with the foregoing, the invention includes replacing a less preferred codon with a preferred codon as well as replacing a non-preferred codon with a preferred or less preferred codon.

By "protein normally expressed in a mammalian cell" is meant a protein that is expressed in mammals under natural conditions. The term includes genes in the mammalian genome such as those encoding Factor VIII, Factor IX, interleukins, and other proteins. The term also includes genes that are expressed in a mammalian cell under disease conditions such as oncogenes as well as genes that are encoded by a virus (including a retrovirus) which are expressed in cells of post-infection mammals. By "protein normally expressed in a eukaryotic cell" is meant a protein that is expressed in a eukaryote under natural conditions. The term also includes genes that are expressed in a mammalian cell under disease conditions. In preferred embodiments in synthetic gene it is capable of expressing the mammalian or eukaryotic protein at a level that is at least 110 percent, 150 percent, 200 percent, 500 percent, 1,000 percent, 5,000 percent or up to 10,000 percent. percent expressed by the "natural" (or "original") gene in a mammalian cell culture system in vitro under identical conditions (ie, the same cell type, the same culture conditions), the same expression vector). Suitable cell culture systems for measuring the expression of the synthetic gene and the corresponding natural gene are described below. Other convenient expression systems employing mammalian cells are well known to those skilled in the art and are described in, for example, the standard molecular biology reference works noted below. By "expression" is meant the expression of protein. The expression can be measured using an antibody specific for the protein of interest. These antibodies and measurement techniques are well known to those skilled in the art. By "natural gene" and "original gene" is meant the gene sequence (including allelic variants that occur naturally) that naturally encodes the protein, i.e., the original or natural coding sequence. In other preferred embodiments at least 10 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, or 90 percent of the codons in the natural gene are non-preferred codons. In other preferred embodiments at least 10 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, or 90 percent of the non-preferred codons in the gene are replaced with preferred codons or less preferred codons. In other preferred embodiments at least 10 percent, 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, or 90 percent of the non-preferred codons in the gene are replaced with preferred codons.

In a preferred embodiment, the protein is a retroviral protein. In a more preferred embodiment the protein is a lentiviral protein. In still a more preferred embodiment the protein is an HIV protein. In other preferred embodiments, the protein is gag, pol, env, gpl20, or gpldO. In other preferred embodiments the protein is a human protein. In more preferred embodiments, the protein is human Factor VIII and the protein is from human Factor VIII with the deleted B region. In another preferred embodiment the protein is green fluorescent protein. In various preferred embodiments at least 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, and 95 percent of the synthetic codons are preferred or less preferred codons. The invention also provides an expression vector comprising the synthetic gene. In another aspect the invention offers a cell that breeds the synthetic gene. In several preferred embodiments the cell is a prokaryotic cell and the cell is a mammalian cell. In preferred embodiments the synthetic gene includes less than 50, less than 40, less than 30, less than 20, less than 10, less than 5, or no "cg" sequence. The invention also provides a method for preparing a synthetic gene that encodes a protein normally expressed by a mammalian cell or other eukaryotic cell. The method includes identifying non-preferred and less preferred codons in the natural gene encoding the protein and replacing one or more of the non-preferred codons and less preferred codons with a preferred codon encoding the same amino acid as the replaced codon. Under some circumstances (for example, to allow the introduction of a restriction site) it may be desirable to replace a non-preferred codon with a less preferred codon instead of a preferred codon. It is not necessary to replace all the less preferred or non-preferred codons with preferred codons. The augmented expression can be carried out even with the partial replacement of less preferred or non-preferred codons with preferred codons. Under certain circumstances it may be desirable to only partially replace non-preferred codons with preferred or less preferred codons in order to obtain an intermediate level of expression. In other preferred embodiments the invention offers vectors (including expression vectors) comprising one or more of the synthetic genes. By "vector" is meant a DNA molecule, derived, for example, from a plasmid, bacteriophage, or virus from mammals or insects, into which fragments of DNA can be inserted or cloned. A vector will contain one or more unique restriction sites and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible. Thus, by "expression vector" is meant any autonomous element capable of directing the synthesis of a protein. These DNA expression vectors include plasmids and mammalian viruses. The invention also provides synthetic gene fragments that encode a desired portion of the protein. These synthetic gene fragments are similar to the synthetic genes of the invention except that they encode only a portion of the protein. These gene fragments preferably encode at least 50, 100, 150, or 500 contiguous amino acids of the protein. In order to construct the synthetic genes of the invention it may be desirable to avoid CpG sequences since these sequences can cause gene silencing. Thus, in a preferred embodiment, the coding region of the synthetic gene does not include the "cg" sequence. The codon bias present in the HIV gpl20 env gene is also present in the gag and pol genes. Thus, replacement of a portion of the non-preferred and less preferred codons found in these genes with preferred codons should produce a gene capable of higher level expression. A large fraction of codons in the human genes encoding Factor VIII and Factor IX are non-preferred codons or less preferred codons. Replacement of a portion of these codons with preferred codons should produce genes capable of higher level expression in mammalian cell cultures. The synthetic genes of the invention can be introduced into the cells of a living organism. For example, vectors (viral or non-viral) can be used to introduce a synthetic gene into cells of a living organism for gene therapy. Conversely, it may be desirable to replace preferred codons in a naturally occurring gene with less preferred codons as a means of decreasing expression. Standard reference works describing the general principles of recombinant DNA technology include Watson et al., Molecular Biology of the Gene, Volumes I and II, the Benjamin / Cummings Publishing Company, Inc., publisher, Menlo Park, CA (1987). ); Darnell et al., Molecular Cell Biology- Scientific American Books, Inc., Publisher, New York, N: Y: (1986); Oíd et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering-2d edition, University of California Press, publisher, Berkeley, CA (1981); Maniatis et al., Molecular Cloning; A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, NY (1989); and Current Protocols in Molecular Biology, Ausubel et al., Wiley Press, New York, NY (1992). By "transformed cell" is meant a cell in which (or in an ancestor of which) a selected DNA molecule, for example a synthetic gene, has been introduced by recombinant DNA techniques. By "set for expression" is meant that a DNA molecule, for example, a synthetic gene, is placed adjacent to a DNA sequence which directs the transcription and translation of the sequence (i.e., facilitates the production of the protein encoded by the synthetic gene). Description of the Drawings Figure 1 represents the sequence of the synthetic gpl20 and a synthetic gpldO gene in which the codons have been replaced by those found in highly expressed human genes. Figure 2 is a schematic drawing of the synthetic gene gpl20 (HIV-1 MN). The shaded portions marked vi to v5 indicate hypervariable regions. The filled box indicates the CD4 link site. A limited number of restriction sites are shown: H (Hind3), Nh (Nhel), P (Pstl), Na (Nael), M (Mlul), R (EcoRl), A (Agel) and No (Notl). Chemically synthesized DNA fragments that serve as tempers of the polymerase chain reaction are shown below the gpl20 sequence, along with the sites of the primers used for amplification. Figure 3 is a photograph of the results of the transient transfection assays used to measure the expression of gpl20. Gel electrophoresis of immuno-precipitated supernatants of 293T cells transfected with plasmids expressing gpl20 encoded by HIV-1 isolate IIIB (gpl20IIIb), by HIV-1 MN isolate (gpl20mn), by HIV MN isolate -l modified by substitution of the endogenous forward peptide with that of the CD5 antigen (gpl20mnCD5L), or by the chemically synthesized gene encoding the MN variant of the HIV-1 with the forward CD5 (syngpl20mn). The supernatants were harvested after a period of 12 hours marked 60 hours after transfection and immunoprecipitated with CD4 fusion protein: IgGl and protein A sepharose. Figure 4 is a graph depicting the results of the immunosorbent assay of linked enzyme (ELISA) used to measure protein levels in supernatants of transiently transfected 293T cells. Supernatants of 293T cells transfected with plasmids expressing gpl20 encoded by HIV-1 isolate IIIB (gpl20IIIb), by the MN isolate of HIV-1 (gpl20mn), by the MN isolate of HIV-1 modified by substitution of the endogenous forward peptide with that of the CD5 antigen (gpl20mnCD5L), or by the chemically synthesized gene encoding the MN variant of HIV-l with the forward CD5 (syngpl20mn) were harvested after 4 days and the enzyme-linked immunosorbent assay was applied in gpl20 / CD4. The level of gpl20 is expressed in nanograms / milliliters). Figure 5A is a photograph of a gel illustrating the results of an immunoprecipitation assay used to measure the expression of the original and synthetic gpl20 in the presence of rev in trans and RRE in cis. In this experiment, 293T cells were transiently transfected by coprecipitation of calcium phosphate of 10 μg of plasmid expressing: (A) the synthetic gpl20MN sequence and RRE in cis, (B) the gpl20 portion of HIV-IIIB, (C) gpl20 portion of HIV-1 IIIB and RRE in cis, all in the presence or absence of the expression rev. The RRE gpl20IIIbRRE and syngpl20mnRRE constructs were generated using an Eagl / Hpal RRE fragment cloned by the polymerase chain reaction of a HIV-1 proviral clone HXB2. Each gpl20 expression plasmid was cotransfected with 10μg of plasmid DNA pCMVrev or CDM7. The supernatants were harvested 60 hours later * of the transfection, immunoprecipitated with CD4 fusion protein: IgG and protein A agarose, and run on a 7% reduction SDS-PAGE. The gel exposure time was extended to allow the induction of gpl20IIIbrre to be demonstrated by rev. 15 Figure 5B is a shorter exposition of a similar experiment in which syngpl20mnrre was co-transfered with or without • pCMVrev. Figure 5C is a schematic diagram of the constructions used in Figure 5A. Figure 6 is a comparison of the rat ratty-1 natural type (wt) rat sequence and a synthetic ratTHY-1 gene (env) constructed by chemical synthesis and having the most prevalent codons found in the HIV env gene -l. Figure 7 is a schematic diagram of the synthetic gene. coratTHY-1. The solid black box denotes the signal peptide. The shaded picture denotes sequences in the precursor that direct the binding of an anchor of phosphatidylinositol glycan. The unique restriction sites used for the assembly of the THY-1 constructs are labeled H (Hind3), M (Mlul), S (Sacl) and No (Notl). The position of the synthetic oligonucleotides used in the construction are shown in the lower part of the figure. Figure 8 is a graph that represents the results of the flow analysis of cytometry. In this experiment 293T cells transiently transfected with an expression plasmid of ratTHY-1 wild-type (thick line), expression plasmid ratTHY-1 with envelope codons (thin line), or only vector (dotted line) by coprecipitation with phosphate calcium. The cells were stained with anti-ratThy-1 0X7 monoclonal antibody followed by a polyclonal anti-mouse IgG FITC antibody 3 days after transfection. Figure 9A is a photograph of a gel illustrating the results of immunoprecipitation analysis of supernatants of human 293T cells transfected with either syngpl20mn (A) or a syngpl20mn construct. rTHY-lenv having the rTHY-lenv gene in the 3 'untranslated region of the syngpl20mn gene (B). The construction syngpl20mn. rTHY-lenv was generated by inserting a Notl adapter into the obtuse Hind3 site of the rTHY-lenv plasmid. Subsequently, a 0.5 kb Notl fragment containing the rTHY-lenv gene was cloned into the Notl site of the syngpl20mn plasmid and tested for correct orientation. Supernatants of 35S-labeled cells were harvested 72 hours post transfection, were precipitated with CD4 fusion protein: IgG and protein A agarose, and were operated on a 7% reduction SDS-PAGE. Figure 9B is a schematic diagram of the constructs used in the experiment depicted in Figure 9A. Figure 10A is a photograph of COS cells transfected with vector only showing no fluorescence of green fluorescent protein (GFP). Figure 10B is a photograph of COS cells transfected with a CDM7 expression plasmid encoding a green overlapping fluorescent protein (GFP) to include a consensus translation initiation sequence. Figure 10C is a photograph of COS cells transfected with an expression plasmid having the same flanking sequences and consensus of initiation as in Figure 10B, but carrying a gene sequence with codons optimized. Figure 10D is a photograph of COS cells transfected with an expression plasmid as in Figure 10C, but carrying a Thr at residue 65 instead of Ser. Figure 11 depicts the sequence of a synthetic gene encoding green fluorescent proteins (SEQ ID NO: 40). Figure 12 represents the sequence of an original human Factor VIII gene lacking central domain B (amino acids 760-1639, inclusive) (SEQ ID NO: 41).

Figure 13 represents the sequence of a synthetic human Factor VIII gene lacking central domain B (amino acids 760-1639, inclusive) (SEQ ID NO: 42). Description of the Preferred Modalities EXAMPLE 1 Construction of a gp! 20 synthetic gene having codons found in highly expressed human genes A codon frequency table for the envelope precursor of the HIV-1 LAV subtype was generated using software developed by the Group. of Genetics Computation of the University of Wisconsin (University of Wisconsin Genetics Computer Group). The results of that tabulation are compared in Table 1 with the codon usage pattern by a collection of highly expressed human genes. For any amino acid encoded by degenerate codons, the most favored codon of the highly expressed genes is different from the most favored codon of the HIV envelope precursor. Moreover, a simple rule describes the pattern of favored envelope codons whenever it is applied: the preferred codons maximize the number of adenine residues in the viral RNA. In all but one case this means that the codon in which the third position is A is the one most frequently used. In the special case of serine, three codons also contribute a residue A for the mRNA; these three together comprise 85 percent of the serine codons actually used in envelope transcripts. A particularly striking example of the preponderance of A lies in the codon choice for arginine, in which the AGA triad comprises 88 percent of the arginine codons. In addition to the preponderance of residues A, a marked preference for uridine is seen between the degenerate codons whose third residue must be a pyrimidine. Finally, inconsistencies between the variants used less frequently can be considered by observing that the CpG dinucleotide is underrepresented; thus the third position is less likely to be G provided the second position is C, as in the codons for alanine, proline, serine and threonine; and the CGX triads for arginine are hardly ever used.

TABLE 1 Codon frequency in HIV-1 Illb env gene and in highly expressed human genes High Env High Env Ala Cys GC C 53 27 TG C 68 16 T 17 18 T 32 84 A 13 50 G 17 5 Gln CA A 12 55 Arg G 88 45 GC C 37 0 T 7 4 Glu A 6 0 GA A 25 67 G 21 0 G 75 33 AG A 10 88 G 18 8 Gl? GG C 50 6 Asn T 12 13 AA C 78 30 A 14 53 T 22 70 G 24 J28 Asp His GA C 75 33 CA C 79 25 T 25 67 T 21 75 lie AT C 77 25 T 18 31 A 5 44 Leu Ser CT C 26 10 TC C 28 8 T 5 7 T 13 8 A 3 17 A 5 22 G 58 17 G 9 0 TT A 2 30 AG C 34 22 G 6 20 T 10 41 Lys Thr AA A 18 68 AC C 57 20 G 82 32 T 14 22 A 14 51 Pro Tyr cc c 48 27 TA C 25 12 T 19 14 T 26 92 A 16 55 G 17 5 Phe Val TC 80 26 GT C 25 12 T 20 74 T 7 9 A 5 62 G 64 18 The codon frequency was calculated using the GCG program established by the University of Wisconsin Genetics Computer Group. The numbers represent the percentage of cases in which the particular codon is used. The codon usage frequencies of the envelope genes or other isolates of the HIV-1 virus are comparable and show a similar bias. In order to produce a gpl20 gene capable of high level expression in mammalian cells, a synthetic gene encoding the gpl20 segment of HIV-1 (syngpl20mn) was constructed based on the sequence of the most common North American subtype, HIV-1 MN (Shaw et al, Science 226: 1165, 1984; Gallo et al., Nature 321: 119, 1986). In this synthetic gpl20 gene almost all original codons have been systematically replaced with codons most frequently used in highly expressed human genes (Figure 1). This synthetic gene was assembled from chemically synthesized oligonucleotides of 150 to 200 bases in length. If oligonucleotides exceeding 120 to 150 bases are chemically synthesized, the percentage of full-length product may be low, and the vast excess of material consists of shorter oligonucleotides. Since these shorter fragments inhibit the polymerase chain reaction and cloning procedures, it can be very difficult to use oligonucleotides that exceed a certain length. In order to use crude synthesis material without prior purification, combinations of single-stranded oligonucleotides were amplified by polymerase chain reaction prior to cloning. The polymerase chain reaction products were purified on agarose gels and used as quenched in the next polymerase chain reaction step. Two adjacent fragments could be co-amplified due to overlap sequences at the end of one of the two fragments. These fragments, which were between 350 and 400 base pairs in size, were subcloned into plasmid derived pCDM7 which contained the forward sequence of the surface molecule CD5 followed by a polylinker Nhel / Pstl / Mlul / EcoRl / BamHl. Each of the restriction enzymes in this polylinker represents a site that is present at the 5 'or 3' end of the fragments generated by polymerase chain reaction. Thus, by sequential subcloning of each of the 4 long fragments, the complete gpl20 gene was assembled. For each fragment three to six different clones were subcloned and sequenced before assembly. A schematic drawing of the method used to construct the synthetic gpl20 is shown in Figure 2. The sequence of the synthetic gpl20 gene (and a synthetic gpl60 gene created using the same approach) is presented in Figure 1. The mutation regimen was considerable. The most commonly found mutations were short (1 nucleotide) and long (up to 30 nucleotides) deletions. In some cases it was necessary to exchange parts with one of the synthetic adapters or pieces of other subclones without mutation in that particular region. Some deviations from strict adherence were made for the use of codon optimized to accommodate the introduction of restriction sites in the resulting gene to facilitate the replacement of several segments (Figure 2). These unique restriction sites were introduced into the gene at intervals of approximately 100 base pairs. The forward sequence of the original HIV was exchanged with the highly efficient forward peptide of the human antigen CD5 to facilitate secretion (Aruffo et al., Cell 61: 1303, 1990). The plasmid used for the construction is a derivative of the mammalian expression vector pCDM7 which transcribes the inserted gene under the control of a strong human CMV immediate early promoter. To compare the coding sequences of the gpl20 of the wild-type and synthetic type, the coding sequence of the synthetic gpl20 was inserted into a mammalian expression vector and tested in transient transfection assays. Several different natural genes gpl20 were used as controls to exclude variations in expression levels between different virus isolates and aberrant particles induced by different forward sequences. The gpl20VIH Illb construct used as control was generated by polymerase chain reaction using a Sall / Xhol HIV-1 HXB2 envelope fragment as annealed. To exclude mutations induced by polymerase chain reaction, a Kpnl / Earl fragment containing approximately 1.2 kb of the gene was changed with the respective sequence of the proviral clone. The wild-type gpl20mn constructs used as controls were cloned by polymerase chain reaction from C8166 cells infected with HIV-lMN (AIDS Repository, Rockville, MD) and expressed gpl20 with either a natural cover or a CD5 forward sequence. . Since proviral clones were not available in this case, two clones of each construct were tested to avoid aberrant particles of the polymerase chain reaction. To determine the amount of secreted gpl20, semi-quantitatively supernatants of transiently transfected 293T cells were immunoprecipitated by coprecipitation of calcium phosphate with CD4 fusion protein: soluble immunoglobulin and protein A sepharose. The results of this analysis (Figure 3) show that the synthetic gene product is expressed at a very high level compared to that of the natural gpl20 controls. The molecular weight of the synthetic gpl20 gene was comparable to the control proteins (Figure 3) and appeared to be in the range of 100 to 110 kd. The slightly faster migration can be explained by the fact that in some tumor cell lines, for example, 293T, the glycosylation is not complete or altered to some extent. To more accurately compare the expression, protein levels of gpl20 were quantified using an enzyme-linked immunosorbent assay of gpl20 with CD4 in the demobilized phase. This analysis shows (Figure 4) that the data from the bound enzyme-linked immunosorbent assay was comparable to the immunoprecipitation data, with a gpl20 concentration of approximately 125 nanograms / milliliter for the synthetic gpl20 gene, and less than the background cut ( 5 nanograms / milliliter) for all natural gpl20 genes. Thus, the expression of the synthetic gene gpl20 appears to be at least an order of magnitude greater than the gpl20 genes of the wild type. In the experiment shown, the increase was at least 25 times. The role of rev in the expression of gpl20 Since rev seems to exert its effect in several steps in the expression of a viral transcription, the possible role of non-translation effects in the improved expression of the synthetic gpl20 gene was tested. First, to rule out the possibility of deleting elements of negative signals that confer either increased mRNA degradation or nucleic retention by changing the nucleotide sequence, cytoplasmic mRNA levels were tested. The cytoplasmic RNA was prepared by NP40 lysis of transiently transfected 293T cells and the subsequent elimination of the nuclei by centrifugation. The cytoplasmic RNA was subsequently prepared from lysates by multiple extractions in phenol and precipitation, stained in nitrocellulose using a slot staining apparatus and finally hybridized with specific probe cover. In summary, cytoplasmic RNA was isolated from 293 cells transfected with CDM & amp; amp;;, gpl20 IIB or syngpl20, 36 hours post transfection. The cytoplasmic RNA of Hela cells infected with wild type vaccinia virus or recombinant virus expressing gpl20 Illb or the synthetic gpl20 gene was under the control of the 7.5 promoter was isolated 16 hours post infection. Equal amounts were stained on nitrocellulose using a slot staining device and hybridized with fragments labeled 1.5 kg of gpl20IIIb and syngpl20 or human beta-actin. The expression levels of the RNA were quantified by scanning the membranes hybridized with a phosphor-imaging agent. The procedures used are described in more detail below. This experiment demonstrated that there was no significant difference in the mRNA levels of cells transfected with natural or synthetic gpl20 gene. In fact, in some experiments the level of cytoplasmic mRNA of the synthetic gpl20 gene was even lower than that of the natural gpl20 gene. These data were confirmed by measuring the expression of recombinant vaccinia viruses. Human 293 cells or Hela cells were infected with vaccinia virus expressing the gpl20 Illb wild-type or syngpl20mn in a multi-infection plicity of at least 10. The supernatants were harvested 24 hours post infection and were immunoprecipitated with protein of fusion of CD4: immunoglobin and protein A sepharose. The procedures used in this experiment are described in greater detail below. This experiment demonstrated that increased expression of the synthetic gene was still observed when the product of the endogenous gene and the product of the synthetic gene were expressed from vaccinia virus recombinants under the control of the early and late promoter of 7.5k strong mixed. Because vaccinia virus mRNAs are transcribed and translated into the cytoplasm, the increased expression of the synthetic envelope gene in this experiment can not be attributed to improved export of the nucleus. This experiment was repeated in two additional human cell types, the cancer cell line of Kidney 293 and HeLa cells. As with the transfected 293T cells, mRNA levels were lar in 293 cells infected with any of the recombinant vaccinia viruses. Codon use in lentiviruses Since the codon usage appears to have a significant impact on expression in mammalian cells, the codon frequency was examined in the envelope genes of other retroviruses. This study found no clear pattern of codon preference among retroviruses in general. However, if viruses of the genus lentivirus, to which HIV-1 belongs, are analyzed separately, a preponderance of codon use was found almost identical to that of HIV-1. A codon frequency table of the envelope glycoproteins of a variety of retroviruses (predominantly type C) excluding lentiviruses was prepared, and a codon frequency table created from the envelope sequences of four not closely related lentiviruses was compared. with HIV-1 (goat arthritis encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, and visna virus) (Table 2). The codon usage pattern for lentiviruses is strikingly lar to that of HIV-1, in all but one case, the preferred codon for HIV-1 is the same as the preferred codon for other lentiviruses. The exception is proline, which is encoded by CCT in 41 percent non-HIV lentiviral envelope waste, and by CCA in 40 percent waste, a situation that clearly also reflects a significant preference for the triad that ends in A. The codon usage pattern by non-lentiviral envelope proteins does not show a lar predominance of A residues, and neither C and G residues are biased towards the third position as the codon usage for highly expressed human genes. In general, non-lentiviral retroviruses seem to exploit the different codons more equally, a pattern they share with less highly expressed human genes.

TABLE 2 Codon frequency in the envelope gene of lentiviruses (lenti) and non-lentiviral retroviruses (other) Other Lenti Other Lenti Cys GC C 45 13 TG C 53 21 T 26 37 T 47 79 A 20 46 G 9 3 Gln CA A 52 69 Arg G 48 31 GC C 14 2 T 6 3 Glu A 16 5 GA A 57 68 G 17 3 G 43 32 AG A 31 51 G 15 26 Gly GG C 21 8 Asn T 13 9 AA C 49 31 A 37 56 T 51 69 G 29 26 Asp His GA C 55 33 CA C 51 38 T 51 69 T 49 62 lie AT C 38 16 T 31 22 A 31 61 Leu Ser CT C 22 8 TC C 38 10 T 14 9 T 17 16 A 21 16 A 18 24 G 19 11 G 6 5 TT A 15 41 AG C 13 20 G 10 16 T 7 25 Lys Thr AA A 60 63 AC C 44 18 G 40 37 T 27 20 A 19 55 G 10 8 Pro CC c 42 14 T 30 41 Tyr A 20 40 TA C 48 28 G 7 5 T 52 72 Phe Val TT C 52 25 GT C 36 9 T 48 75 T 17 10 A 22 54 G 25 27 The codon frequency was calculated using the GCG program established by the University of Wisconsin Genetics Computer Group. The numbers represent the percentage of cases in which a particular codon is used. The use of the non-lentiviral retrovirus codon was compiled from the bovine leukemia virus, feline leukemia virus, human T cell leukemia virus type I, human T cell lymphotropic virus type II, isolated isolates that form the envelope precursor sequences of the bovine leukemia virus. focus of the murine leukemia virus isolated murine cell (MuLV), the isolate that forms the Rauscher -bacus focus, the isolate 10A1, the amphotropic isolate 4070A and the isolate of the myeloproliferative leukemia virus, and the leukemia virus of rat, simian sarcoma virus, simian T cell leukemia virus, leuko-mogenic retrovirus T1223 / B and gibbon monkey leukemia virus. The codon frequency tables for the non-HIV, non-SIV lentiviruses were compiled from the envelope precursor sequences for the goat arthritis encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus, and visna virus. In addition to the prevalence of codons containing an A, the lentiviral codons adhere to the HIV pattern of strong CpG under representation, so that the third position for the triads of alanine, proline, serine and threonine is rarely G. The triads of Retroviral envelopes show a sub-presentation of CpG similar, but less pronounced. The most obvious difference between lentiviruses and other retroviruses with respect to the CpG prevalence lies in the use of the CGX variant of the arginine triads, which is often reasonably represented among the retroviral envelope coding sequences, but it is almost never present among comparable lentivirus sequences. Differences in rev dependence between natural and synthetic gp! 20 To examine whether regulation by rev is linked to the use of the HIV-1 codon, the influence of rev on the expression of both the natural and synthetic gene was investigated. Since rev-regulation requires the RRE binding site in cis, constructs were made in which this binding site was cloned in the 3'-untranslated region of both the natural and the synthetic gene. These plasmids were co-transfected with rev or a trans-control plasmid in 293T cells, and the gpl20 expression levels in the supernatants were measured semi-quantitatively by immunoprecipitation. The procedures used in this experiment are described in more detail below. As shown in Figure 5A and Figure 5B, rev upregulates the natural gpl20 gene, but has no effect on the expression of the synthetic gpl20 gene. Thus, the action of rev is not apparent in a substrate which lacks the coding sequence of endogenous viral envelope sequences. Expression of a synthetic ratTHY-1 gene with HIV envelope codons The experiment described above suggests that in fact the "envelope sequences" have to be present for the rev regulation. In order to test this hypothesis, a synthetic version of the gene encoding the small cell surface protein typically highly expressed was prepared, ratTHY-1 antigen. The synthetic version of the ratTHY-1 gene was designed to have a codon usage like that of HIV gpl20. To design this synthetic gene, the AUUUA sequences were abolished, which are associated with the instability of the mRNA. In addition, two restriction sites were introduced to simplify the manipulation of the resulting gene (Figure 6). This synthetic gene with the use of the HIV envelope codon (rTHY-lenv) was generated using three oligonucleotides of 150 to 170 mer (Figure 7). In contrast to the syngpl20mn gene, the polymerase chain reaction products were directly cloned and assembled into pUC12, and subsequently cloned into pCDM7. The expression levels of natural rTHY-1 and rTHY-1 with the HIV envelope codons were quantified by immuno-fluorescence of transiently transfected 293T cells. Figure 8 shows that the expression of the natural THY-1 gene is almost two orders of magnitude above the background level of the transfected control cells (pCDM7). In contrast, the expression of the synthetic ratTHY-1 is substantially less than that of the natural gene (shown by the change of the peak towards a lower channel number). To prove that negative sequence elements promoting mRNA degradation were not introduced inadvertently, a construct was generated in which the rTHY-lenv gene was cloned as the 3"end of the synthetic gpl20 gene (Figure 9B). transfected 293T cells with either the syngpl20mn gene or the syngpl20 / ratTHY-l env fusion gene (syngpl20mn, rTHY-lenv) Expression was measured by immunoprecipitation with CD4 fusion protein: IgG and protein A agarose. In this experiment, they were described in more detail below: Since the synthetic gpl20 gene has a stop codon UAG, rTHY-lenv does not translate from this transcript If there were negative elements that confer increased degradation in the sequence, the levels of gpl20 protein expressed from this construction should decrease compared to the construction of syngpl20mn without rTHY-lenv.Figure 9A, shows that the expression of both constructions is similar, indicating that the low expression must be linked to the translation. Rev dependent expression of the synthetic ratTHY-1 gene with envelope codons To explore whether rev is capable of regulating the expression of a ratTHY-1 gene having env codons, a construct with a rev binding site was made at the 3 'end of the open reading frame rTHYlenv. To measure the response rev of a ratTHY-lenv construct having a 3 'RRE, human 293T cells were cotransfected with ratTHY-lenvrre and CDM7 or pCMVrev. At 60 hours post transfection the cells were discarded with 1 mM EDTA in phosphate buffered saline (PBS) and stained with the mouse monoclonal antibody OX-7 anti rTHY-1 and a secondary antibody FITC-conjugated. The intensity of the fluorescence was measured using EPICS XL cytofluorometer. These procedures are described in more detail later. In repeated experiments, a slight increase in rTHY-lenv expression was detected if rev was cotransfected with the rTHY-lenv gene. To further increase the sensitivity of the assay system, a construct expressing a secreted version of the rTHY-lenv was generated. This construction should produce more reliable data because the accumulated amount of protein secreted in the supernatant reflects the result of protein production over an extended period, in contrast to the surface expressed protein, which seems to reflect more closely the current production regime . A gene capable of expressing a secreted form was prepared by polymerase chain reaction using forward and backward primers 3 'of the endogenous forward sequence and 5' of the sequence motif required for phosphatidylinositol glycan anchor respectively. The polymerase chain reaction product was cloned into a plasmid that already contained a CD5 forward sequence, thus generating a construction in which the anchor of the membrane has been deleted and the forward sequence exchanged by a heterologous (and probably more efficient) forward peptide. The responsivity rev of the secreted form of the ratTHY-lenv was measured by immunoprecipitation of supernatants from human 293T cells cotransfected with a plasmid expressing a secreted form of ratTHY-lenv and the RRE sequence in cis (rTHY-lenvPI-rre) and CDM7 or pCMVrev. The rTHY-lenvPI-RRE construct was made by polymerase chain reaction using the oligonucleotide: cgcggggctagcgcaaagagtaataagtttaac (SEQ ID NO: 38) as the forward primer, the oligonucleotide: cgcggatcccttgtattttgtactaata (SEQ ID NO: 39) as the reverse primer, and the synthetic rTHY-lenv construction as a tempered. After digestion with Nhel and Notl the polymerase chain reaction fragment was cloned into a plasmid containing the forward CD5 and RRE sequences. Supernatants of 35S-labeled cells were harvested 72 hours post transfection, were precipitated with a mouse monoclonal antibody 0X7 against rTHY-1 and anti-mouse IgG sepharose, and run on a SDS-PAGE with 12 percent reduction. In this experiment the induction of rTHY-lenv by rev was much more prominent and clear cut than in the experiment described above and strongly suggests that rev is able to regulate transcriptionally-like transcripts that are suppressed by codons of low use. Rev independent expression of a rTHY-lenv fusion protein: immunoglobulin To test if low-use codons must be present throughout the entire coding sequence or if a short region is sufficient to confer rev response, a fusion protein was generated rTHY-lenv: immunoglobulin. In this construct the rTHY-lenv gene (without the motif sequence responsible for anchoring phosphatidylinositol glycan) binds to the human IgG1 joint, domains CH2 and CH3. This construction was generated by anchor polymerase chain reaction using primers with Nhel and BamHl and rTHY-lenv restriction sites as annealing. The polymerase chain reaction fragment was cloned into a plasmid containing the forward sequence of the surface molecule CD5 and the joint, CH2 and CH3 parts of human IgGl immunoglobulin. A Hind3 / Eagl fragment containing the rTHY-lenvl insert was subsequently cloned into a derived pCDM7 plasmid with RRE sequence. To measure the response of the rTHY-lenv / immunoglobin fusion gene (rTHY-lenveglrre) to human 293T cells rev cotransfected with rTHY-lenveglrre and either pCDM7 or pCMVrev. The rTHY-lenveglrre construct was made by annealing polymerase chain reaction using forward and backward primers with restriction sites Nhel and BamHl respectively. The polymerase chain reaction fragment was cloned into a plasmid containing a forward CD5 and human IgG1 linkage, CH2 and CH3 domains. The supernatants of 35S-labeled cells were harvested 72 hours post transfection, were precipitated with a mouse monoclonal antibody 0X7 against rTHY-1 and anti-mouse IgG sepharose, and run on a SDS-PAGE at 12 percent reduction. The procedures used are described in more detail below. As with the rTHY-lenvPI gene product, this rTHY-lenv / immunoglobulin fusion protein is secreted into the supernatant. Thus, this gene must respond to the induction rev. However, unlike rTHY-lenvPI-, the cotransfection of rev in trans did not induce or only induced a negligible increase in the expression of rTHY-lenvegl. The expression of the fusion protein rTHY-1: immunoglobulin with natural rTHY-1 or envelope codons of HIV was measured by immunoprecipitation. Briefly, human 293T cells transfected with rTHY-lenvegl (env codons) or rTHY-lwtegl (original codons). The construction rTHY-lwtegl was generated in a similar way to that used for the construction rTHY-lenvegl, with the exception that a plasmid containing the native rTHY-1 gene was used as a template. Supernatants from 35S-labeled cells were harvested 72 hours post-transfection, precipitated with a mouse monoclonal antibody 0X7 against rTHY-1 and anti-mouse IgG sepharose, and run on a SDS-PAGE at 12 percent reduction. The procedures used in this experiment are described in more detail below. The expression levels of rTHY-lenvgl decreased compared to a similar construction with rTHY-1 of the wild type as the fusion partner, but were still considerably higher than rTHY-env. In accordance with the above, both parts of the fusion protein influenced expression levels. The addition of the rTHY-lenv did not restrict the expression to an equal level as seen for rTHY-lenv alone. Thus, the regulation by rev seems to be ineffective if the expression of the protein is not almost completely suppressed. Codon preference in HIV-1 envelope genes The direct comparison between the frequency of codon usage of the HIV envelope and highly expressed human genes reveals a striking difference for the twenty amino acids. A simple measure of the statistical significance of this codon preference is the finding that among nine amino acids with twice codon degeneracy, the third favored residue is A or U in the nine. The probability that the nine of two equiprobable choices will be the same is approximately 0.004, and therefore by any conventional measure the choice of the third residue can not be considered random. Further evidence of a biased codon preference is found among the more degenerate codons, where a strong selection of adenine-bearing triads can be seen. This contrasts with the pattern for highly expressed genes, which favor codons carrying C, or less commonly G, in the third position of codons with three or more times of degeneration. The systematic exchange of native codons with codons of highly expressed human genes markedly increased the expression of gpl20. A quantitative analysis by enzyme-linked immunosorbent assay showed that the expression of the synthetic gene was at least 25 times higher compared to the native gpl20 after transient transfection in human 293 cells. The concentration levels in the bound enzyme immunosorbent assay experiment shown were quite low. Since an enzyme-linked immunosorbent assay was used for the quantification based on gpl20 that binds to CD4, only natural, non-denatured material was detected. This may explain the apparent low expression. Measurement of cytoplasmic mRNA levels showed that the difference in protein expression is due to translation differences and not to the stability of the mRNA. Retroviruses in general do not show a similar preference towards A and T as found for HIV. But if this family is divided into two subgroups, lentiviruses and non-lentiviral retroviruses, a similar preference for A and, less frequently, T, was detected at the position of the third codon for lentivirus. Thus the available evidence suggests that lentiviruses retain a characteristic pattern of envelope codons not because of an inherent advantage of reverse transcription or replication of these residues, but rather for some reason peculiar to the physiology of that class of virus. The major difference between lentiviruses and non-complex retroviruses are additional regulatory genes and not essentially accessory, as already mentioned. Thus, a simple explanation of the restriction of the envelope expression could be that a regularly important mechanism of one of these additional molecules is based on it. In fact, it is known that one of these proteins, rev, which most likely has homologs in all lentiviruses. Thus the use of codon in viral mRNA is used to create a class of transcripts that is susceptible to the rev stimulatory action. This hypothesis was tested using a similar strategy as the previous one, but this time the codon usage was changed to the reverse direction. The codon usage of a highly expressed cellular gene was replaced with the codons most frequently used in the HIV envelope. As expected, the expression levels were considerably lower compared to the native molecule, almost two orders of magnitude analyzed by immunofluorescence of the molecule expressed on the surface. If rev was coexpressed in trns and an RRE element was present in cis, only a slight induction was found for the surface molecule.

However, if THY-1 was expressed as a secreted molecule, rev induction was much more prominent, supporting the previous hypothesis. This can probably be explained by the accumulation of the protein secreted in the supernatant, which considerably amplifies the rev effect. If rev induces only a smaller increase for surface molecules in general, the induction of the HIV envelope by rev can not have the purpose of an increased surface abundance, but rather of an increased intracellular gpldO level. It is completely unclear at the moment because this should be the case. To test whether the subtotal of elements of a gene are sufficient to restrict expression and make it dependent on rev, rTHYlenv: immunoglobulin fusion proteins were generated, in which only about one third of the total gene had the use of envelope codon . The expression levels of this construct were at an intermediate level, indicating that the negative sequence element rTHY-lenv is not dominant on the immunoglobulin part. This fusion protein was not or was only slightly responsive to rev, indicating that only genes almost completely suppressed can be responsive to rev. Another characteristic aspect that was found in the codon frequency tables is a striking under-representation of CpG triads. In a comparative study of codon usage in E. coli, yeast, drosophila and primates showed that in a large number of primate genes analyzed the 8 least used codons all contained codons with the CpG dinucleotide sequence. Avoiding codons that contain this dinucleotide motif was also found in the sequence of other retroviruses. It seems plausible that the reason for underrepresentation of triads carrying CpG has something to do with preventing silencing of the gene by methylation of CpG cytokines. The expected number of CpG dinucleotides for HIV as a whole is approximately one fifth of that expected based on the base composition. This could indicate that the possibility of high expression is restored, and that the gene indeed has to be highly expressed at some point during viral pathogenesis. The results presented herein clearly indicate that the preference of condoms has a severe effect on protein levels, and suggests that the lengthening of the translation is to control the expression of the mammalian gene. However, other factors can play a role. First, the abundance of non-maximally loaded mRNA in eukaryotic cells indicates that the onset is limiting the rate for translation in at least some cases, since otherwise all transcripts would be completely covered by ribosomes. Furthermore, if ribosome arrest and subsequent degradation of the mRNA were the mechanism, deletion by rare codons could more likely not be reversed by any regulatory mechanism such as that presented herein. A possible explanation for the influence of both the initiation and the lengthening of translation activity is that the initiation regime, or access to ribosomes, is controlled in part by indications distributed throughout the RNA, so that lentiviral codons They predispose RNA to accumulate in a combination of poorly initiated RNA. However, this limitation does not need to be kinetic; for example, the choice of codons could influence the likelihood that a given translation product, as soon as it is started, will be properly completed. Under this mechanism, the abundance of less favored codons could incur a significantly cumulative failure probability to complete the nascent polypeptide chain. An improved rate of initiation would then be lent to the spacer RNA by the action of rev. Since adenine residues are abundant in transcripts that respond to, it could be that RNA methylation of adenine mediates this suppression of translation. Detailed procedures The following procedures were used in the experiments described above. Sequence analysis Sequence analyzes used software developed by the University of Wisconsin Computer Group. Plasmid constructions The plasmid constructs employed the following methods. The vectors and the insert DNA were digested at a concentration of 0.5 μg / 10 μl in the appropriate restriction regulator for 1-4 hours (total reaction volume of approximately 30 μl). The digested vector was treated with 10 percent (volume / volume) of 1 μg / ml calf intestine alkaline phosphatase for 30 minutes before gel electrophoresis. Both the vector and insert digests (5 to 10 μl each) were operated on a 1.5% low melting point agarose gel with TAE regulator. Gel sections containing bands of interest were transferred to a 1.5 milliliter reaction tube, melted at 65 ° C and added directly to the ligation without removal of the agarose. Ligations were typically made in a total volume of 25 μL in Lx Regulator Under lx additions of ligations with 200-400 U of ligase, 1 μl of vector, and 4 μl of insert. When necessary, the overclocked 5 'ends were filled by adding 1/10 volume of 250 μM dNTP and 2-5 U of Klenow polymerase to heat inactivated digests or extracted by phenol and incubating for 20 minutes at room temperature. When necessary, the 3 'overclocked ends were filled by adding 1/10 volume of 2.5 mM dNTP and 5-10 U of T4 DNA polymerase to heat inactivated digests or extracted by phenol, followed by incubation at 37 ° C for 30 minutes. The following regulators were used in these reactions: lOx Regulator Low (60 mM Tris HCl, pH 7.5, 60 mM MgCl2, 50 mM NaCl, 4 mg / ml BSA, 70 mM β-mercaptoethanol, 0.02 percent NaN3); lOx Medium regulator (60 mM Tris HCl, pH 7.5, 60 mM MgCl2, 50 mM NaCl, 4 mg / ml BSA, 70 mM β-mercaptoethanol, 0.02 percent NaN3); lOx High regulator (60 mM Tris HCl, pH 7.5, 60 mM MgCl2, 50 mM NaCl, 4 mg / ml BSA, 70 mM β-mercaptoethanol, 0.02 percent NaN3); 10 Additions of Ligations (1 mM ATP, 20 mM DTT, 1 mg / ml BSA, 10 mM spermidine); 50x TAE (2 M Tris acetate, 50 mM EDTA). Synthesis and purification of oligonucleotides The oligonucleotides were produced on a Milligen 8750 synthesizer (Millipore). The columns were eluted with 1 ml of 30 percent ammonium hydroxide, and the eluted oligonucleotides were deblocked at 55 ° C for 6 to 12 hours. After deblocking, 150 μg of oligonucleotide was precipitated with 10 x unsaturated n-butanol volume in 1.5 milliliter reaction tubes, followed by centrifugation at 15,000 rpm in a microfuge. The pellet was washed with 70 percent ethanol and resuspended in 50 μl of H20. The concentration was determined by measuring the optical density at 260 nm at a dilution of 1: 333 (1 OD260 = 30 μg / milliliter). The following oligonucleotides were used for the construction of the synthetic gpl20 gene (all sequences shown in this text are in the 5 'to 3' direction). Oligo 1 in front (Nhel): cgc ggg cta gcc acc gag aag ctg (SEQ ID No: l). Oligo 1: acc gag aag ctg tgg gtg acc gtg tac tac ggc gtg ecc gtg tgg ag ag gcc acc acc acct ctg ttc tgc gcc age gac gcc aag gcg tac gac acc gag gtg cac aac gtg tgg gcc acc cag gcg tgc gtg ecc acc gac ecc aac ecc cag gag gtg gag ctc gtg aac gtg acc gag aac ttc aac at (SEQ ID No: 2). Oligo 1 reverse: cea cea tgt tgt tct tec ac t tt tga agt tct c (SEQ ID No: 3). Oligo 2 in front: gac cga gaa ctt caa cat gtg gaa gaa cat cat (SEQ ID No. 4). Oligo 2: tgg aag aac aac atg gtg gag cag atg cat gag gac ate ate age ctg tgg gac cag age ctg aag ecc tgc gtg aag ctg acc cc ctg tgc gtg acc tg aac tgc acc gac ctg agg aac acc ac aac ac aac ac age acc gcc aac aac aac age aac age gag ggc acc ate aag ggc ggc gag atg (SEQ ID No: 5). Oligo 2 reverse (Ptsl): gtt gaa get gca gtt ctt cat ctc gcc gcc ctt (SEQ ID No: 6). Oligo 3 in front (Pstl): gaa gaa ctg cag ctt caa cat cac cac cag (SEQ ID No: 7). Oligo 3: aac ate acc acc age ate cgc gac aag atg cag aag gag tac gcc ctg ctg tac aag ctg ate ate gtg ate ate gac aac gac age acc agec tac cgc ctg ate tec tgc aac acc age gtg ate acc gcc tgc ccc ecc aag ate age tcc gag ecc ate ecc ate cac tac tcc gcc ecc gcc gcc ttc gcc (SEQ ID No: 8). Oligo 3 reverse: gaa ctt ctt gtc ggc ggc gaa gcc ggc ggg (SEQ ID No: 9). Oligo 4 front: gcg ecc ceg ceg get teg cea tec tga agt gca acg aga aga agt tc (SEQ ID NO: 10). Oligo 4: gcc gac aag aag ttc age ggc aag ggc age tgc aag aac gtg age acc gtg cag tgc acc cac ggc ate cgg ceg gtg gtg age acc cag ctc ctg ctg aac ggc age ctg gcc gag gag gag gtg gtg ate cgc age gag aac ttc acc gac aac gcc aag acc ate ate gtg cac ctg aat gag age gtg cag ate (SEQ ID No: 11) Oligo 4 reverse (Mlul): agt tgg gac gcg tgc agt tga tct gca cgc tct c (SEQ ID No: 12). Oligo 5 in front (Mlul): gag age gtg cag ate aac tgc acg cgt ecc (SEQ ID No: 13). Oligo 5: aac tgc acg cgt ecc aac tac aac c a g c a c a c c a cc a gc c e c tc gc cc cc gc cc gc tc cc tc acc aac aac ate ate ggc acc ate ctc cag gcc cac tgc aac atect aga (SEQ ID NO: 14 ). Oligo 5 reverse: gtc gtt cea ctt ggc tct aga gat gtt gca (SEQ ID No: 15). Oligo 6 front: gca here tct cta gag cea agt gga act ac (SEQ ID NO: 16). Oligo 6: gcc aag tgg aac gac acc ctg cgc cag ate gtg age aag ctg aag gag cag ttc aag aac aag a gtg gtg ac tc g age gcg gac ecc gag ate gtg atg cac age tcc aac tgc ggc ggc (SEQ ID No: 17). Oligo 6 reverse: (EcoRl): gca gta gaa gaa tcc gcc gcc gca gtt ga (SEQ ID NO: 18). Oligo 7 in front (EcoRl): tea act gcg gcg gcg aat tct tct act gc (SEQ ID NO: 19). Oligo 7: ggc gaa ttc tcc tac tcc aacccc ccccc tcc tcc tcc tcc tcc tcc aac tcc cc tcc aac acc tc tc tc tc tc tc tc tc tc tc tc tc tc tc tccc tcc tcc tcc tcc tcc tcc tcc ctc cag tgc aag ate aag cag ate ate aac atg tgg cag gag gtg ggc aag gcc atg tac gcc ecc ecc ate gag ggc cag ate cgg tgc age age (SEQ ID No: 20). Oligo 7 reverse: gca gac cgg tga tgt tgc tgc tgc acc gga tct ggc ect c (SEQ ID No: 21). Oligo 8 in front: cga ggg cea gat ceg gtg cag cag caa cat cac cgg tct g (SEQ ID No: 22). Oligo 8: aac ate acc ggt ctg ctg ctg acc cgc gac ggc ggc aag gac acc gac acc aac gac acc gaa ate tcc cgc ecc ggc ggc ggc gac atg cgc gac aac tgg aga tct gag ctg tac aag tac aag gtg gtg acg ate gag ecc ctg ggc gtg gcc ecc acc aag gcc aag cgc cgc gtg gtg cag cgc gag aag cgc (SEQ ID No: 23). Oligo 8 reverse (Notl): cgc ggg cgg ceg ctt tag cgc ttc teg cgc tgc acc ac) SEQ ID No: 24).

The following oligonucleotides were used for the construction of the ratTHY-lenv gene. Oligo 1 front) BamHl / Hind3): cgc ggg gga tec aag ctt acc atg att cea gta ata agt (SEQ ID No: 25). Oligo 1: atg aat cea gta ata agt ata here tta tta tta agta gta tta caa atg agt aga gga caa aga gta ata agt tta here gca tct tta gta aat caat aat ttg aga tta gat tgt aga cat gaa aat aat aca aat ttg cea ata cat cat gaa ttt tea tta act (SEQ ID No: 26). Oligo 1 reverse (EcoRl / Mlul): cgc ggg gaa ttc acg cgt taa tga aaa ttc atg ttg (SEQ ID NO.27). Oligo 2 in front (BamHl / Mlul): cgc gga tec acg cgt gaa aaa aaa aaa cat (SEQ ID No: 28). Oligo 2: cgt gaa aaa aaa aaa cat gta tta agt gga here tta gga gta cea gaa cat here tat aga agt aga gta aat ttg ttt agt gat aga aga ttc ata aaa gta tta here tta gca aat ttt here aaa gat gaa gga gat tat atg tgt gat (SEQ ID No: 29). Oligo 2 reverse (EcoRl / Sacl): cgc gaa ttc gag ctc here cat ata ate tec (SEQ ID No: 30). Oligo 3 front) BamHl / Sacl): cgc gga tec gag ctc aga gta agt gga caá (SEQ ID No: 31). Oligo 3: ctc aga gta agt gga caa aat cea ag ag agat aat aaa ata ata aat gta ata aga gat aaa tta gta aaa tgt ga gga ata agt tta tta gta caat aat here agt tgg tta tta tta tta tta tta agt tta agt ttt tta caca gca here gat ttt ata agt tta tga (SEQ ID No: 32). Oligo 3 reverse (EcoRl / Notl): cgc gaa ttc gcg gcc get tea taa act tat aaa ate (SEQ ID No.33). Polymerase Chain Reaction Short overlapping oligonucleotides of 15 to 25 mer were used by quenching at both ends to amplify the long oligonucleotides by polymerase chain reaction (PCR). Typical conditions of the polymerase chain reaction were: 35 cycles, annealing temperature 55 ° C, extension time 0.2 seconds. The products were gel purified, extracted with phenol, and used in a polymerase chain reaction to generate longer fragments consisting of two adjacent small fragments. These longer fragments were cloned into a plasmid derived from CDM7 which contained a forward sequence of the surface molecule CD5 followed by a polylinker Nhel / Pstl / Mlul / EcoRl / BamHl. The following solutions were used in these reactions: lOx PCR regulator (500 mM KCl, 100 mM Tris HCl, pH 7.5, 8 mM MgCl 2, 2 M each dNTP). The final regulator was complemented with 10 percent DMSO to increase the fidelity of the Taq polymerase. Small scale DNA preparation Transformed bacteria were cultured in 3 milliliters of LB cultures for more than 6 hours or overnight. Approximately 1.5 milliliters of each culture was poured into 1.5 milliliter microfuge tubes, centrifuged for 20 seconds to agglomerated cells and resuspended in 200 μl of solution I. Subsequently, 400 μl of solution II and 300 μl of the solution were added. III. Microfuge tubes were capped, mixed and centrifuged for more than 30 seconds. The supernatants were transferred to new tubes and extracted with phenol once. The DNA was precipitated by filling the tubes with isopropanol, mixed and centrifuged in a microfuge for more than 2 minutes. The agglomerates were rinsed in 70 percent ethanol and resuspended in 50 μl of dH20 containing 10 μl of RNAse A. The following media and solutions were used in these procedures: Medium LB (1.0 percent NaCl, 0.5 percent extract of yeast, 1.0 percent tripthon); solution I (10 mM EDTA pH 8.0); solution II (0.2 M NaOH, 1.0 percent SDS); solution III (2.5 M KOAc, 2.5 M glacial acetic acid); phenol (pH adjusted to 6.0, covered with TE); TE (10 mM Tris HCl, pH 7.5, 1 mM EDTA pH 8.0). DNA preparation on a large scale One liter of cultures of transformed bacteria were grown 24 to 36 hours (MC1061p3 transformed with pCDM derivatives) or 12 to 16 hours (MC1061 transformed with pUC derivatives) to 37 ° C in either the bacterial medium M9 (derivatives pCDM) or LB (derived pUC). The bacteria were centrifuged in 1 liter bottles using a Beckman J6 centrifuge at 4, 200 rpm for 20 minutes. The agglomerate was resuspended in 40 milliliters of solution I. Subsequently, 80 milliliters of solution II and 40 milliliters of solution III were added and the bottles shaken semi-vigorously until lumps of 2 to 3 millimeters in size were developed. The bottle was centrifuged at 4,200 rpm for 5 minutes and the supernatant was poured through cloth in a 250 milliliter bottle. Isopropanol was added to the top and the bottle was centrifuged at 4,200 rpm for 10 minutes. The agglomerate was resuspended in 4.1 milliliters of solution I and 4.5 grams of cesium chloride, 0.3 milliliters of 10 milligrams / milliliter of ethidium bromide, and 0.1 milliliter of 1 percent Triton XlOO solution were added. The tubes were centrifuged in a high speed Beckman J2 centrifuge at 10,000 rpm for 5 minutes. The supernatant was transferred to Beckman Quick Seal ultracentrifuge tubes, which were sealed and centrifuged in a Beckman ultracentrifuge using a NVT90 fixed angle rotor at 80,000 rpm for more than 2.5 hours. The band was extracted by visible light using a 1 milliliter syringe and 20 gauge needle. An equal volume of dH20 was added to the extracted material. The DNA was extracted once with n-butanol saturated with 1 M sodium chloride, followed by the addition of an equal volume of 10 M ammonium acetate / 1 mM EDTA. The material was poured into a 13 milliliter holding tube which was then filled up with absolute ethanol, mixed, and centrifuged in a Beckman J2 centrifuge at 10,000 rpm for 10 minutes. The agglomerate was rinsed with 70 percent ethanol and resuspended in 0.5 to 1 milliliter of H20. The concentration of DNA was determined by measuring the optical density at 260 nm at a dilution of 1: 200 (1 OD260 = 50 μg / ml). The following media and regulators were used in these procedures: M9 bacterial medium (10 grams of M9 salts, 10 grams of casamino acids (hydrolysates), 10 milliliters of M9 additions, 7.5 μg / milliliter of tetracycline (500 μl of a solution of broth from 15 milligram / milliliter), 12.5 μg / milliliter of ampicillin (125 μl of a 10 mg / ml broth solution); M9 additions (10 mM CaCl2, 100 mM MgSO4, 200 μg / ml thiamine, 70 percent glycerol); medium LB (1.0 percent NaCl, 0.5 percent yeast extract, 1.0 percent tripthon); Solution I (10 mM EDTA pH 8.0); Solution II (0.2 M NaOH 1.0 percent SDS); Solution III (2.5 M KOAc 2.5 M HOAc) Sequencing The synthetic genes were sequenced by the dideoxynucleotide method of Sanger. Briefly, 20 to 50 μg of double-stranded plasmid DNA was denatured in 0.5 M NaOH for 5 minutes. Subsequently the DNA was precipitated with 1/10 volume of sodium acetate (pH 5.2) and 2 volumes of ethanol and centrifuged for 5 minutes. The granule was washed with 70 percent ethanol and resuspended at a concentration of 1 μg / milliliter. The quenching reaction was carried out with 4 μg of tempered DNA and 40 ng of primer in the tempered regulator in a final volume of 10 μl. The reaction was heated to 65 ° C and cooled slowly to 37 ° C. In a separate tube, 1 μl of 0.1 M DTT, 2 μl of labeling mixture, 0.75 μl of dH20, 1 μl of [35S] dATP (10 μCi), and 0.25 μl of Sequenase ™ (12 U / μl) were added for each reaction Five μl of this mixture was added to each annealed annealed primer tube and incubated for 5 minutes at room temperature. For each labeling reaction, 2.5 μl of each of the 4 finishing mixtures was added to a Terasaki plate and preheated to 37 ° C. At the end of the incubation period, 3.5 μl of stop solution was added to each of the 4 termination mixtures. After 5 minutes, 4 μl of stop solution was added to each reaction and the Terasaki plate was incubated at 80 ° C for 10 minutes in an oven. Sequencing reactions were run on a 5 percent denaturing polyacrylamide gel. An acrylamide solution was prepared by adding 200 milliliters of regulator TBE lOx and 957 milliliters of dH20 to 100 grams of acrylamide: bisacrylamide (29: 1). 5% polyacrylamide gel was prepared 46 percent urea and lx TBE combining 38 milliliters of acrylamide solution and 28 grams of urea. Polymerization was initiated by the addition of 400 μl of 10 percent ammonium peroxydisulfate and 60 μl TEMED. The gels were poured using silanized glass plates and shark tooth combs and run in the TBE regulator at 60 to 100 W for 2 to 4 hours (depending on the region to be read). The gels were transferred to Whatman stain paper, dried at 80 ° C for about 1 hour, and exposed to X-ray film at room temperature. The exposure time was typically 12 hours. The following solutions were used in these procedures: 5x annealing buffer (200 mM Tris HCl, pH 7.5, 100 mM MgCl 2, 250 mM NaCl); Labeling mixture (7.5 μM each of dCTP, dGTP, and dTTP); Termination mixtures (80 μM of each dNTP, 50 mM NaCl, 8 μM ddNTP (one each)); Stop solution (95 percent formamide, 20 mM EDTA, 0.05 percent bromophenol blue, 0.05 xylene cyanol) 5x TBE (0.9 M Tris borate, 20 mM EDTA); Polyacrylamide solution (96.7 grams of polyacrylamide, 3.3 grams of bisacrylamide, 200 milliliters Ix TBE, 957 milliliters dH20). Isolation of RNA Cytoplasmic RNA was isolated from 293T cells transfected by calcium phosphate 36 hours post transfection and from Hela cells infected with cowpox 16 hours post-infection essentially as described by Gilman. (Gilman Preparation of cytoplasmic RNA from tissue culture cells [Gilman's preparation of cytoplasmic RNA from tissue culture cells], in Current Protocols in Molecular Biology, Ausubel et al., Editors, Wiley &Sons, New York, 1992) . Briefly, the cells are lysed in 400 μl of lysis buffer, the nuclei are centrifuged outward, and SDS and Proteinase K are added at 0.2 percent and 0.2 milligrams / milliliter respectively. The cytoplasmic extracts were incubated at 37 ° C for 20 minutes, extracted twice with phenol / chloroform, and precipitated. The RNA was dissolved in 100 μl of buffer I and incubated at 37 ° C for 20 minutes. The reaction was stopped by adding 25 μl of stop buffer and precipitated again. The following solutions were used in this procedure: Lysis regulator (TRUSTEE regulator containing 50 mM Tris pH 8.0, 100 mM NaCl, 5 mM MgCl2, 0.5 percent NP40); Regulator I (TRUSTEE regulator with 10 mM MgCl2, 1 mM DTT, 0.5 U / μl placental RNAse inhibitor, 0.1 U / μl DNase-free RNAse I); detention regulator (50 mM EDTA 1.5 M NaOAc 1.0 percent SDS). Slit stain analysis For slit stain analysis, 10 μg of cytoplasmic RNA was dissolved in 50 μl dH20 to which 150 μl of lOx SSC / 18 percent formaldehyde was added. The solubilized RNA was then incubated at 65 ° C for 15 minutes and splashed on with a slot staining apparatus. Radioactively labeled probes of gbl20IIIb and syngpl20mn fragments of 1.5 kb were used for hybridization. Each of the two fragments was randomly labeled in a 50 μl reaction with 10 μl of 5x oligo-labeling buffer, 8 μl of 2.5 mg / ml BSA, 4 μl of [23P] -dCTP (20 uCi / μl, 6000 Ci / mmol), and 5 U of Klenow fragment. After 1 to 3 hours of incubation at 37 ° C 100 μl of TRUSTEE was added and the unincorporated [OÍ 23 P] -dCTP was removed using G50 spin column. The activity was measured in a Beckman beta counter, and identical specific activities were used for hybridization. The membranes were prehybridized for 2 hours and hybridized for 12 to 24 hours at 42 ° C with 0.5 x 106 cpm probe per milliliter of hybridization fluid. The membrane was washed twice (5 minutes) with wash buffer I at room temperature, for one hour in wash buffer II at 65 ° C, and then exposed to x-ray film. Similar results obtained were obtained using a Not 1.1 / Sfil fragment of pCM7 that contained the 3 untranslated region. Control hybridizations were done in parallel with a randomly-labeled human beta-actin probe. RNA expression was quantified by scavenging the nitrocellulose membranes hybridized with a Magnetic Dynamics phosphor-imaging. The following solutions were used in this procedure: 5x Oligo-labeling buffer (250 mM Tris HCl, pH 8.0, 25 mM MgCl 2, 5 mM β-mercaptoethanol, 2 mM dATP, 2 mM dGTP, mM dTTP, 1 M Hepes pH 6.6 , 1 mg / ml hexanucleotides [dNTP] 6); Hybridization solution (0.05 M sodium phosphate, 250 mM NaCl, 7 percent SDS, 1 mM EDTA, 5 percent dextran sulfate, 50 percent formamide, 100 μg / ml denatured salmon sperm DNA); Wash regulator I (2x SSC, 0.1 percent SDS); wash regulator II (0.5x SSC, 0.1 percent SDS); 20 x SSC (3 M NaCl, 0.3 M Na 3 citrate, pH adjusted to 7.0). Recombination of vaccine Recombination of vaccine used a modification of the method described by Romeo and Seed (Romeo and Seed, Cell, 64: 1037, 1991). Briefly, CV1 cells at a confluence of 70 to 90 percent were infected with 1 to 3 μl of a wild type WR vaccine broth (2 x 108 pfu / ml) for 1 hour in culture medium without calf serum. After 24 hours, the cells were transfected with calcium phosphate with 25 μg of plasmid TKG DNA per dish. After an additional 24 to 48 hours the 6 cells were scraped off the dish, centrifuged, and resuspended in a volume of 1 milliliter. After 3 freeze / thaw cycles, trypsin was added at 0.05 mg / ml and lysates were incubated for 20 minutes. A series of dilutions of 10, 1 and 0.1 μl of this lysate was used to infect small dishes (6 centimeters) of CV1 cells, which had been pretreated with 12.5 μg / ml of mycophenolic acid, 0.25 mg / ml of xanthine 1.36 mg / hypoxanthine milliliter for 6 hours. The infected cells were cultured for two to three days, and subsequently stained with monoclonal antibody NEA9301 against gpl20 and a conjugated secondary antibody of alkaline phosphatase. Cells were incubated with 0.33 mg / ml NBT and 0.16 mg / ml BCIP in AP buffer and finally covered with 1 percent agarose in phosphate buffered saline. Positive plaques were collected and resuspended in 100 μl Tris pH 9.0. The purification of the plate was repeated once. In order to produce broths with higher titers the infection rose step by step slowly. Finally, a large dish of Hela cells was infected with half of the viruses from the previous round. The infected cells were discarded in 3 milliliters of phosphate buffered saline, used with a Dounce homogenizer and rinsed from the larger debris by centrifugation. Recombinant VPE-8 vaccine broths were kindly provided by the AIDS Repository, Rockville, MD, and expressed HIV-IIIBpl gpl20 under the mixed early / late promoter 7.5 (Earl et al., J. Virol., 65:31, 1991). In all experiments with recombinant vaccine, the cells were infected at a multiplicity of infection of at least 10. The following solution was used in this procedure: AP regulator (100 mM Tris HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) Cell culture The monkey kidney carcinoma cell lines CV1 and Cos7, the 293T kidney carcinoma cell line, and the Hela human cervical carcinoma cell line were obtained from the American Tissue Typing Collection and maintained in supplemented IMDM. . They were maintained in 10 cm tissue culture plates and were typically divided 1: 5 to 1:20 every 3 to 4 days. The following medium was used in this procedure: supplemented IMDM (90 percent Iscove's modified Dulbecco's medium, 10 percent calf serum, supplemented with iron, heat-inactivated 30 minutes at 56 ° C, 0.3 mg / ml L- glutamine, 25 μg / ml gentamicin 0.5 mM ß-mercaptoethanol (adjusted pH with 5 M NaOH, 0.5 ml)). Transfection The calcium phosphate transfection of 293T cells was performed by adding slowly and under centrifugation 10 μg of plasmid DNA in 250 μl 0.25 M CaCl2 to the same volume of 2x HEBS regulator at the same time as stirring. After incubation for 10 to 30 minutes at room temperature, the DNA precipitate was added to a small dish of 50 to 70 percent confluent cells. In co-transfection experiments with rev, cells were transfected with 10 μg gpl20IIIb, gpl20IIIb-rre, siyngpl20mnrre or rTHY-lenveglrre and 10 μg of pCMVrev or plasmid DNA CDM7. The following solutions were used in this procedure: 2x HEBS regulator (280 mM NaCl, 10 mM KCl, 1.5 mM sterile filtrate); 0.25 mM CaCl2 (sterilized in autoclave). Immunoprecipitation After 48 to 60 hours the medium was changed and the cells were incubated for an additional 12 hours in Cys / Met free medium containing 200 μCi of 35S trans marker. The supernatants were harvested and centrifuged for 15 minutes to remove debris. After the addition of protease inhibitors leupeptin, aprotinin and PMSF at 2.5 μg / ml, 50 μg / ml, lOOμ / ml respectively, 1 ml of supernatant was incubated with either 10 μl of packed protein A sepharose alone ( rTHY-lenveglrre) or with protein Sepharose A and 3 μg of a purified CD4 fusion protein / immunoglobulin (gently provided by Behring) (all gpl20 constructs) at 4 ° C for 12 hours on a rotator. Subsequently the protein A beads were washed 5 times for 5 to 15 minutes each time. After the final wash 10 μl of charge buffer was added, the samples were boiled for 3 minutes and applied on 7 percent (all gpl20 constructs) or 10 percent (rTHY-lenveglrre) SDS polyacrylamide gels (regulator TRIS pH 8.8 regulator in the resolution, regulator TRIS pH 6.8 in the piling gel, TRIS-glycine operation regulator, Maniatis et al., supra 1989). The gels were fixed in 10 percent acetic acid and 10 percent methanol, incubated with Amplify for 20 minutes, dried and exposed for 12 hours. The following regulators and solutions were used in this procedure: Washing regulator (100 mM Tris, pH 7.5, 150 mM NaCl, 5 mM CaCl 2, 1 percent NP-40); 5x Opération Regulator (125 mM Tris, 1.25 M Glycine, 0.5 percent SDS); Charge regulator (10 percent glycerol, 4 percent SDS, 4 percent β-mercaptoethanol, 0.02 percent blue bromophenol). Immunofluorescence 293T cells were transfected by calcium phosphate coprecipitation and analyzed for THY-1 surface expression after 3 days. After detachment with 1 mM EDTA / PBS, the cells were stained with monoclonal antibody OX-7 at a dilution of 1: 250 at 4 ° C for 20 minutes, washed with phosphate buffered saline and subsequently incubated with a 1: 500 dilution of an FITC-conjugated goat anti-mouse immunoglobulin antiserum. The cells were washed again, resuspended in 0.5 milliliters of a fixation solution, and analyzed in an EPICS XL cytofluorometer (Coulter). The following solutions were used in this procedure: Saline regulated phosphate solution (137 mM NaCl, 2.7 mM KCl, 4.3 Na2HP04, 1.4 mM KH2P04, pH adjusted to 7.4); Fixation solution (2 percent formaldehyde in phosphate buffered saline). Linked enzyme immunosorbent assay (ELISA) The concentration of gp 120 in culture supernatants was determined using ELISA plates coated with CD4 and goat anti-gpl20 antibody in the soluble phase. The supernatants of 293T cells transfected by calcium phosphate were harvested after 4 days, centrifuged at 3000 rpm for 10 minutes to remove the debris and incubated for 12 hours at 4 ° C in the dishes. After 6 washes with phosphate buffered saline 100 μl of goat anti-gpl20 antiserum diluted 1: 200 were added for 2 hours. The dishes were washed again and incubated for 2 hours with mouse anti-goat IgG antiserum conjugated with peroxidase 1: 1000. The plates were then washed and incubated for 30 minutes with 100 μl of substrate solution containing 2 mg / ml o-phenylenediamine in sodium citrate buffer. The reaction was finally stopped with 100 μl of 4 M sulfuric acid. The plates were read at 490 nm with a Coulter microplate reader. The purified recombinant gpl20IIIb recombinant was used as a control. The following regulators and solutions were used in this procedure: Washing regulator (0.1 percent NP40 in phosphate buffered saline); Substrate solution (2 mg / ml o-phenylenediamine in sodium citrate regulator). EXAMPLE 2 A synthetic green fluorescent protein gene The codon replacement efficiency for gpl20 suggests that replacing non-preferred codons with less preferred codons or preferred codons (and replacing less preferred codons with preferred codons) will increase expression in mammalian cells or other proteins , for example, other eukaryotic proteins. The green fluorescent protein (GFP) of the jellyfish Aequorea victoria (Ward, Photochem, Photobiol 4: 1, 1979, Prasher et al, Gene 111: 229, 1992, Cody et al, Biochem 32: 1212, 1993) has attracted attention recently for its possible usefulness as a marker or reporter for transfection and lineage studies (Chalfie et al., Science 263: 802, 1994). Examination of a codon usage table constructed from the native coding sequence of the green fluorescent protein showed that the codons of the green fluorescent protein favored either A or U in the third position. The bias in this case favors A less than what the bp20 bias does, but it is substantial. A synthetic gene was created in which the sequence of the natural green fluorescent protein was delayed in a similar manner as for the gpl20 (Figure 11; SEQ ID NO: 40). In addition, the translation start sequence of the green fluorescent protein was replaced with sequences corresponding to the consensus of translation initiation. Expression of the resulting protein was compared to that of the natural type sequence, similarly overlapped to bring an optimized translation initiation consensus (Figure 10B and Figure 10C). In addition, the effect of the inclusion of the Ser 65-> mutation was examined. Thr, reported to improve the excitation efficiency of green fluorescent protein at 490 nm and then preferred for fluorescence microscopy (Heim et al., Nature 373: 663, 1995) (Figure 10D). The codon overlap conferred a significant increase in the efficiency of expression (a concomitant percentage of apparently positive cells for transfection), and the combination of the Ser 65-> mutation. Thr and codon optimization resulted in the DNA segment encoding a highly visible mammalian marker protein (Figure 10D). The sequence encoding the synthetic green fluorescent protein described above was assembled in a similar manner as for gpl20 from six fragments of approximately 120 base pairs each, using a strategy to assemble which depends on the capacity of restriction enzymes Bsal and Bbsl to segment out of its recognition sequence. Long oligonucleotides were synthesized which contained portions of the coding sequence for the green fluorescent protein embedded in flanking sequences encoding EcoRI and Bsal at one end, and BamHl and Bbsl at the other end. Thus, each oligonucleotide has the EcoRI / Bsal / GFP / BbsI / BamHI fragment. The ends of the restriction site generated by the Bsal and Bbsl sites were designed to produce compatible ends that could be used to join adjacent green fluorescent protein fragments. Each of the compatible ends was designed to be unique and non-self-complementary. The crude synthetic DNA segments were amplified by polymerase chain reaction, inserted between EcoRI and BamHl in pUC9, and sequenced. Subsequently, the intact coding sequence was assembled in a ligation of six fragments, using fragments of inserts prepared with Bsal and BbsI. Two of six plasmids resulting from the ligation drill an insert of the correct size, and one contained the desired full-length sequence. The Ser65 mutation in Thr was carried out by standard mutagenesis based on polymerase chain reaction, using a primer that overlapped a unique BssSI site in the green synthetic fluorescent protein. Codon Optimization as a Strategy for Improved Expression in Mammalian Cells The data presented here suggests that re-overlapping the coding sequence may have a general utility for the enhancement of mammalian and non-mammalian eukaryotic gene expression in mammalian cells. The results obtained here with three unrelated proteins: HIV Gpl20, the rat cell surface antigen Thy-1 and the green fluorescent protein of Aequorea victoria, and human Factor VIII (see below) suggest that codon optimization may prove to be a successful strategy to improve expression in mammalian cells of a wide variety of eukaryotic genes. EXAMPLE III Design of an optimized codon gene expressing human Factor VIII lacking central domain B A synthetic gene encoding mature human Factor VIII lacking amino acid residues 760 to 1639, inclusive (residues 779 to 1658, was designed. , inclusive, of the precursor). The synthetic gene was created by choosing codons corresponding to those favored by highly expressed human genes. Some deviation from strict adherence to the favored residue pattern was made to allow unique restriction enzyme cleavage sites to be introduced throughout the gene to facilitate future manipulations. For the preparation of the synthetic gene the sequence was then divided into 28 segments of 150 base pairs, and a 29 segment of 161 base pairs. A synthetic gene expressing the gene was constructed as follows Human Factor VIII lacking the central domain B. Twenty-nine pairs of tempered oligonucleotides were synthesized (see below). The 5 'tempered oligos were 105 bases long and the 3' oligos were 104 bases long (except for the last oligo 3 ', which was 125 residues long). The tempered oligos were designed so that each annealed pair composed of an oligo 5 'and an oligo 3', created a double chain region of 19 base pairs. To facilitate the polymerase chain reaction and subsequent manipulations, the 5 'ends of the oligo pairs were designed to be invariant over the first 18 residues, allowing a common pair of polymerase chain reaction primers to be used for the amplification , and allowing the same polymerase chain reaction conditions to be used for all pairs. The first 18 residues of each member of the hardened pair were cgc gaa ttc gga aga ecc (SEQ ID NO: 110) and the first 18 residues of each member 3 'of the pair used were: ggg gat ect cac gtc tea (SEQ ID NO: 43). Oligo pairs were annealed and then extended and amplified by polymerase chain reaction in a reaction mixture as follows: the annealed ones were annealed at 200 μg / ml each in polymerase chain reaction buffer (10 mM Tris-HCl , 1.5 mM MgCl2, 50 mM KCl, 100 μg / ml gelatin, pH 8.3). The polymerase chain reactions contained 2 ng of the annealed tempered oligos, 0.5 μg of each of the two 18-mer primers (described below), 200 μM of each of the deosinucleoside triphosphates, 10 percent by volume of DMSO and polymerase chain reaction regulator as supplied by Boehringer Mannheim Biochemicals, in a final volume of 50 μl. After the addition of Taq polymerase (2.5 units, 0.5 μl, Boehringer Mannheim Biochemicals) amplifications were performed on a Perkin-Elmer Thermal Cycler for 25 cycles (94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds). The final cycle was followed by a 10 minute extension at 72 ° C. The amplified fragments were digested with EcoRI and BamHI (cleaving at the 5 'and 3' ends of the fragments respectively) and ligated to a cut derived pUC9 with EcoRI and BamHI. Individual clones were sequenced and a collection of plasmids corresponding to the entire desired sequence was identified. The clones were assembled by multifragment ligation using the restriction sites at the 3 'ends of the polymerase chain reaction primers immediately adjacent to the amplified sequence. The 5 'polymerase chain reaction primer contained a Bbsl site, and the 3' polymerase chain reaction primer contained a BsmBI site, positioned so that cleavage by the respective enzymes preceded the first nucleotide of the amplified portion and he left an overclocked 5 'of 4 bases created by the first 4 bases of the amplified portion. Simultaneous digestion with Bbsl and BsmBI thus freed the amplified portion with unique 5 'overcups from 4 bases at each end that did not contain any of the primer sequences. In general these overcrowded were not self-complementary, allowing multiple fragment ligation reactions to produce the desired product very efficiently. The single portion of the first 28 pairs of amplified oligonucleotides was then 154 base pairs, and after digestion each gave rise to a fragment of 150 base pairs with single ends. The first and last fragments were not manipulated in this way, however, since they had other restriction sites designated therein to facilitate the insertion of the assembled sequence into a suitable mammalian expression vector. The actual assembly process proceeded as follows. Assembling the synthetic gene of Factor VIII Step 1: 29 fragments assembled to form 10 fragments. The 29 pairs of oligonucleotides, which formed segments of 1-29 when the bases matched, are described below. The plasmids carrying segments 1, 5, 9, 12, 16, 20, 24 and 27 were digested with EcoRl and BsmBI and the fragments of 170 base pairs were isolated; the plasmids carrying segments 2, 3, 6, 7, 10, 13, 17, 18, 21, 25, and 28 were digested with Bbsl and BsmBI and the fragments of 170 base pairs were isolated; and the plasmids carrying segments 4, 8, 11, 14, 19, 22, 26 and 29 were digested with EcoRI and Bbsl and the vector fragment of 2440 base pairs was isolated. The fragments carrying the segments 1, 2, 3 and then linked to generate the segments "A"; the fragments carrying segments 5, 6, 7 and 8 were ligated to generate segment "B"; the fragments carrying segments 9, 10 and 11 were ligated to generate segment "C"; the fragments carrying segments 12, 13, and 14 were ligated to generate segment "D"; the fragments carrying segments 16, 17, 18 and 19 were ligated to generate segment "F"; the fragments carrying segments 20, 21 and 22 were ligated to generate segment "G"; the fragments carrying the segments 24, 25 and 26 were ligated to generate the segment "I"; and the fragments carrying segments 27, 28 and 29 were ligated to generate segment "J". Step 2: Assemble the 10 fragments resulting from step 1 to three fragments. The plasmids carrying segments "A", "D" and "G" were digested with EcoRI and BsmBI, the plasmids carrying segments B, 15, 23, and I were digested with Bbsl and BsmBI, and the plasmids carrying segments C, F, and J were digested with EcoRI and BbsI. The fragments carrying the segments A, B, and C were ligated to generate the "K" segment; the fragments carrying the segments D, 15, and F were ligated to generate the "O" segment; and the fragments carrying the segments G, 23, I, and J were ligated to generate the "P" segment. Step 3: Assemble the final three pieces. The plasmid carrying the K segment was digested with EcoRI and BsmBI, the plasmid carrying the O segment was digested with Bbsl and BsmBI, and the plasmid carrying the P segment was digested with EcoRI and BbsI. The three resulting fragments were ligated to generate segments. Step 4: Insertion of the synthetic gene into a mammalian expression vector The plasmid carrying the S segment was digested with Nhel and Notl and was inserted between the Nhel and Eagl sites of the plasmid CD51NEgl to generate the plasmid cd51sf8b-. Sequencing and Correction of the Factor VIII Synthetic Gene After assembly of the synthetic gene it was discovered that there were two unwanted residues encoded in the sequence. One was an Arg residue in 749, which is present in the GenBank sequence entry originating from Genentech but is not in the sequence reported by Genentech in the literature. The other was a Wing residue at 146, which should have been Pro. This mutation arises at an unidentified step subsequent to the sequencing of the 29 constituent fragments. The Pro749Arg mutation was corrected by incorporating the desired change in a polymerase chain reaction primer (ctg ctt ctg acg cgt get ggg gtg gcg gga gtt; SEQ ID NO: 44) which included the Mlul site at position 2335 of the longest sequence forward (sequence from the HindIII to NotI segment) and amplify between that primer and a primer (ctg ctg aaa gtc tec age tgc; SEQ ID NO: 44) 5 'to the SgrAI site at 2225. The fragment from SgrAI to Mlul was then inserted into the expression vector at the known sites in the vector, and the resulting correct sequence change was verified by sequencing. The Prol46AIa mutation was corrected by incorporating the desired sequence change into an oligonucleotide (ggc agg tgc tta agg aga acg gcc cta tgg cea, SEQ ID NO: 46) bringing the AflII site to residue 504, and amplifying the fragment resulting from the reaction of polymerase chain between that oligo and the primer having the sequence cgt tgt tct tea tac gcg tct ggg get ect cg ggc (SEQ ID NO: 109), cutting the resulting polymerase chain reaction fragment with AflII and Avrll in (residue 989), inserting the corrected fragment into the expression vector and confirming the construction by sequencing. Construction of a paired native gene expressing human Factor VIII lacking the central domain B. A paired B-domain deletion expression plasmid of Factor VIII having the original codon sequence was constructed by introducing Nhel at the 5 'end of the mature coding sequence using the primer cgc ca ggg cta gcc gcc acc aga tac tac ctg ggt (SEQ ID NO: 47), amplifying between that primer and the primer att cgt agt tgg ggt tec tct gga cag (corresponding to residues 1067 to 1093 of the sequence shown below), cutting with Nhel and AflII (residue 345 in the sequence shown below) and inserting the resulting fragment into a suitably segmented plasmid carrying the native Factor VIII. The deletion of domain B was created by overlapping polymerase chain reaction using ctg tat ttg atg aga acc g, (corresponding to residues 1813 to 1831 below) and ca gac tgg tgg ggt ggc att aaa ttg ctt t (SEQ ID NO: 48) (2342 to 2372 in supplement below) for the 5 'end of the overlap, and aat gcc acc cea cea gtc ttg aaa cgc ca (SEQ ID NO: 49) (2352 to 2380 in the sequence below) and cat ctg gat att gca ggg ag (SEQ ID NO: 50) (3145 to 3164). The products of two individual polymerase chain reactions were then mixed and reamplified by the use of the outermost primers, the resulting fragment was segmented by Asp718 (Kpnl isosqui-zomer, 1837 in the sequence below) and PflMI (3100 in the sequence below), and inserted into the suitably segmented expression plasmid carrying the original Factor VIII. The complete sequence (SEQ ID NO: 41) of the deleted human factor VIII human gene for the central region B is presented in Figure 12. The complete sequence (SEQ ID NO: 42) of the synthetic factor VIII gene deleted for the region Central B is presented in Figure 13. Preparation and testing of expression plasmids Two independent plasmid isolates from the original, and four isolates independent of the synthetic Factor VIII expression plasmid were propagated separately in bacteria and their DNA prepared by density centrifugation buoyant CsCl followed by extraction by phenol. Analysis of the supernatants of COS cells transfected with the plasmids showed that the synthetic gene gave rise to approximately four times as much Factor VIII as did the native gene.

COS cells were transfected with 5 μg of each factor VIII construct per 6 cm dish using the DEAE-dextran method. At 72 hours post-transfection, 4 milliliters of fresh medium containing 10 percent calf serum was added to each dish. A sample of the medium was taken from each plate 12 hours later. The mixtures were tested by the enzyme-linked immunosorbent assay using a light chain monoclonal antibody of mouse anti-human factor VIII and goat anti-human monoclonal antibody VIII conjugated to peroxidase. Factor VIII of purified human plasma was used as a standard. Cells transfected with the synthetic Factor VIII gene construct expressed 138 + 20.2 ng / ml (equivalent ng / ml Factor VIII not deleted) Factor VIII (n = 4) while cells transfected with the native Factor VIII gene expressed 33.5 ± 0.7 ng / ml (equivalent ng / ml Factor VIII not deleted) Factor VIII (n = 2). The following tempered oligonucleotides were used for the construction of the synthetic Factor VIII gene. Rl bbs 1 for (gcta) cgc gaa ttc gga aga ecc get age cgc cac 1 rl ceg ceg cta cta ect ggg cgc cgt gga get gtc ctg gga cta cat gca gag cga ect ggg cga get ecc cgt gga (SEQ ID No: 51 ) ggg gat ect cac gtc tea ggt ttt ctt gta 1 bam cac cac get ggt gtt gaa ggg gaa get ctt ggg cac gcg ggg ggg gaa gcg ggc gtc cac ggg gag ctc gcc ca (SEQ ID No: 52) Rl bbs 2 for (aacc) cgc gaa ttc gga aga ecc aac ect gtt cgt 2 rl gga gtt cac cga cea ect gtt caa cat tgc ca gcc gcg ecc ecc ctg gat ggg ect get ggg ecc cac cat cea (SEQ ID No: 53 ) ggg gat ect cac gtc tea gtg cag get gac 2 bam ggg gtg get ggc cat gtt ctt cag ggt gat cac cac ggt gtc gta cac ctc ggc ctg gat ggt ggg gcc cag ca (SEQ ID No: 54) Rl bbs 3 for (gcac) cgc gaa ttc gga aga ecc gca cgc cgt ggg 3 rl cgt gag cta ctg gaa ggc cag cga ggg cgc cga gta cga cga cea gac gtc cea gcg cga gaa gga gga cga caa (SEQ ID No: 55 ) ggg gat ect cac gtc tea get ggc cat agg 3 bam gcc gtt ctc ctt aag cac ctg cea cac gta ggt gtg get ecc ecc cgg gaa cac ctt gtc gtc ctc ctt ctc gc (SEQ ID No: 56) rl bbs 4 for (cagc ) cgc gaa ttc gga aga ecc cag cga ecc ect 4 rl gtg ect gac cta cag cta ect gag cea cgt gga ect ggt gaa gga tct gaa cag egg get gat cgg cgc ect get (SEQ ID No: 57) ggg gat ect cac gtc tea gaa cag cag gat 4 bam gaa ctt gtg cag ggt ctg ggt ttt ctc ctt ggc cag get gcc ctc gcg here cac cag cag ggc gcc gat cag cc (SEQ ID No: 58) cgc gaa ttc gga aga ecc gtt cgc cgt gtt 5rl cga cga gga gag gag ctg gca cag cga gac taa gaa cag ect gat gca gga ceg cga cgc cgc cag cgc ceg cgc (SEQ ID No: 59) ggg gat ect cac gtc tea gtg gca gcc gat 5 bam cag gcc ggg cag get gcg gtt cac gta gcc gtt aac ggt gtg cat ctt ggg cea ggc gcg ggc get ggc ggc gt (SEQ ID No: 60) Rl bbs 6 for (ccac) cgc gaa ttc gga aga ecc cea ceg caa gag 6rl cgt gta ctg gca cgt cat cgg cat ggg cac cac ecc tga ggt gca cag cat ctt ect gga ggg cea cac ctt ect (SEQ ID No: 61) ggg gat ect cac gtc tea cag ggt ctg ggc 6 bam agt cag gaa ggt gat ggg get gat ctc cag get ggc ctg gcg gtg gtt gcg cac cag gaa ggt gtg gcc ctc ca (SEQ ID No: 62) Rl bbs 7 for (ectg) cgc gaa ttc gga aga ecc ect get gat gga 7rl ect agg cea gtt ect get gtt ctg cea cat cag cag cea cea gca cga egg cat gga ggc tta cgt gaa ggt gga (SEQ ID No: 63) ggg gat ect cac gtc gtc tea gtc gtc gtc gtc gtc gtc gtc gtc ctc ctc gtt gtt ctt cat gcg cag ctg ggg ctc ctc ggg gca get gtc cac ctt cac gta agc_ct (SEQ ID No: 64) rl bbs 8 for (cgac) cgc gaa ttc gga aga ecc cga ect gac cga 8 rl cag cga gat gga tgt cgt acg ctt cga cga cga caga cag ecc cag ctt cat cea gat ceg cag cgt ggc caa gaa (SEQ ID No: 65 ) ggg gat ect cac gtc tea tac tag cgg ggc 8 bam gta gtc cea gtc ctc ctc ctc ggc ggc gat gta gtg cac cea ggt ctt agg gtg ctt ctt ggc cac get gcg ga (SEQ ID No .: 66) Rl bbs 9 for (agta) cgc gaa ttc gga aga ecc agt act ggc ecc 9 rl cga cga ceg cag cta caa gag cea gta ect gaa caa cgg ecc cea gcg cat cgg ceg caa gta caa gaa ggt gcg (SEQ ID No: 67 ) ggg gat ect cac gtc tea gag gat gcc gga 9 bam ctc gtg ctg gat ggc ctc gcg ggt ctt gaa agt ctc gtc ggt gta ggc cat gaa gcg cac ctt ctt gta ctt gc (SEQ ID No: 68) rl bbs 10 for (cetc) cgc gaa ttc gga aga ecc ect cgg ecc ect 10 rl get gta cgg cga ggt ggg cga cc cact ect get cat cat ctt caa gaa cea ggc cag cag gcc cta cat cat cta cat ecc (SEQ ID No: 69 ) ggg gat ect cac gtc tea ctt cag gtg ctt 10 bam cac gcc ctt ggg cag gcg gcg get gta cag ggg gcg cac gtc ggt gat gcc gtg ggg gta gat gtt gta ggg cc (SEQ ID No: 70) rl bbs 11 for (gaag ) cgc gaa ttc gga aga ecc gaa gga ctt ecc 11 rl cat ect gcc cgg cga gat ctt ca gta ca gtg gac cgt gac cgt gga gga cgg ecc cac ca ga ga cga ecc ceg (SEQ ID No: 71) ggg gat ect cac gtc tea gcc gat cag tec 11 bam gga ggc cag gtc gcg ctc cat gtt cac gaa get get gta gta gcg ggt cag gca gcg ggg gtc get ctt ggt gg (SEQ ID No: 72) rl bbs 12 for (cggc) cgc gaa ttc gga aga ecc cgg ecc ect get 12 rl gat ctg cta ca gga gag cgt gga cea gcg cgg caa cea gat cat gag cga caa gcg caa cgt gat ect gtt cag (SEQ ID No: 73 ) ggg gat ect cac gtc tea age gg gtt ggg 12 bam cag gaa gcg ctg gat gtt ctc ggt cag ata cea get gcg gtt ctc gtc gaa cac get gaa cag gat cac gtt gc (SEQ ID No: 74) rl bbs 13 for (eget) cgc gaa ttc gga aga ecc cgc tgg cgt gca 13 rl get gga aga tec cga gtt cea ggc cag caa cat cat gca cag cat cag cgg cta cgt gtt cga cag ect gca get (SEQ ID No: 75 ) ggg gat ect cac gtc tea cag gaa gtc ggt 13 bam ctg ggc gcc gat get cag gat gta cea gta ggc cac ctc atg cag gca cac get cag ctg cag get gtc gaa ca (SEQ ID No: 76) rl bbs 14 for (ectg) cgc gaa ttc gga aga ecc ect gag cgt gtt 14 rl ctt ctc cgg gta tac ctt ca gca ca g gat gta cga gga cac ect gac ect gtt ecc ctt ctc cgg cga gac (SEQ ID No: 77 ) ggg gat ect cac gtc tea gtt gcg gaa gtc 14 bam get gtt gtg gca gcc cag aat cea cag gcc ggg gtt ctc cat aga cat gaa cac agt ctc gcc gga gaa ggg ga (SEQ ID No: 78) rl bbs 15 for (caac) cgc gaa ttc gga aga ecc caa ceg egg cat 15 rl gac tgc ect get gaa agt ctc cag ctg cga caa gaa cac cgg cga cta cta cga cga gga cag cta cga gga cat ctc (SEQ ID No: 79 ) ggg gat ect cac gtc tea gcg gtg gcg gga 15 bam gtt ttg gga gaa gga gcg ggg ctc gat ggc gtt gtt ctt gga cag gg gta ggc gat gtc ctc gta get gt (SEQ ID No: 80) rl bbs 16 for (cege) cgc gaa ttc gga aga ecc ceg cag cac gcg 16 rl tea gaa gca gtt cac cgc cac ecc ecc cgt get gaa gcg cea cea gcg cga gat cac ceg cac cac ect gca aag (SEQ ID No: 81 ) ggg gat ect cac gtc tea gat gtc gaa gtc 16 bam ctc ctt ctt cat ctc cac get gat ggt gtc gtc gta gtc gat ctc ctc ctg gtc get tgg ggt ggt ggg gg gg (SEQ ID No: 82) rl bbs 17 for (cat) cgc gaa ttc gga aga ecc cat cta cga cga 17 rl gga cga gaa _ cea gag ecc ceg ctc ctt cea aaa gaa aac ceg cea cta ctt cat cgc cgc cgt gga gcg ect gtg (SEQ ID No: 83) ggg gat ect cac gtc tea ctg ggg cac get 17 bam gcc get ctg ggc gcg gtt gcg cag gac gtg ggg get get get cat gcc gta gtc cea cag gcg ctc cac ggc gg (SEQ ID No: 84) rl bbs 18 for (ccag) cgc gaa ttc gga aga ecc cea gtt caa gaa 18 rl ggt ggt gtt cea gga gtt cac cga cgg cag ctt cac cea gcc ect gta ceg cgg cga get gaa cga gca ect ggg (SEQ ID No: 85 ) ggg gat ect cac gtc tea ggc ttg gtt gcg 18 bam gaa ggt cac cat gat gtt gtc ctc cac ctc ggc gcg gat gta ggg gcc gag cag gcc cag gtg ctc gtt cag ct (SEQ ID No: 86) rl bbs 19 for (agee) cgc gaa ttc gga aga ecc age ctc ceg gcc 19 rl cta ctc ctt cta ctc ctc ect gat cag cta cga gga gga cea gcg cea ggg cgc cga gcc ceg caa gaa ctt cgt (SEQ ID No: 87 ) ggg gat ect cac gtc tea ctc gtc ctt ggt 19 bam gg ggc cat gtg gtg ctg cac ctt cea gaa gta gtt ctt agt ctc gtt ggg ctt cac gaa gtt ctt gcg ggg ct (SEQ ID No: 88) rl bbs 20 for (cgag) cgc gaa ttc gga aga ecc cga gtt cga ctg 20 rl ca ggc ctg ggc cta ctt cag cga cgt gga ect gga gaa gga cgt gca cag cgg ect gat cgg ecc ect get ggt (SEQ ID No: 89 ) ggg gat ect cac gtc tea gaa cag ggc aaa 20 bam ttc ctg cac agt cac ctg ect ecc gtg ggg ggg gtt cag ggt gtt ggt gtg gca cag cag cag ggg gcc gat ca (SEQ ID No: 90) rl bbs 21 for (gtte) cgc gaa ttc gga aga ecc gtt ctt cac cat 21 rl ctt cga cga gac taa gag ctg gta ctt cac cga gaa cat gga gcg caa ctg ceg cgc ecc ctg caa cat cea gat (SEQ ID No: 91 ) ggg gat ect cac gtc tea cag ggt gtc cat 21 bam gat gta gcc gtt gat ggc gtg gaa gcg gta gtt ctc ctt gaa ggt ggg ate ttc cat ctg gat gtt gca ggg gg (SEQ ID No: 92) rl bbs 22 for (ectg) cgc gaa ttc gga aga ecc ect gcc egg ect 22 rl ggt gat ggc cea gga cea gcg cat ceg ctg gta ect get gtc tat ggg cag caga cga gaa cat cea cag cat cea (SEQ ID No: 93 ) ggg gat ect cac gtc gta gta cata ca gtt gta 22 bam cag ggc cat ctt gta ctc ctc ctt ctt gcg cac ggt gaa aac gtg gcc get gaa gtg gat get gtg gat gtt ct (SEQ ID No: 94) rl bbs 23 for (gtac) cgc gaa ttc gga aga ecc gta ecc cgg cgt 23 rl gtt cga gac tgt gga gat get gcc cag cag ggc cgg gat ctg gcg cgt gga gtg ect gat cgg cga gca ect gca (SEQ ID No: 95 ) ggg gat ect cac gtc tea get ggc cat gcc 23 bam cag ggg ggt ctg gca ctt gtt get gta cag cag gaa cag ggt get cat gcc ggc gtg cag gtg ctc gcc gat ca (SEQ ID No: 96) rl bbs 24 for (cage) cgc gaa ttc gga aga ecc cag cgg cea cat 24 rl ceg cga ctt cea gat cac cgc cag cgg cea gta cgg cea gtg ggc tec caa get ggc ceg ect gca cta cag cgg (SEQ ID No: 97 ) ggg gat ect cac gtc tea cat ggg ggc cag 24 bam cag gtc cac ctt gat cea gga gaa ggg ctc ctt ggt cga cea ggc gtt gat get gcc get gta gtg cag gcg gg (SEQ ID No: 98) rl bbs 25 for (catg) cgc gaa ttc gga aga ecc cat gat cat cea 25 rl cgg cat ca gac cec ggg cgc ceg cea gaa gtt cag cag ect gta cat cag cea gtt cat cat cat gta ctc tct (SEQ ID No: 99 ) ggg gat ect cac gtc tea gtt gcc gaa gaa 25 bam cac cat cag ggt gcc ggt get gtt gcc gcg gta ggt ctg cea ctt ctt gcc gtc tag aga gta cat gat gat ga (SEQ ID No: 100) rl bbs 26 for (caac) cgc gaa ttc gga aga ecc caa cgt gga cag 26 rl cag cgg cat caca gca ca cat cat cat ecc ecc cat cat cgc ceg cgta cat ceg ect gca ecc cac cea cta cag (SEQ ID No: 101 ) ggg gat ect cac gtc tea gcc cag ggg cat 26 bam get gca get gtt cag gtc gca gcc cat cag ctc cat gcg cag ggt get gcg gat get gta gtg ggt ggg gtg ca (SEQ ID No: 102) rl bbs 27 for (gggc ) cgc gaa ttc gga aga ecc ggg cat gga gag 27 rl ca ggc cat cag cga cgc cea gat cac cgc ctc cag cta ctt cac ca cat gtt cgc cac ctg gag ecc cag caa (SEQ ID No: 103) ggg gat ect cac gtc tea cea ctc ctt ggg 27 bam gtt gtt cac ctg ggg gcg cea ggc gtt get gcg gcc ctg cag gtg cag gcg ggc ctt get ggg get cea ggt gg (SEQ ID No: 104) rl bbs 28 for (gtgg) cgc gaa ttc gga aga ecc gtg get gca ggt 28 rl gga ctt cea gaa aac cat gaa ggt gac tgg cgt gac cac cea ggg cgt ca ga ga ect get gac cag cat gta cgt (SEQ ID No: 105 ) ggg gat ect cac gtc tea ctt gcc gtt ttg 28 bam gaa gaa cag ggt cea ctg gtg gcc gtc ctg get get get gat cag gaa ctc ctt cac gta cat get ggt cag ca (SEQ ID No: 106) rl bbs 29 for (caag) cgc gaa ttc gga aga ecc ca ggt gaa ggt 29 rl gtt cea ggg caa cea gga cag ctt cac acc ggt cgt gaa cag ect gga ecc ecc ect get gac ceg cta ect gcg (SEQ ID No: 107 ) ggg gat ect cac gtc tea gcg gcc get tea 29 bam gta cag gtc ctg ggc ctc gca gcc cag cac ctc cat gcg cag ggc gat ctg gtg cac cea get ctg ggg gtg gat gcg cag gta gcg ggt cag ca (SEQ ID No: 108 ) The codon usage for the original and synthetic genes described above are presented in Tables 3 and 4, respectively. TABLE 3: Codon frequency in the synthetic gene of the Factor VIII with Domain B deleted AA Codon Number / 1000 Fraction Gly GGG 7.00 4.82 0.09 Gly GGA 1.00 0.69 0.01 Gly GGT 0.00 0.00 0.00 Gly GGC 74.00 50.93 0.90 Glu GAG 81.00 55.75 0.96 Glu GAA 3.00 2.06 0.04 Asp GAT 4.00 2.75 0.05 Asp GAC 78.00 53.68 0.95 Val GTG 77.00 52.99 0.88 Val GTA 2.00 1.38 0.02 Val GTT 2.00 1.38 0.02 Val GTC 7.00 4.82 0.08 Ala GCG 0.00 0.00 0.00 Ala GCA 0.00 0.00 0.00 Ala GCT 3.00 2.06 0.04 Ala GCC 67.00 46.11 0.96 Arg AGG 2.00 1.38 0.03 Arg AGA 0.00 0.00 0.00 Ser AGT 0.00 0.00 0.00 Being AGC 97.00 66.76 0.81 Lys AAG 75.00 51.62 0.94 Lys AAA 5.00 3.44 0.06 Asn AAT 0.00 0.00 0.0000 43.36 1.00 Met ATG 43.00 29.59 1.00 lie ATA 0.00 0.00 0.00 lie ATT 2.00 1.38 0.03 lie ATC 72.00 49.55 0.97 Thr ACG 2.00 1.38 0.02 Thr ACA 1.00 0.69 0.01 Thr ACT 10.00 6.88 0.12 Thr ACC 70.00 48.18 0.84 Trp TGG 28.00 19.27 1.00 End TGA 1.00 0.69 1.00 Cys TGT 1.00 0.69 0.05 Cys TGC 18.00 12.39 0.95 End TAG_0_.00 0.00 0.00 End TAA 0.00 0.00 0.00 Tyr TAT 2.00 1.38 0.03 Tyr TAC 66.00 45.42 0.97 Leu TTG 0.00 0.00 0.00 Leu TTA 0.00 0.00 0.00 Phe TTT 1.00 0.69 0.01 Phe TTC 76.00 52.31 0.99 Be TCG 1.00 0.69 Q.01 Ser TCA 0.00 0.00 0.00 Be TCT 3.00 2.06 0.03 Ser TCC 19.00 13.08 0.16 Arg CGG 1.00 0.69 0.01 Arg CGA 0.00 0.00 0.00 Arg CGT 1.00 0.69 0.01 Arg CGC 69.00 47.49 0.95 Gln CAG 62.00 42.67 0.93 Gln CAA 5.00 3.44 0.07 His CAT 1.00 0.69 0.02 His CAC 50.00 34.41 0.98 Leu CTG 118.00 81.21 0.94 Leu CTA 3.00 2.06 0.02 Leu CTT 1.00 0.69 0.01 Leu CTC 3.00 2.06 0.02 Pro CCG 4.00 2.75 0.05 Pro CCA 0.00 0.00 0.00 Pro CCT 3.00 2.06 0.04 Pro CCC 68.00 46.80 0.91 TABLE 4: Codon frequency table for the native Factor VIII gene with the deleted B domain AA Codon Number / 1000 Fraction Gly GGG 12.00 8.26 0.15 Gly GGA 34.00 23.40 0.41 Gly GGT 16.00 11.01 0.20 Gly GGC 20.00 13.76 0.24 Glu GAG 33.00 22.71 0.39 Glu GAA 51.00 35.10 0.61 Asp GAT 55.00 37.85 0.67 Asp GAC 27.00 18.58 0.33 Val GTG 29.00 19.96 0.33 Val GTA 19.00 13.08 0.22 Val GTT 17.00 11.70 0.19 Val GTC 23.00 15.83 0.26 Ala GCG 2.00 1.38 0.03 GCA wing 18.00 12.39 0.25 GCT wing 31.00 21.34 0.44 GCC wing 20.00 13.76 0.28 Arg AGG 18.00 12.39 0.25 Arg AGA 22.00 15.14 0.30 Be AGT - 22.00 15.14 0.18 Be AGC 24.00 16.52 0.20 Lys AAG 32.00 22.02 0.40 Lys AAA 48.00 33.04 0.60 Asn AAT 38.00 26.15 0.60 Asn AAC 25.00 17.21 1.40 Met ATG 43.00 29.59 1.00 lie ATA 13.00 8.95 0.18 I have ATT 36.00 24.78 0.49 He has ATC 25.00 17.21 0.34 Thr ACG 1.00 0.69 0.01 Thr ACA 23.00 15.83 0.28 Thr ACT 36.00 24.78 0.43 Thr ACC 23.00 17.21 0.28 Trp TGG 28.00 19.27 1.00 End TGA 1.00 0.69 1.00 Cys TGT 7.00 4.82 0.37 Cys TGC 12.00 8.26 0.63 End TAG_0_.00 0.00 0.00 End TAA 0.00 0.00 0.00 Tyr TAT 41.00 28.22 0.60 Tyr TAC 27.00 18.58 0.40 Leu TTG 20.00 13.76 0.16 Leu TTA -10.00 6.88 0.08 Phe TTT 45.00 30.97 0.58 Phe TTC 32.00 22.02 0.42 Be TCG 2.00 1.38 0.02 Ser TCA 27.00 18.58 0.22 Be TCT 27.00 18.58 0.22 Ser TCC 18.00 12.39 0.15 Arg CGG 6.00 4.13 0.08 Arg CGA 10.00 6.88 0.14 Arg CGT 7.00 4.82 0.10 Arg CGC 10.00 6.88 0.14 Gln CAG 42.00 28.91 0.63 Gln CAA 25.00 17.21 0.37 His CAT 28.00 19.27 0.55 His CAC 23.00 15.83 0.45 Leu CTG 36.00 24.78 0.29 Leu CTA 15.00 10.32 0.12 Leu CTT 24.00 16.52 0.19 Leu CTC 20.00 13.76 0.16 Pro CCG 1.00 0.69 0.01 Pro CCA_ 32.00 22.02 0.43 Pro CCT 2 6 .. 00 17 .. 8 9 0 .. 35 Pro CCC 15. . 00 10. . 32 0,. twenty Use The synthetic genes of the invention are useful for expressing the protein normally expressed in mammalian cells in cell culture (eg, for the commercial production of human proteins such as hGH, TPA, Factor VIII, and Factor IX). The synthetic genes of the invention are also useful for gene therapy. For example, a synthetic gene encoding a selected protein can be introduced into a cell that can express the protein to create a cell which can be administered to a patient in need of the protein. These cell-based gene therapy techniques are well known to those skilled in the art, see, e.g., Anderson et al., U.S. Patent Number: 5,399,349; Mulligan and Wilson, Patent of the United States of America Number: 5,460,959.

LIST OF SEQUENCES NUMBER OF SEQUENCES: 37 (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: CGCGGGCTAG CCACCGAGAA GCTG 24 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 196 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ACCGAGAAGC TGTGGGTGAC CGTGTACTAC GGCGTGCCCG TGTGGAAGAG AGGCCACCAC 60 CACCCTGTTC TGCGCCAGCG ACGCCAAGGC GTACGACACC GAGGTGCACA ACGTGTGGGC 120 CACCCAGGCG TGCGTGCCCA CCGACCCCAA CCCCCAGGAG GTGGAGCTCG TGAACGTGAC 180 CGAGAACTTC AACATG 196 (2) INFORMATION FOR SEQ ID NO: 3: (i ) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CCACCATGTT GTTCTTCCAC ATGTTGAAGT TCTC 34 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) ) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: GACCGAGAAC TTCAA CATGT GGAAGAACAA CAT 33 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 192 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: TGGAAGAACA ACATGGTGGA GCAGATGCAT GAGGACATCA TCAGCCTGTG GGACCAGAGC 60 CTGAAGCCCT GCGTGAAGCT GACCCCCTGT GCGTGACCTG AACTGCACCG ACCTGAGGAA 120 CACCACCAAC ACCAACACAG CACCGCCAAC AACAACAGCA ACAGCGAGGG CACCATCAAG 180 GGCGGCGAGA TG 192 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: GTTGAAGCTG CAGTTCTTCA TCTCGCCGCC CTT 33 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: GAAGAACTGC AGCTTCAACA TCACCACCAG C 31 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 195 base pairs (B) TYPE : nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 8: AACATCACCA CCAGCATCCG CGACAAGATG CAGAAGGAGT ACGCCCTGCT GTACAAGCTG 60 GATATCGTGA GCATCGACAA CGACAGCACC AGCTACCGCC TGATCTCCTG CAACACCAGC 120 GTGATCACCC AGGCCTGCCC CAAGATCAGC TTCGAGCCCA TCCCCATCCA CTACTGCGCC 180 CCCGCCGGCT TCGCC 195 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 9: GAACTTCTTG TCGGCGGCGA AGCCGGCGGG 30 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: GCGCCCCCGC CGGCTTCGCC ATCCTGAAGT GCAACGACAA GAAGTTC 47 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 198 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GCCGACAAGA AGTTCAGCGG CAAGGGCAGC TGCAAGAACG TGAGCACCGT GCAGTGCACC 60 CACGGCATCC GGCCGGTGGT GAGCACCCAG CTCCTGCTGA ACGGCAGCCT GGCCGAGGAG 120 GAGGTGGTGA TCCGCAGCGA GAACTTCACC GACAACGCCA AGACCATCAT CGTGCACCTG 180 198 AATGAGAGCG TGCAGATC (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: AGTTGGGACG CGTGCAGTTG ATCTGCACGC TCTC 34 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: GAGAGCGTGC AGATCAACTG CACGCGTCCC 30 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 120 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: AACTGCACGC GTCCCAACTA CAACAAGCGC AAGCGCATCC ACATCGGCCC CGGGCGCGCC 60 TTCTACACCA CCAAGAACAT CATCGGCACC ATCCTCCAGG CCCACTGCAA CATCTCTAGA 120 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid ( C) CHAIN TYPE: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: GTCGTTCCAC TTGGCTCTAG AGATGTTGCA 30 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: GCAACATCTC TAGAGCCAAG TGGAACGAC 29 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 131 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: GCCAAGTGGA ACGACACCCT GCGCCAGATC GTGAGCAAGC TGAAGGAGCA GTTCAAGAAC 60 AAGACCATCG TGTTCACCAG AGCAGCGGCG GCGACCCCGA GATCGTGATC CACAGCTTCA 120 ACTGCGGCGG C 131 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: GCAGTAGAAG AATTCGCCGC CGCAGTTGA 29 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TCAACTGCGG CGGCGAATTC TTCTACTGC 29 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 195 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: GGCGAATTCT TCTACTGCAA CACCAGCCCC CTGTTCAACA GCACCTGGAA CGGCAACAAC 60 ACCTGGAACA ACACCACCGG CAGCAACAAC AATATTACCC TCCAGTGCAA GATCAAGCAG 120 ATCATCAACA TGTGGCAGGA GGTGGGCAAG GCCATGTACG CCCCCCCCAT CGAGGGCCAG 180 ATCCGGTGCA GCAGC - 195 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: GCAGACCGGT GATGTTGCTG CTGCACCGGA TCTGGCCCTC 40 (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: CGAGGGCCAG ATCCGGTGCA GCAGCAACAT CACCGGTCTG 40 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 242 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: AACATCACCG GTCTGCTGCT GCTGCTGACC CGGACGGCGG CAAGGACACC GACACCAACG 60 ACACCGAAAT CTTCCGCGAC GGCGGCAAGG ACACCAACGA CACCGAAATC TTCCGCCCCG 120 GCGGCGGCGA CATGCGCGAC AACTGGAGAT CTGAGCTGTA CAAGTACAAG GTGGTGACGA 180 TCGAGCCCCT GGGCGTGGCC CCCACCAAGG CCAAGCGCGC GGTGGTGCAG CGCGAGAAGC 240 GC 242 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24: CGCGGGCGGC CGCTTTAGCG CTTCTCGCGC TGCACCAC 38 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: CGCGGGGGAT CCAAGCTTAC CATGATTCCA GTAATAAGT 39 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 165 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 26: ATGAATCCAG TAATAAGTAT AACATTATTA TTAAGTGTAT TACAAATGAG TAGAGGACAA 60 AGGATAATAA GTTTAACAGC ATCTTTAGTA AATCAAAATT TGAGATTAGA TTGTAGACAT 120 GAAAATAATA CAAATTTGCC AATACAACAT GAATTTTCAT TAACG 165 (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH : 36 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 27: CGCGGGGAAT TCACGCGTTA ATGAAAATTC ATGTTG 36 (2) INFORMATION FOR SEQ ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple OR (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 28: CGCGGATCCA CGCGTGAAAA AAAAAAACAT 30 (2) INFORMATION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 149 base pairs (B) TYPE : nucleic acid (C) STRING TYPE: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 29: CGTGAAAAAA AAAAACATGT ATTAAGTGGA ACATTAGGAG TACCAGAACA TACATATAGA 60 AGTAGAGTAA TTTGTTTAGT GATAGATTCA TAAAAGTATT AACATTAGCA AATTTTACAA 120 CAAAAGATGA AGGAGATTAT ATGTGTGAG 149 (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30: CGCGAATTCG AGCTCACACA TATAATCTCC 30 (2) INFORMATION FOR SEQ ID NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 31: CGCGGATCCG AGCTCAGAGT AAGTGGACAA 30 (2) INFORMATION FOR SEQ ID NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 170 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 32: CTCAGAGTAA GTGGACAAAA TCCAACAAGT AGTAATAAAA CAATAAATGT AATAAGAGAT 60 AAATTAGTAA AATGTGAGGA ATAAGTTTAT TAGTACAAAA TACAAGTTGG TTATTATTAT 120 TATTATTAAG TTTAAGTTTT TTACAAGCAA CAGATTTTAT AAGTTTATGA 170 (2) INFORMATION FOR SEQ ID NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 33: CGCGAATTCG CGGCCGCTTC ATAAACTTAT AAAATC 36 (2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1632 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: CTCGAGATCC ATTGTGCTCT AAAGGAGATA CCCGGCCAGA CACCCTCACC TGCGGTGCCC 60 AGCTGCCCAG GCTGAGGCAA GAGAAGGCCA GAAACCATGC CCATGGGGTC TCTGCAACCG 120 CTGGCCACCT TGTACCTGCT GGGGATGCTG GTCGCTTCCG TGCTAGCCAC CGAGAAGCTG 180 TGGGTGACCG TGTACTACGG CGTGCCCGTG TGGAAGGAGG CCACCACCAC CCTGTTCTGC 240 GCCAGCGACG CCAAGGCGTA CGACACCGAG GTGCACAACG TGTGGGCCAC CCAGGCGTGC 300 GTGCCCACCG ACCCCAACCC CCAGGAGGTG GAGCTCGTGA ACGTGACCGA GAACTTCAAC 360 ATGTGGAAGA ACAACATGGT GGAGCAGATG CATGAGGACA GTG TCATCAGCCT GGACCAG 420 AGCCTGAAGC CCTGCGTGAA GCTGACCCCC CTGTGCGTGA CCCTGAACTG CACCGACCTG 480 AGGAACACCA CCAACACCAA CAACAGCACC GCCAACAACA ACAGCAACAG CGAGGGCACC 540 ATCAAGGGCG GCGAGATGAA CAACTGCAGC TTCAACATCA CCACCAGCAT CCGCGACAAG 600 ATGCAGAAGG AGTACGCCCT GCTGTACAAG CTGGATATCG TGAGCATCGA CAACGACAGC 660 ACCAGCTACC GCCTGATCTC CTGCAACACC AGCGTGATCA CCCAGGCCTG GCCCAAGATC 720 AGCTTCGAGC CCATCCCCAT CCACTACTGC GCCCCCGCCG GCTTCGCCAT CCTGAAGTGC 780 AACGACAAGA AGTTCAGCGG CAAGGGCAGC TGCAAGAACG TGAGCACCGT GCAGTGCACC 840 CACGGCATCC GGCCGGTGGT GAGCACCCAG CTCCTGCTGA ACGGCAGCCT * GGCCGAGGAG 900 GAGGTGGTGA TCCGCAGCGA GAACTTCACC GACAACGCCA AGACCATCAT CGTGCACCTG 960 AATGAGAGCG TGCAGATCAA CTGCACGCGT CCCAACTACA ACAAGCGCAA GCGCATCCAC 1020 ATCGGCCCCG GGCGCGCCTT CTACACCACC AAGAACATCA TCGGCACCAT CCGCCAGGCC 1080 CACTGCAACA TCTCTAGAGC CAAGTGGAAC GACACCCTGC GCCAGATCGT GAGCAAGCTG 1140 AAGGAGCAGT TCAAGAACAA GACCATCGTG TTCAACCAGA GCAGCGGCGG CGACCCCGAG 1200 ATCGTGATGC ACAGCTTCAA CTGCGGCGGC GAATTCTTCT ACTGCAACAC CAGCCCCCTG 1260 TT CAACAGCA CCTGGAACGG CAACAACACC TGGAACAACA CCACCGGCAG * CAACAACAAT 1320 ATTACCCTCC AGTGCAAGAT CAAGCAGATC ATCAACATGT GGCAGGAGGT GGGCAAGGCC 1380 ATGTACGCCC CCCCCATCGA GGGCCAGATC CGGTGCAGCA GCAACATCAC CGGTCTGCTG 1440 CTGACCCGCG ACGGCGGCAA GGACACCGAC ACCAACGACA CCGAAATCTT CCGCCCCGGC 1500 GGCGGCGACA TGCGCGACAA CTGGAGATCT GAGCTGTACA AGTACAAGGT GGTGACGATC 1560 GAGCCCCTGG GCGTGGCCCC CACCAAGGCC AAGCGCCGCG TGGTGCAGCG CGAGAAGCGC 1620 TAAAGCGGCC GC 1632 (2) INFORMATION FOR SEQ ID NO: 35: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 2481 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY : linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 35: ACCGAGAAGC TGTGGGTGAC CGTGTACTAC GGCGTGCCCG TGTGGAAGGA GGCCACCACC 60 ACCCTGTTCT GCGCCAGCGA CGCCAAGGCG TACGACACCG AGGTGCACAA CGTGTGGGCC Í20 ACCCAGGCGT GCGTGCCCAC CGACCCCAAC CCCCAGGAGG TGGAGCTCGT GAACGTGACC 180 GAGAACTTCA ACATGTGGAA GAACAACATG CTGGAGCAGA TGCATGAGGA CATCATCAGC 240 CTGTGGGACC AGAGCCTGAA GCCCTGCGTG AAGCTGACCC CCCTGTGCGT GACCCTGAAC 300 TGCACCGACC TGAGGAACAC CACCAACACC AACAACAGCA CCGCCAACAA CAACAGCAAC 360 AGCGAGGGCA CCATCAAGGG CGGCGAGATG AAGAACTGCA GCTTCAACAT CACCACCAGC 420 ATCCGCGACA AGATGCAGAA GGAGTACGCC CTGCTGTACA AGCTGGATAT CGTGAGCATC 480 CACAACGACA GCACCAGCTA CCGCCTGATC TCCTGCAACA CCAGCGTGAT CACCCAGGCC 540 TGCCCCAAGA TCAGCTTCGA GCCCATCCCC ATCCACTACT GCGCCCCCGC CGGCTTCGCC 600 ATCCTGAAGT GCAACGACAA GAAGTTCAGC GGCAAGGGCA GCTGCAAGAA CGTGACCACC 660 GTGCAGTGCA CCCACGGCAT CCGGCCGGTG GTGAGCACCC AGCTCCTGCT GAACGGCAGC 720 CTGGCCGAGG AGGAGGTGGT GATCCGCAGC GAGAACTTCA CCGACAACGC CAAGACCATC 780 ATCGTGCACC TGAATGAGAG CGTGCAGATC AACTGCACGC GTCCCAACTA CAACAAGCGC 840 AAGCGCATCC ACATCGGCCC CGGGCGCGCC TTCTACACCA CCAAGAACAT CATCGGCACC 900 ATCCGCCAGG CCCACTGCAA CATCTCTAGA GCCAAGTGGA ACGACACCCT GCGCCAGATC 960 GTGAGCAAGC TGAAGGAGCA GTTCAAGAAC AAGACCATCG TGTTCAACCA GAGCAGCGGC 1020 GGCGACCCCG AGATCGTGAT GCACAGCTTC AACTGCGGCG GCGAATTCTT CTACTGCAAC 1080 ACCAGCCCCC TGTTCAACAG CACCTGGAAC GGCAACAACA CCTGGAACAA CACCACCGGC 1140 AGCAACAACA ATATTACCCT CCAGTGCAAG ATCAAGCAGA TCATCAACAT GTGGCAGGAG 1200 GTGGGCAAGG CCATGTACGC CCCCCCCATC GAGGGCCAGA TCCGGTGCAG CAGCAACATC 1260 ACCGGTCTGC TGCTGACCCG CGACGGCGGC AAGGACACCG ACACCAACGA CACCGAAATC 1320 TTCCGCCCCG GCGGCGGCGA CATGCGCGAC AACTGGAGAT CTGAGCTGTA CAAGTACAAG 1380 GTGGTGACGA TCGAGCCCCT GGGCGTGGCC CCCACCAAGG CCAAGCGCCG CGTGGTGCAG 1440 CGCGAGAAGC GGGCCGCCAT CGGCGCCCTG TTCCTGGGCT TCCTGGGGGC GGCGGGCGC 1500 ACCATGGGGG CCGCCAGCGT GACCCTGACC GTGCAGGCCC GCCTGCTCCT GAGCGGCATC 1560 GTGCAGCAGC AGAACAACCT CCTCCGCGCC ATCGAGGCCC AGCAGCATAT GCTCCAGCTC 1620 ACCGTGTGGG GCATCAAGCA GCTCCAGGCC CGCGTGCTGG CCGTGGAGCG CTACCTGAAG 1680 GACCAGCAGC TCCTGGGCTT CTGGGGCTGC TCCGGCAAGC TGATCTGCAC CACCACGGTA 1740 CCCTGGAACG CCTCCTGGAG CAACAAGAGC CTGGACGACA TCTGGAACAA CATGACCTGG 1800 ATGCAGTGGG AGCGCGAGAT CGATAACTAC ACCAGCCTGA TCTACAGCCT GCTGGAGAAG 1860 AGCCAGACCC AGCAGGAGAA GAACGAGCAG GAGCTGCTGG AGCTGGACAA CTGGGCGAGC 1920 CTGTGGAACT GGTTCGACAT CACCAACTGG CTGTGGTACA TCAAAATCTT CATCATGATT 1980 GTGGGCGGCC TGGTGGGCCT CCGCATCGTG TTCGCCGTGC TGAGCATCGT GAACCGCGTG 2040 CGCCAGGGCT ACAGCCCCCT GAGCCTCCAG ACCCGGCCCC CCGTGCCGCG CGGGCCCGAC 2100 CGCCCCGAGG GCATCGAGGA GGAGGGCGGC GAGCGCGACC GCGACACCAG CGGCAGGCTC 2160 GTGCACGGCT TCCTGGCGAT CATCTGGGTC GACCTCCGCA GCCTGTTCCT GTTCAGCTAC 2220 CACCACCGCG ACCTGCTGCT GATCGCCG-CC CGCATCGTGG AACTCCTAGG CCGCCGCGGC 2280 TGGGAGGTGC TGAAGTACTG GTGGAACCTC CTCCAGTATT GGAGCCAGGA GCTGAAGTCC 2340 AGCGCCGTGA GCCTGCTGAA CGCCACCGCC ATCGCCGTGG CCGAGGGCAC CGACCGCGTG 2400 ATCGAGGTGC TCCAGAGGGC CGGGAGGGCG ATCCTGCACA TCCCCACCCG CATCCGCCAG 2460 GGGCTCGAGA GGGCGCTGCT G 2481 (2) INFORMATION FOR SEQ ID NO: 36: (i) CHARACTERISTICS THE SEQUENCE: (A) LENGTH: 486 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 36: ATGAATCCAG TAATAAGTAT AACATTATTA TTAAGTGTAT TACAAATGAG TAGAGGACAA 60 AGAGTAATAA GTTTAACAGC ATGTTTAGTA AATCAAAATT TGAGATTAGA TTGTAGACAT 120 GAAAATAATA CACCTTTGCC AATACAACAT GAATTTTCAT TAACGCGTGA AAAAAAAAAA 180 CATGTATTAA GTGGAACATT AGGAGTACCA GAACATACAT ATAGAAGTAG AGTAAATTTG 240 TTTAGTGATA GATTCATAAA AGTATTAACA TTAGCAAATT TTACAACAAA AGATGAAGGA 300 GATTATATGT GTGAGCTCAG AGTAAGTGGA CAAAATCCAA CAAGTAGTAA TAAAACAATA 360 AATGTAATAA GAGATAAATT AGTA AAATGT GGAGGAATAA GTTTATTAGT ACAAAATACA 420 AGTTGGTTAT TATTATTATT ATTAAGTTTA AGTTTTTTAC AAGCAACAGA TTTTATAAGT 480 TTATGA 486 (2) INFORMATION FOR SEQ ID NO: 37: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 485 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: ATGAACCCAG TCATCAGCAT CACTCTCCTG CTTTCAGTCT TGCAGATGTC CCGAGGACAG 60 AGGGTGATCA GCCTGACAGC CTGCCTGGTG AACAGAACCT TCGACTGGAC TGCCGTCATG 120 AGAATAACAC CAACTTGCCC ATCCAGCATG AGTTCAGCCT GACCCGAGAG AAGAAGAAGC 180 ACGTGCTGTC AGGCACCCTG GGGGTTCCCG AGCACACTTA CCGCTCCCGC GTCAACCTTT 240 TCAGTGACCG CTTTATCAAG GTCCTTACTC TAGCCAACTT GACCACCAAG GATGAGGGCG 300 ACTACATGTG TGAACTTCGA GTCTCGGGCC AGAATCCCAC AAGCTCCAAT AAAACTATCA 360 ATGTGATCAG AGACAAGCTG GTCAAGTGTG GTGGCATAAG CCTGCTGGTT CAAAACACTT 420 CCTGGCTGCT GCTGCTCCTG CTTTCCCTCT CCTTCCTCCA AGCCACGGAC TTCATTTCTC TGTGA 480 485

Claims (19)

1. CLAIMS 1. A synthetic gene encoding a green fluorescent protein, wherein at least one non-preferred or less preferred codon in a natural gene encoding said protein has been replaced by a preferred codon encoding the same amino acid, said synthetic gene being capable of expressing said protein at a level that is at least 110% of that expressed by said natural gene in a mammalian cell culture system in vitro under identical conditions.
2. 2. A synthetic gene encoding a factor VIII protein that lacks the central region B domain, where at least one non-preferred or less preferred codon in a natural gene encoding said protein has been replaced by a preferred codon encoding the same amino acid, said synthetic gene being able to express said protein at a level that is at least 110% of that expressed by said natural gene in a mammalian cell culture system in vi tro under identical conditions.
3. 3. The synthetic gene of claim 1 or 2, wherein said synthetic gene is capable of expressing said protein at a level that is at least 150% that expressed by said natural gene in a cell culture system in vi tro under identical conditions .
4. 4. The synthetic gene of claim 1 or 2, wherein said synthetic gene is capable of expressing said protein at a level that is at least 200% of that expressed by said natural gene in a cell culture system in vi tro under identical conditions .
5. 5. The synthetic gene of claim 1 or 2, wherein said synthetic gene is capable of expressing said protein at a level that is at least 500% that expressed by said natural gene in a cell culture system in vi tro under identical conditions .
6. 6. The synthetic gene of claim 1 or 2, wherein said synthetic gene is capable of expressing said protein at a level that is at least 1,000% of that expressed by said natural gene in a mammalian cell culture system in vi tro under identical conditions.
7. 7. The synthetic gene of claim 1 or 2, wherein said synthetic gene comprises less than five occurrences of the CG sequence.
8. 8. The synthetic gene of claim 1 or 2, wherein at least 10% of the codons in said wild-type gene are non-preferred codons.
9. 9. The synthetic gene of claim 1 or 2, wherein at least 50% of the codons in said wild-type gene are non-preferred codons.
10. The synthetic gene of claim 1 or 2, wherein at least 50% of the non-preferred codons and the less preferred codons present in said natural gene have been replaced by preferred codons.
11. 11. The synthetic gene of claim 1 or 2, wherein at least 90% of the non-preferred codons and the less preferred codons present in said natural gene have been replaced by preferred codons.
12. 12. The synthetic gene of claim 1 or 2, wherein 20% of the codons are preferred codons.
13. The synthetic gene of claim 1, wherein said gene has the coding sequence present in SEQ ID NO: 40.
14. The synthetic gene of claim 2, wherein said gene has the coding sequence present in SEQ ID NO: 42.
15. 15. An expression vector comprising the synthetic gene of claim 1 or 2.
16. 16. The expression vector of claim 15, said expression vector being a mammalian expression vector.
17. 17. A mammalian cell harboring the synthetic gene of claim 1 or 2.
18. 18. A method for preparing a synthetic green fluorescent gene, which comprises identifying or preferred and less preferred codons in the natural green fluorescent protein gene and replacing one or more of said non-preferred and less preferred codons with a preferred codon encoding the same amino acid as the replaced codon.
19. 19. A method for preparing a synthetic gene encoding a factor VIII protein that lacks the B-region domain, which comprises identifying non-preferred and less-preferred codons in the natural gene encoding said factor VIII protein that lacks the domain of the central region B and replacing one or more of said non-preferred and less preferred codons with a preferred codon encoding the same amino acid as the replaced codon.