Glycosylated analogues of Flavivirus E proteins and their use in diagnostic methods
11401307 · 2022-08-02
Assignee
Inventors
Cpc classification
C12N2770/24122
CHEMISTRY; METALLURGY
C12N2770/24134
CHEMISTRY; METALLURGY
G01N2333/185
PHYSICS
C07K14/1825
CHEMISTRY; METALLURGY
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
A61K39/00
HUMAN NECESSITIES
Abstract
The invention relates to isolated recombinant analogues of flavivirus E-proteins comprising an analogue of a flavivirus E-protein fusion loop, wherein the analogue of the flavivirus E-protein fusion loop comprises at least one glycosylation site for an N-linked glycan that is not present in a natural flavivirus E-protein fusion loop sequence, wherein the at least one glycosylation site is an N-linked glycosylation sequon (Asn-X-Ser/Thr) and the Asn (N) residue of the sequon occupies any of positions 98-110 (DRGWGNGCGLFGK) of the natural flavivirus E-protein fusion loop amino acid sequence, wherein X is any amino acid residue except proline and Ser/Thr denotes a serine or threonine residue for use in an in vitro method for specific detection of anti-flavivirus antibody, diagnosis of flavivirus infection and/or to investigate exposure to flavivirus.
Claims
1. An isolated recombinant analogue of a flavivirus E-protein comprising an analogue of a flavivirus E-protein fusion loop, wherein the analogue of the flavivirus E-protein fusion loop comprises at least one glycosylation site for an N-linked glycan that is not present in a natural flavivirus E-protein fusion loop sequence, wherein the at least one glycosylation site is an N-linked glycosylation sequon (Asn-X-Ser/Thr) and the Asn (N) residue of the sequon occupies any of positions 98-110 (DRGWGNGCGLFGK; SEQ ID NO: 1) of the natural flavivirus E-protein fusion loop amino acid sequence, wherein X is any amino acid residue except proline and Ser/Thr denotes a serine or threonine residue, wherein the analogue is configured for use in an in vitro method for specific detection of anti-flavivirus antibody, diagnosis of flavivirus infection and/or to investigate exposure to flavivirus.
2. The isolated recombinant analogue of a flavivirus E-protein according to claim 1, wherein the analogue of the flavivirus E-protein fusion loop comprises two glycosylation sites that are not present in a natural flavivirus E-protein fusion loop.
3. The isolated recombinant analogue of a flavivirus E-protein of claim 1 which is glycosylated with a glycan at one or at both of the introduced glycosylation sites in the analogue of the flavivirus E-protein fusion loop.
4. The isolated recombinant analogue of a flavivirus E-protein of claim 1 wherein the glycan is an N-linked glycan.
5. The isolated recombinant analogue of a flavivirus E-protein of claim 1, comprising an N-linked glycosylation sequon (Asn-X-Ser/Thr) such that an Asn (N) residue of the sequon occupies any of positions 98-101 and/or 106-110.
6. The isolated recombinant analogue of a flavivirus E-protein of claim 1, wherein X is selected from any of the following 13 amino acid residues Gly, His, Asn, Gln, Tyr, Val, Ala, Met, Ile, Lys, Arg, Thr or Ser.
7. The isolated recombinant analogue of a flavivirus E-protein of claim 1, wherein the flavivirus E-protein is a dengue virus E-protein and the Asn (N) residue of the sequon occupies position 101, 108 or both 101 and 108 of the amino acid sequence of the flavivirus E-protein fusion loop or the flavivirus E-protein is a Zika E-protein and the Asn (N) residue of the sequon occupies position 100 of the amino acid sequence of the flavivirus E-protein fusion loop.
8. The isolated recombinant analogue of a flavivirus E-protein of claim 1, wherein the flavivirus is a dengue virus and the amino acid sequence of the analogue flavivirus E-protein fusion loop 98-110 is selected from: DRGNGSGCGLNGS (SEQ ID NO: 2), DRGNGSGCGLFGK (SEQ ID NO: 3) and DRGWGNGCGLNGS (SEQ ID NO: 4).
9. The isolated recombinant analogue of a flavivirus E-protein of claim 1, wherein the flavivirus is a Zika virus and the amino acid sequence of the analogue flavivirus E-protein fusion loop 98-110 is DRNHTNGCGLFGK (SEQ ID NO: 5).
10. A test kit comprising an isolated recombinant analogue of a flavivirus E-protein of claim 1 and a reagent capable of detecting an immunological (antigen-antibody) complex which contains said isolated analogue or binding molecule.
11. The test kit according to claim 10, wherein said analogue and/or binding molecule is immobilized on a solid support.
12. The test kit according to claim 11, wherein the solid support is a microplate well.
13. The test kit according to claim 10, wherein said immunological complex which contains said isolated analogue or binding molecule is detected by lateral flow.
14. The test kit according to claim 10, wherein said kit comprises a test device comprising lateral flow test strip comprising: a sample pad for application of a liquid sample, a conjugate pad comprising a detector conjugate for conjugation of anti-flavivirus antibody in the liquid sample, a capture strip comprising a capture means to capture detector conjugate-anti-flavivirus antibody complex and an absorbent pad, the pads and strip being arranged to permit capillary flow communication with each other.
15. The test kit according to claim 10, wherein said kit comprises a test device comprising a lateral flow test strip comprising: a sample pad for application of a liquid sample, said sample pad comprising a first test antigen with a first tag and one or more second pre-absorbing antigen(s) optionally with a second tag, a conjugate pad comprising a detector conjugate for conjugation of anti-flavivirus antibody in the liquid sample, a capture strip comprising a capture means to capture detector conjugate-anti-flavivirus antibody complex via the first tag, and an absorbent pad, the pads and strip being arranged to permit capillary flow communication with each other, wherein the first antigen comprises a glycosylated analogue of a Zika E-protein fusion loop of claim 1 and the second pre-absorbing antigen(s) comprises one or more glycosylated analogue of a Dengue E-protein fusion loop of claim 1, or wherein the first antigen comprises a glycosylated analogue of a Dengue E-protein fusion loop of claim 1 and the second pre-absorbing antigen comprises a glycosylated analogue of a Zika E-protein fusion loop of claim 1.
16. The test kit according to claim 14 wherein the capture means for capture of the detector conjugate-anti-flavivirus antibody complex is an antigen comprising a recombinant analogue of a flavivirus E-protein of claim 1.
17. The test kit according to claim 14, wherein the capture means is provided as a line on the capture strip.
18. The test kit according to claim 14, wherein the liquid sample is a biological sample.
19. The test kit according to claim 18, wherein the liquid sample is a biological sample selected from blood, plasma, serum, saliva, oral fluid and CSF.
20. A method for detection of a flavivirus antibody in a sample comprising contacting the sample with a) a recombinant analogue of a flavivirus E-protein of claim 1 and b) a reagent capable of detecting an immunological (antigen-antibody) complex which contains the recombinant analogue.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1) The invention will now be described with reference to the accompanying drawing in which:
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(6) 1: pSF236 transfected cells WT, 2: pCRO21 transfected cells, 3: pSF237 transfected cells WT, 4: pCRO22 transfected cells, 5: pSF238 transfected cells WT, 6: pCRO23 transfected cells, 7: pSF239 transfected cells WT, 8: pCRO24 transfected cells, 9: pSF233 transfected cells WT, 10: pCRO25 transfected cells. 11: pSF236 transfected cells WT, 12: pCRO21 transfected cells, 13: pSF237 transfected cells WT, 14: pCRO22 transfected cells, 15: pSF238 transfected cells WT, 16: pCRO23 transfected cells, 17: pSF239 transfected cells WT, 18: pCRO24 transfected cells, 19: pSF233 transfected cells WT, 20: pCRO25 transfected cells. For lanes 1 to 10, the supernatant concentrate was 1 ul/1.1 ml, for lanes 11 to 20 the supernatant concentrate Talon eluate concentration was 26 ul/400 ul.
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(20) The x-axis shows the number of days after immunisation and the y-axis shows the IgG antibody titre. Three doses were given on days 0, 14 and 21. Dosages are indicated in Table 9. Antibody responses were measured in individual mice against all five antigens as wild-type VLPs on the ELISA solid phase as indicted: top row left Den 1 VLP antigen, top row right Den 2 VLP antigen, middle row left Den 3 VLP antigen, middle row right Den 4 VLP antigen, bottom row left Zika VLP antigen. Immunogens (as distinct from antigens uses for assay above) were Penta-DNA (a combination of each of the Den1-4 and Zika DNAs of the invention) shown as an open circle, Penta-Prot (a combination of each of the Den1-4 and Zika proteins of the invention) is shown as an filled square, Monovalent Zika is shown as a filled triangle, Penta VLP (a combination of each of the Den1-4 and Zika VLPs of the invention) is shown as a filled inverted triangle. PBS control is shown as an open inverted triangle.
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(22) In order to further characterize the hyperglycosylated antigens of the present disclosure, comparing them to wild-type equivalent antigens, an ELISA assay was established to measure antibody binding to diverse wild-type and recombinant exodomains (as distinct from the VLP antigens of
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(32) Upper panel shows ELISA reactivity of antibodies in a dengue convalescent serum with immobilized Zika and dengue wild-type (WT) and hyperglycosylated (HX) exodomain proteins oriented on the solid phase by capture with a rabbit anti-His-tag monoclonal antibody, in the presence (grey bars, right of each pair) and absence (black bars, left of each pair) of competing mouse monoconal flavivirus fusion loop antibody 4G2 (an anti-dengue-serotype-2 cross-reactive monoclonal antibody) at a concentration of 10 ug/ml during serum incubation. Human sera were tested at a constant concentration of 1/1000.
(33) Lower panel shows ELISA reactivity of antibodies in a Zika convalescent serum with immobilized Zika and Dengue wild-type (WT) and hyperglycosylated (HX) exodomain proteins in the presence (grey bars) and absence (black bars) of competing mouse monoclonal flavivirus fusion loop antibody 4G2. Conditions and labelling are the same as for the upper panel. Error bars are standard error of duplicate determinations.
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(47) (B) With a high threshold (set at greater than 3=positive), Zika LF of the invention markedly outperforms commercial (Euroimmun/Perkin Elmer) and bespoke (DABA) Zika ELISA assays based on measuring the NS1 protein (a test for acute Zika infection), the NS1 ELISA tests score half of this BOB-negative sample group as positive for Zika (an implausible result given the sample collection date—before significant Zika circulation had occurred) whereas the Excivion Zika LF scores 2/50=4% as positive—a more plausible result (Zika was present in Brazil in 2014 but the precise exent of its circulation is unknown);
(48) (C) dengue LF test of the invention (setting a threshold of greater than 3=positive, as in B) scores 92% of Rio 2014 endemic sera as dengue +ve, which tallies with the 90-95% accepted value based on several published studies of dengue seropositivity in Brazil at this time. The dengue LF of the invention tallies well 92% compared to the 90-95% dengue seropositivity in this 2014 sample group from Brazil, demonstrating excellent sensitivity of the dengue-LF (confirming results from the Pune tests, which demonstrated superior sensitivity of IgG detection over the Standard Diagnostics Duo LF test, p<0.0000001).
(49) (D) Frequency-distribution of LF scores in the Zika LF and dengue LF tests of the invention in the Rio 2014 sample set (before significant Zika circulation had occurred); one subject had a zero score in the dengue LF test and exemplifies the utility of the test in identifying dengue-negative subjects for companion-diagnostic use of the test with dengue vaccines (e.g. Dengvaxia), i.e., in order to avoid priming for dengue haemorrhagic fever this subject would be spared vaccination, the two subjects who were LF-positive for Zika in this sample set (scoring ‘4’) may represent genuine Zika cases and the ability of the Zika LF of the invention to be used as a ‘sentinel test’ providing early warning of a Zika outbreak.
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EXAMPLES
Example 1 Design of New Vaccine Immunogens Designed to Avoid the Elicitation or Stimulation of Infection-Enhancing Antibodies
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(52) Conversely, ‘B’ illustrates a vaccine immunogen designed in accordance with the invention. The novel immunogen, containing an E-protein wherein the fusion loop sequence has been modified and has been designed to be substituted with a glycan with the aim to generate neutralising antibodies against the E-proteins of the vaccine without generating infection-enhancing antibodies. ‘D’ represents occasional failure of the vaccine of the invention to elicit a protective level of antibody response in some subjects (e.g., the immunosuppressed), however, unlike other vaccine designs known in the art, the vaccine of the invention is designed to not render immunosuppressed subjects susceptible to enhanced infection with dengue or Zika viruses. Immunogens and vaccines of the present design are thereby designed to be safer on an individual subject basis and moreover to lack the potential to facilitate the epidemic spread of Zika by creating a population of subjects that have Zika-infection-enhancing antibodies, in the absence of neutralising antibodies. (WT=wild type).
Example 2 (FIG. 2) Recombinant Expression of Glycoengineered (Hyperglycosylated) Forms of Dengue and Zika Exodomain Proteins
(53) Plasmid inserts encoding various novel recombinant forms of the natural wild type (WT) exodomain sequences representative of the four dengue serotypes and of Zika and containing an E. coli origin of replication and a cytomegalovirus (CMV) promoter, as well as a hexahistidine C-terminal tag, were made by de novo gene synthesis (Thermofisher, GeneArt). Where two glycosylation sequons were inserted in the DNA sequence, the sequence was changed ‘manually’ to avoid the creation of direct DNA sequence repeats that might otherwise allow undesirable homologous recombination events.
(54) Plasmid expression vectors pCRO21 (SEQ ID NO: 13), pCRO22 (SEQ ID NO: 14), pCRO23 (SEQ ID NO: 15), pCRO24 (SEQ ID NO: 16) and pCRO28 (SEQ ID NO: 17), coding for the mutated exodomain of the Envelope proteins of DENV1, DENV2, DENV3, DENV4 and ZIKV, respectively, were ultimately selected and produced by The Native Antigen Company, Oxford, as follows: expression cassettes were synthesized de novo to contain a 5′ NotI site followed by a consensus Kozak sequence followed by the coding sequence for the first 17 amino acids of the influenza-A virus haemagglutinin protein acting as secretion signal. The Envelope protein coding sequences used, (numbering relative to the polyprotein), were 280-675 (NCBI ACA48859.1), 281-676 (NCBI ADK37484.1), 281-673 (NCBI AIH13925.1), 280-675 (NCBI ANK35835.1) and 291-696 (NCBI ARB07957.1), respectively. [Elsewhere, for ease of reference, numbering is expressed according to residue number in the E-protein, with W at 101 of the fusion loop as a reference point]. Each construct contained coding sequences for a glycine-serine linker 7 to 8 amino acids in length followed by a 6× His-tag and a stop codon. The stop codon is followed by a NheI site in each expression cassette. The mammalian expression vector pSF-CMV (Oxford Genetics, Oxford) was digested with NotI and NheI, and the 4.2 kb fragment was ligated to the 1.3 kb NotI and NheI fragments of the expression cassette harbouring maintenance vectors (pUC57). In each case, one or two additional sequons of the general formula (NXS/T) was introduced into the fusion loop of the E-protein exodomain, capable (theoretically) of encoding a functional N-linked glycosylation site. The wild-type dengue proteins naturally already have two glycosylation sites, and Zika one. None of the natural glycans are found in the fusion loop.
(55) For small-scale preparation 15 ml aliquots of HEK293FT cells at 3e6/ml were individually transfected with pCRO21, pCRO22, pCRO23, pCRO24 or pCRO25 (SEQ ID NO: 18), 4 control transfections were performed using pSF233, pSF236, pSF237, pSF238 or pSF239. After a day, 15 ml of rescue medium was added to each transfection. At day 3 after transfection each of the 10 transfections was treated the same way as follows: 30 ml of suspension was spun at 4,000 g for 7 minutes. The resulting supernatant was filtered using a 0.22 um disc filter. The pellet was resuspended in 1 ml of PBS. The filtered supernatant was then concentrated using a Vivaspin20 (30,000 Da cutoff) as per manufacturer's instructions. Concentrate volumes ranged from 0.6 ml to 1.2 ml. All concentrates were brought up to 1.2 ml with PBS. The concentrated supernatants were subjected to Talon purification as per manufacturer's instructions using Talon HiTrap Spin (GE). Buffers for Talon capture were: Equilibration Buffer: 50 mM phosphate pH7.8, 300 mM NaCl; Wash Buffer: 50 mM phosphate pH78, 300 mM NaCl, 5 mM imidazole; Elution Buffer: 50 mM phosphate pH7.8, 300 mM NaCl, 150 mM imidazole.
(56) Characterisation of the resulting proteins by coomassie-blue staining (
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(59) For scale-up production, the novel hyperglycosylated proteins were expressed recombinantly in human embryonic kidney cells (HEK 293) by transient transfection with linear polyethyleneimine (PEI), and purified by metal chelate affinity chromatography with a cobalt chelate (TALON®, Clontech/GE), as described as follows for the dengue-1 hyperglycosylated construct based on pCRO21. 20×1 L of HEK293 cells were transfected with DEN V1_Eexo_2xglyco expression vector pCRO21. 3 days post transfection, the supernatant was harvested by centrifugation, and the cleared supernatant was 0.2 um filtered and concentrated to ˜200 ml by tangential flow filtration (TFF). Immobilised metal affinity chromatography (IMAC) was performed on the TFF retentate using 5 ml HiTRAP Talon pre-packed column (GE) according to manufacturer's instructions using 20 mM sodium phosphate pH7.8 based buffer systems. DENV1_Eexo_2xglyco protein containing fractions were pooled and dialysed against 20 mM TRIS-HCl pH7.8 10 mM NaCl. Ion exchange chromatography was performed using a pre-packed 5 ml HiTrap Q HP column according to manufacturer's instructions. DENV1_Eexo_2xglyco were pooled and dialysed against DPBS pH7.4. The dialysed solution was 0.22 um filtered and vialled under sterile conditions. BCA assay and SDS-PAGE were performed according to manufacturer's instructions (Bio-Rad).
(60) Note that three of the hyperglycosylated constructs express at levels much higher than wild type (these are the hyperglycosylated dengue serotypes 2, 3 and 4 corresponding to plasmids pCRO22, pCRO23 and pCRO24). Zika plasmid, pCRO25 did not give rise to detectable secreted protein (
(61) Therefore a further round of constructs was made (see
(62) The hyperglycosylated forms chosen were pCRO21, pCRO22, pCRO23, pCRO24 (for dengue serotypes 1-4 respectively) and pCRO28 for Zika. Hyperglycosylated exodomains D1, D2, D3, D4 and Zika correspond to plasmids pCRO21, pCRO22, pCRO23, pCRO24 and pCRO28, respectively (SEQ ID NO: 24, 25, 26, 27 and 28 respectively). Molecular weight increments due to glycosylation are apparent, higher for the +2 dengue constructs than for the Zika +1 construct.
(63) In all, eleven plasmid constructs were made and tested for protein expression and five were selected for further investigation, based on equivalent or (in most cases) superior levels of expression compared to wild type (pCRO21, pCRO22, pCRO23, pCRO24 representing the four serotypes of dengue, and pCRO28 representing Zika).
(64) Surprisingly, given the extremely hydrophobic nature of the fusion loop (which features the residues W, F and L exposed at the tip of the E protein in close juxtaposition at its distal end in three dimensional space) in the case of dengue, all four representative serotypes tolerated substitution of two glycans (which are hydrophilic, and radically transform the topography of this part of the protein to an extent that mere amino-acid substitutions cannot) with no penalty to levels of expression (i.e., all expressed as well as the wild type sequence, in some cases markedly better). An objective had been set of ‘no less than wild type’ for levels of expression in order to ensure that the proteins were not misfolded which would have resulted in eradication from the endoplasmic reticulum via the ERAD channel for proteasomal degradation. Examples of the dengue serotype-1 sequence with a single glycan in the fusion loop were also made, but it did not express any better than wild type or the species with two glycans. In the case of Zika, attempts to generate variants with two glycosylation sites into the fusion loop (following the method established for dengue) were not successful, resulting in less secretion of the recombinant protein into the culture medium than for wild type.
(65) In the case of the Zika E-protein exodomain we therefore explored the generation of variants with a single glycan at various sites in the fusion loop. Substitution of the tryptophan (W101), as for one of the dengue sequons, with an asparagine (the N of the sequon at 101 in place of W), resulted in a level of expression of the construct that was less than for wild type. Likewise, insertion of a glycan at F108 (i.e. the N of the sequon at 108, in place of F), resulted in a level of expression of the construct that was less than for wild type. We concluded that the Zika fusion loop was less tolerant to glycan insertion, and sought a more conservative way to allow it.
(66) Having established, in the case of Zika, that neither the W101 nor the following F of the fusion loop could be replaced with the N of an N-linked glycosylation sequon, an alternative strategy was developed, which was not modeled on the approach taken for dengue. We sought to place a single glycan as near as possible to the end of the fusion loop (based on the 3D structure PDB 5IRE). Rather than go through the process of systematically making and testing the hundreds of possible variants that might allow glycan insertion (which would have been arduous by gene synthesis or by library technologies), we contrived a hypothetical solution and tested it. We contrived to straddle the W at the apex of the fusion loop with an N-linked glycosylation sequon. However, we reasoned that may have been infeasible by insertion of the classical NXS/T sequon, because W is not tolerated at the X position of a sequon. However, although W is not tolerated in the ‘X’ position in the centre of a sequon, H (histidine, a relatively conserved replacement for W, having a hydrophobic-aromatic/cationic dual character) can be tolerated in the X-position. We therefore substituted the 100 position with an N, used a H in place of the W for the X-position, and used a T (which we find works better with H than S), to make a single sequon that read ‘NHT’ (i.e. residues 100, 101, 102, using the E-protein numbering convention rather than the polyprotein numbering convention). The resulting protein, made from plasmid pCRO28, was found to express as well as wild type, and gave greater yield on purification than wild type, indicating no impediment to expression. The other variants of Zika that we explored gave rise to low level or no secreted protein in the expression systems used.
Example 3 (FIG. 3) Characterisation of Glycans Present on the Glycoengineered Dengue Serotype-2 and Zika Proteins
(67) Glycan compositional analysis (GlycoThera, Germany) was performed on two of the selected proteins from Example 2, the dengue-2 serotype product of pCRO22 (representative of the selected dengue constructs that were all designed to carry two glycans in the fusion loop) and that of Zika (the product of pCRO28, designed to carry one glycan in the fusion loop) obtained from transfections of HEK 293.
(68) The results of SDS-PAGE analysis of dengue and Zika samples prior to and after digestion with polypeptide N-glycosidase F (PNGase, Prozyme Inc.) are shown in
(69) Glycans were released from the hyperglycosylated protein products and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and normal-phase HPLC with fluorescence detection of 2-AB-labelled N-glycans, along with specific exoglycosidase treatment (
(70) TABLE-US-00003 TABLE 2 Sample DENV2_ENV_2xGlyco Zika_ENV_recombinant recombinant Antigen; Antigen; Lot #20161026 Lot #20161213 Structure mol (%) mol (%) neutral 16.9 17.0 monosialylated 30.7 36.9 disialylated 26.6 32.0 trisialylated 15.0 8.4 tetrasialylated 9.5 5.1 pentasialylated/ 1.3 0.6 sulphated sum 100.0 100.0
(71) Quantitative HPAEC-PAD analysis of native oligosaccharides was performed on an ICS 5000+ ion chromatography system of the Thermo Fisher Scientific Inc. (Waltham, Mass., USA; GlycoThera device-ID: HPAEC-7) using high resolution CarboPac PA200 columns. Injection of appropriate oligosaccharide reference standards was included in the analytical sequence. N-glycans were detected via electrochemical detection. The data were collected and the chromatograms were acquired by using Chromeleon Chromatography Management System Version 6.8. Native N-glycans were analyzed via HPAEC-PAD revealing mainly neutral, monosialylated, disialylated and trisialylated oligosaccharides in both preparations according to GlycoThera's reference oligosaccharide standards. (
(72) Desialylated N-glycans were analyzed via NP-HPLC after 2-AB labelling revealing predominantly complex-type N-glycans with significant permutational diversity, having proximal a 1,6-linked fucose in both samples (CV94=dengue-2, and CV95=Zika) according to GlycoThera's reference oligosaccharide standards. HPAEC-PAD mapping of native N-glycans released from dengue and Zika preparations CV94 (dengue 2 pCRO22 protein) and CV95 (pCRO28 protein) Zika (as shown in Table 2) revealed the presence of predominantly neutral (16.9% and 17.0%, respectively), monosialylated (30.7% and 36.9%, respectively), disialylated (26.6% and 32.0%, respectively) and trisialylated (15.0% and 8.4%, respectively) oligosaccharides in both samples. Significant amounts of tetrasialylated N-glycans (9.5% and 5.1%, respectively) as well as low proportions of pentasialylated/sulphated oligosaccharides (1.3% and 0.6%, respectively) were found in dengue and Zika samples CV94 and CV95; phosphorylated N-glycan structures such as oligomannosidic Man5-6GlcNAc2 glycan chains with one phosphate residue were not detected in either of the samples analyzed.
(73) TABLE-US-00004 TABLE 3 N-glycan mapping of 2-AB labelled desialylated N-glycans, according to standard procedures at GlycoThera, from Dengue and Zika preparations CV94 and CV95 after sialidase treatment using normal-phase HPLC with fluorescence detection revealed the following compositions for the two proteins. Sample code CV94 CV95 Sample code DENV2_ENV_2xGlyco Zika_ENV_recombinant recombinant Antigen; Antigen; Lot #20161026 Lot #20161213 # N-glycan structure mol (%) mol (%) complex-type N-glycans 61.4 56.6 1 diantennary w/o 2 β-Gal w/o 1 0.1 0.2 GlcNAc with α1,6-Fuc 2 diantennary w/o 2 β-Gal with 0.9 1.2 α1,6-Fuc 3 diantennary w/o 1 β-Gal with 3.1 4.4 α1,6-Fuc 4 diantennary w/o 1 β-Gal w/o 0.4 0.8 α1,6-Fuc 5 diantennary with α1,6-Fuc 8.1 8.8 6 diantennary with α1,6-Fuc with 5.0 6.1 1x α1,3-Fuc 7 triantennary w/o 3 β-Gal with 0.6 0.4 α1,6-Fuc 8 triantennary w/o 2 β-Gal with 1.6 2.9 α1,6-Fuc 9 triantennary w/o 1 β-Gal with 3.9 7.5 α1,6-Fuc 10 triantennary with α1,6-Fuc 8.8 7.3 11 tetraantennary w/o 4 β-Gal with 1.0 1.9 α1,6-Fuc 12 tetraantennary w/o 3 β-Gal with 1.4 2.7 α1,6-Fuc 13 tetraantennary w/o 2 β-Gal with 3.8 6.0 α1,6-Fuc 14 tetraantennary w/o 1 β-Gal with 4.9 3.3 α1,6-Fuc 15 tetraantennary with α1,6-Fuc 15.8 2.6 16 tetraantennary with one 2.0 0.5 LacNAc repeat with α1,6-Fuc oligomannosidic N-glycans 0.1 0.8 17 Man5GlcNAc2 0.1 0.8 hybrid-type N-glycans n.d.* n.d.* not identified 38.5 42.6 X1 — 0.1 0.1 X2 — 0.4 1.5 X3 — 1.0 2.3 X4 — 3.9 8.8 X5 — 4.0 8.2 X6 — 2.5 6.5 X7 — 1.1 1.1 X8 — 2.4 3.7 X9 — 7.4 4.4 X10 — 12.9 5.0 X11 — 2.8 1.0 sum 100.0 100.0 *n.d. = not detected.
Site Occupancy Analysis of the Glycans:
(74) Site occupancy was determined by LC-MS measurement of tryptic peptides. The analysis was based on the LC-MS measurement of tryptic or Endo Lys-C generated peptides liberated from proteins de-N-glycosylated enzymatically by PNGase F. Since PNGaseF is a glycoamidase, the asparagine (N) becomes converted to an aspartic acid residue (D). Quantification was done by creation of extracted ion chromatograms (EICs). The EICs were generated using the theoretical m/z values of differently charged target peptides within a mass window of +/−m/z of 0.01. In order to compare the peptide intensity with the specifically modified counterpart generated by de-N-glycosylation, the area of the peak of the EIC was used. The ratio/extent of modification was then calculated as follows: extent of modification=[area under EIC of modified peptide]/([area under EIC of modified peptide]+[area under EIC of unmodified peptide]).
(75) Sequence numbering is by protein rather than the polyprotein sequence numbering convention, with W101 (at the very tip of the fusion loop) as a useful reference point. Sites are numbered according to their appearance in the linear sequence starting at the N-terminus, such that in dengue (pCRO22, GlycoThera sample number CV94) there were two additional sequons comprising sites 2 and 3. The Occupancy of the natural WT N-glycosylation sites was confirmed to be 100% and 99% for site 1 and site 4, respectively. The added N-glycosylation sites 2 and 3 (in the fusion loop) are located on one tryptic peptide (T15) and the occupancy was 38% (both sites) and additional 51% where only one of the two sites were N-glycosylated. In all 89% of the fusion loops had at least one glycan.
(76) In the case of Zika, the occupancy of the N-glycosylation sites was confirmed to be 99.5% and 100% for the added ‘site1’ (residue 100, fusion loop) and site 2 (residue 154 the glycan naturally present), respectively. Site occupancy of the programmed glycosylation sequons was deduced from PNGase digestion and its effects on the mass of tryptic peptide fragments (whereby the amide NH.sub.2 group of the asparagine side chain is lost and converted to a hydroxyl group). (In the following sequences programmed sequons are in bold). In the hyperglycosylated dengue 2 exodomain the relevant tryptic peptide was T15, i.e., the 15th tryptic peptide (GN.sub.101GSGCGLN.sub.108GSGGIVTCAMFTCK.sub.122 (SEQ ID NO: 35)—containing the substituted N residues at 101 and 108. In the hyperglycosylated Zika exodomain (with a single introduced glycosylation sequon ‘NHT’) the relevant peptide was T10 (N.sub.100HTNGCGLFGK.sub.110 (SEQ ID NO: 36)).
(77) These findings of efficient introduction of large and complex glycans into the fusion loop of dengue and Zika exodomain proteins strengthened our expectation that these proteins would neither bind to the fusion loop, nor elicit fusion-loop antibodies, giving confidence that B-cells or antibodies capable of recognising the wild type versions of the fusion loop would not engage with the glycosylated forms of the invention. This scenario is markedly different from mere introduction of mutations into the fusion loop, because by imposing one or more large additional glycan structures into the fusion loop, the resulting variant fusion loop cannot bind antibodies or B-cell receptors or generate fusion loop antibodies reactive with the wild type versions of the fusion loop. This was fully confirmed in later examples. This strategy may also be contrasted to deleting domains I and II from the structure of the protein, as these domains also contribute neutralising epitopes and T-cell epitopes useful for anamnestic immune responses upon encounter with flaviviruses in the wild, while pre-conditioning the immune system in such a way as to avoid the dangerous dominance of the fusion loop in immune responses to natural virus infections or to other vaccines.
(78) TABLE-US-00005 TABLE 4 list of m/z values used for creating Extracted-Ion-Chromatograms (EIC) for N-glycosylation-site occupancy for dengue-2 Amino Theor. m/z values Acid mass used for ID Range Amino acid sequence in Da EIC [M + n H].sup.n+ Site 1 T10 [65-73] L65TN67TTTESR73 (SEQ ID NO: 37) 1022.511 1022.511; T10 [65-73] L65TD67TTTESR73(SEQ ID NO: 38) 1023.495 1023.495; Site 2 + 3 T15 [100-122] G100N101GSGCGLN108GSGGIVTCAMFTCK122 2304.983 1152.995; (SEQ ID NO: 39) 768.999 T15 [100-122] G100D101GSGCGLN108GSGGIVTCAMFTCK122 2305.967 1153.487; 1x (SEQ ID NO: 40) OR 769.327 de-N G100N101GSGCGLD108GSGGIVTCAMFTCK122 (SEQ ID NO: 41) T15 [100-122] G100D101GSGOGLD108GSGGIVTCAMPTCK122 2306.951 1153.979; 2x (SEQ ID NO: 42) 769.655 de-N Site 4 T18 [129-157] V129VQPENLEYTIVITPHSGEEHAVGN153DTGK157 3133.544 1567.276; (SEQ ID NO: 43) 1045.186; 784.142; 627.515 T18 [129-157] V129VQPENLEYTIVITPHSGEEHAVGD153DTGK157 3134.528 1567.768; de-N (SEQ ID NO: 44) 1045.514; 784.388; 627.712
(79) TABLE-US-00006 TABLE 5 list of m/z values used for creating Extracted-Ion-Chromatograms (EIC) for N-glycosylation-site occupancy for Zika Amino Theor. m/z values Acid mass used for ID Range Amino acid sequence in Da EIC [M + n H].sup.n+ Site 1 L4 [94-110] R94TLVDR99N100HTNGCGLFGK110 1944.982 1944.98 (SEQ ID NO: 45) 972.99 648.92; 5; 99; L4 [94-110] R94TLVDR99D100HTNGCGLFGK110 1945.966 1945.96 (SEQ ID NO: 46) 973.48 649.36; 7; 27; Site 2 T16 [32-164]] I.sub.139MLSVHGSQHSGMIVN.sub.154DTGHETDENR.sub.164 2864.305 1432.65 (SEQ ID NO: 47) 955.44 716.86; 0; 32; T16 [139-164]] I.sub.139MLSVHGSQHSGMIVD.sub.154DTGHETDENR.sub.164 2865.289 1433.14 de-N (SEQ ID NO: 48) 955.76 717.08; 8; 78;
(80) TABLE-US-00007 TABLE 6 site occupancy (% occupation) for dengue-2 (sites 2 and 3 are in the fusion loop) Rate of N-glycosylation site occupancy [%] N-glycosylation site [peptide] Site 1 Site 2 + 3 Site 2 or 3 Site 4 N.sub.67 N.sub.101; N.sub.108 N.sub.101 or N.sub.108 N.sub.153 Sample GT-code [T10] [T15] [T15] [T15] DENV2_ENV CV94 100 38 51 99
(collectively, 89% of molecules have a glycan or two in the fusion loop. N101 replaced W101 of the WT sequence; N108 replaced F108 of the wild type sequence)
(81) TABLE-US-00008 TABLE 7 site occupancy (% occupation) for Zika (site 1 is in the fusion loop) Rate of N-glycosylation site occupancy [%] N-glycosylation site [peptide] Site 1 Site 2 N.sub.100 N.sub.154 Sample GT-code [L4] [T16] Zika_ENV CV95 99.5 100
(99.5% of molecules have a single glycan in the fusion loop; N100 replaced G100 of the WT sequence)
Example 4 (FIG. 4) Immunogenicity of Select Glycoengineered Dengue Proteins 1, 2, 3 and 4 and Zika in Direct ELISA
(82) Female Balb-c mice were immunized with PBS (negative control) and various dengue and Zika formulations of the hyperglycosylated exodomain proteins on Alhydrogel, alone (Zika mono) and in combination (Penta-) and as naked DNA (DNA). Alhydrogel formulations of proteins were injected subcutaneously (s.c.) in a total volume of 200 ul and naked DNA (comprising plasmids pCRO21, pCRO22, pCRO23 and pCRO24 of dengue plus pCRO28 representing Zika) was injected intramuscularly (i.m.) in a total volume of 50 ul for pentavalent DNA (representing 5 micrograms of each plasmid immunogen). Pentavalent protein combinations contained 5 ug amounts per dose of each hyperglycosylated exodomain, and monovalent (Zika) contained 10 ug per dose. Mice were dosed three times, once at each of day 0, day 14 and day 21. The legend at the bottom right of
(83) The Balb-c Mice were immunized with DNA and protein representations of the glycoengineered exodomains and with the corresponding VLPs (i.e. VLPs representing the wild type sequences) from The Native Antigen Company Ltd, Oxford, UK (with no extra glycans, and exposed fusion loops) as positive control. These VLPs (see Table 8, used as both immunogens and also as test antigens in the ELISA tests of
(84) TABLE-US-00009 TABLE 8 Alhydrogel* adjuvant Group (2% w/v (n = 5) aqueous female Route of alhydrogel Balb-c immu- Injectate suspension) mice Immunogen nization Doe volume (ul) 1 Pentavalent i.m., in 50 ug of each 50 ul None glycoengineered DNA 10 mM Tris- plasmid (250 (‘Penta-DNA’ in figures) HCl pH 7.4 ug total) 2 Pentavalent s.c. 5 ug of each 200 ul 50 glycoengineered proteins protein (25 ug (Penta-Prot) in total) 3 Monovalent Zika s.c. 10 ug of Zika 80 ul 20 glycoengineered protein protein (Zika-mono) 4 Pentavalent wild type s.c. 5 ug of each 200 ul 50 VLP (Penta VLP) VLP (25 ug in total) 5 PBS s.c. 0 200 ul none
(85) There was little antibody response to naked DNA representing the five exodomains—as expected in the absence of delivery assistance from liposomal formulation, gene-gun or electroporation technology. Antibody responses to naked DNA were evident against dengue 1, 2 and 3 native VLPs, and not against Zika and dengue 4 VLPs. However these results served to demonstrate the potential utility of these DNA encoded antigens (all of them) with appropriate delivery systems. The assay is naturally more sensitive to detect immune responses to VLPs, due to the presence of additional epitopes (noted above), such that, as expected, antibody responses to the VLP antigens were uniform and very strong in the VLP-immunised ‘Group 4’. However, so too were responses to the novel glycoengineered exodomain proteins of the present invention, which gave strong, balanced immune responses against all five components (dengue serotypes 1,2,3 and 4 plus Zika) with the pentavalent immunogen formulation. Responses were uniformly high to the exodomain immunogens (pentavalent protein and monovalent Zika) and there were no non-responders. Also, the response to Zika in the monovalent-Zika-hyperglycosylated-exodomain-immunized group (10 μg dose) was modestly higher than that in the pentavalent protein group where the same exodomain was used at half the dose. This finding indicates a favorable lack of competition among the serotypes in the generation of type specific immune responses (this is a known problem with live attenuated flavivirus vaccine approaches, such as Dengvaxia, where immune responses to dengue serotype 2 are problematically low).
(86) For direct ELISA (
(87) Antibody responses were calibrated against fusion loop antibody 4G2 (The Native Antigen Company Ltd, Oxford) with dengue VLP representing serotype 2 on the solid phase at 0.5 micrograms per ml coating concentration. Units of antibody measurement “IgG antibody titre” are micrograms per ml 4G2-equivalent in undiluted serum, determined by interpolation of the standard curve using a four-component polynomial regression fit (AssayFit, IVD Tools). At day 42, antibody responses reached 10.sup.4-10.sup.5 for the hyperglycosylated exodomain immunogens (a notional 10 mg per ml-100 mg per ml in neat serum). These concentrations (taken literally) are unattainably high since the IgG concentration of mouse serum is only 2-5 mg per ml, and probably reflect the higher affinity or avidity of the antibodies generated compared to the antibody, 4G2, used for standardization, or may reflect better epitope exposure (4G2's fusion loop epitope being semi-crytpic in the structure of VLPs and virions). Nevertheless the 4G2 calibration serves a useful purpose allowing the assay to be run from time to time, controlling for such variables as batch to batch variation in the conjugate—(an anti-IgG-Fc horseradish peroxidase conjugate made from polyclonal antibodies which vary by batch). This is more reliable than quoting antibody ‘titres’ based on a threshold absorbance value which are very conjugate-batch and antigen-batch dependent, and may vary further among conjugates sourced by different manufacturers.
(88) A further aspect of these observations is that the antibodies generated are of the IgG class demonstrating class-switching (even at day 14) from IgM, for all of the protein immunogens. This is an essential component of the B-cell memory response, important for the development of vaccines. A further aspect of these findings is that the antibodies generated by exodomain protein immunogens (and to some extent the DNA immunogens) strongly recognize the native form of the VLP antigens, which also lack His tags, ruling out the possibility of false positives due to anti-His-tag responses. This proves that both the dengue and Zika exodomain materials represent native epitopes of the exodomain proteins that are immunogenic in generating antiviral (VLP) antibodies. These results suggest that other nucleic acid encoded forms of the hyperglycosylated exodomain species, e.g., liposomal RNA or lipoplex RNA, would also generate desirable antibody responses against virions (VLPs) and viruses.
(89) There was specificity in the immune response to the Zika monovalent hyperglycosylated exodomain, which generated higher antibody titres against the homologous Zika VLP than to other VLPs, despite the known cross-reactivity of these various viruses with antibodies. This is a favourable result since type-specific anti-Zika antibodies are known to have better neutralizing activity generally than dengue-cross-reactive ones. Also, as seen in the antibody-responses to the Zika-monovalent hyperglycosylated exodomain at the later time points (after two or three doses), there was a degree of cross-reactivity against dengue strains that developed over time, raising the potential for generation of beneficial cross-reactive neutralizing responses, excluding the fusion loop epitope (which was not recognized by antibodies generated by hyperglycosylated exodomain species as demonstrated in the data that follows in later examples).
Example 5 (FIG. 5) Avoidance of Recognition of the Glycoengineered Proteins by Fusion Loop Antibodies, and Retention of Neutralizing Epitopes
(90) An ELISA test (of
(91) Unless otherwise specified, conditions were the same as for the ELISA test of Example 4 and
(92) Antigens were as follows: wild type dengue exodomains representing dengue serotypes 2 and 4 were from The Native Antigen Company (DENV2-ENV, DENV4-ENV); ‘HX’ designated exodomains (hyperglycosylated exodomains) were the selected set of Excivion exodomains of the present disclosure (pCRO21-24 for dengue, pCRO28 for Zika). Prospec Zika was a non-glycosylated bacterial exodomain from Prospec of Israel (zkv-007-a), and Aalto Zika was an insect (Sf9 cell) derived Zika exodomain (AZ6312—Lot3909). Mouse monoclonal antibodies against Zika virus exodomain were as follows: Aalto Bioreagents AZ1176-0302156-Lot3889; Z48 and Z67 were neutralizing antibodies described by Zhao et al, Cell 2016 (The Native Antigen Company ZV67 MAB12125 and ZV48 MAB12124). Antibody 4G2 is an anti-dengue-serotype-2 antibody recognizing the fusion loop (The Native Antigen Company AbFLAVENV-4G2).
(93)
(94) The data of
(95) The data of
(96) A further aspect of the data of
Example 6 (FIG. 6) Avoidance of Generation of Fusion-Loop Antibodies by the Glycoengineered Proteins
(97) An ELISA test was established to measure the binding of polyclonal antibodies against the fusion loop (represented in this example by dengue serotype-3 VLP on solid phase ELISA plates).
(98) A competition ELISA was set up using biotinylated 4G2 (Integrated Biotherapeutics) which was detected using streptavidin-horseradish peroxidase conjugate. Dengue serotype 3 VLP (The Native Antigen Company) which reacts with 4G2 slightly better than the immunizing serotype dengue-2 VLP was used as antigen coated at 0.5 ug per ml on the solid phase. Pooled sera (from the groups of
(99) In this assay (
(100)
(101) The data of
Example 7 (FIG. 7) Generation of Neutralising Antibodies by the Glycoengineered Dengue and Zika Proteins
(102) Serum pools from Example 4 were tested for their ability to neutralize dengue serotype 2 and Zika viruses using Vero cells in plaque reduction neutralization tests (PRNT).
(103) In the case of dengue, the dengue serotype 2 strain used to infect the Vero cells (D2Y98P) was a different serotype-2 strain (non-homologous) from the sequence of the immunizing dengue 2 strain of the VLPs and exodomains. In the groups expected (from Example 4) to generate dengue neutralizing antibodies (namely pentavalent protein and pentavalent VLPs, Groups 2 & 4) there was potent neutralization of the ‘off target’ dengue test virus. In the case of Zika there was significant (albeit partial) neutralization as expected from the results of Example 4, in groups shown to contain antibodies that recognized native Zika VLPs (namely pentavalent protein and pentavalent VLPs, Groups 2, 3 & 4). Due to limitations on sample volume, the maximum concentration of serum that was tested was 1/50, such that in interpreting these results this factor needs to be taken into consideration (i.e. that there would be higher neutralizing capability in the blood of the immunized animals).
(104) TABLE-US-00010 TABLE 9 Immunogenicity Study Design Group Vaccine (n = 5) Vaccine* Schedule Dosage Bleeds Readout 1 Pentavalent On days 0, 250 μg total Test bleed Measurement glycoengineered 14, & 21 via DNA (50 μg of for serum on of antibodies DNA IM route each) Days 14 & against ZIKV 2 Pentavalent 25 μg total 21. Terminal & DENV 1-4 glycoengineered protein (5 μg bleed on via ELISA proteins on each) Day 42. Alhydrogel 3 Monovalent Zika 10 μg protein glycoengineered protein on Alhydrogel 4 Pentavalent wild 25 μg total type VLP on VLPs (5 μg Alhydrogel each) 5 PBS —
(105) PRNT Assay was performed as follows. Five mouse serum samples were pooled by taking an equal volume of individual samples in each group (sample description in next slide) and were then tested against ZIKV and DENV, respectively. Twelve two-fold serial dilutions of each serum sample in duplicates starting at 1:50 were prepared for the two-hour inoculation with virus. The serum-virus mix was then added to Vero cells seeded in 24-well culture plates and incubated at 37° C. in a humidified 5% CO.sub.2 atmosphere. The Vero cells were fixed on 3 days post incubation (dpi) for ZIKV PRNT and 4 dpi for DENV PRNT. Viral plaque was determined by crystal violet staining.
(106) Potent inhibition of infection by dengue was observed in the group immunized with hyperglycosylated exodomain proteins of the present disclosure (Penta-prot). Zika immunized animals generated antibodies that did not prevent dengue infection of Vero cells, illustrating the type-specific nature of antibodies generated by these novel immunogens. These Zika antibodies (from the Zika monovalent group and from the pentavalent proteins group) were significantly protective of infection of Vero cells by Zika virus. As expected, PBS-sham-immunised animals did not give rise to protective antibodies, nor did pentavalent DNA administered intramuscularly. This latter result may have been due to the low concentrations of antibodies generated by naked DNA, as expected from intramuscular injection (as distinct from gene-gun or electroporation strategies, or strategies incorporating encoded proteins as molecular adjuvants).
(107) The results of Example 6 (generation of neutralizing antibodies) combined with those of Example 5 (lack of recognition by or generation of fusion loop antibodies) by the hyperglycosylated Exodomain proteins of the invention strongly suggest that these proteins can form the basis of a protective vaccine for dengue or Zika viruses (or, in combination, for both viruses) without the generation of fusion loop antibodies, which are particularly implicated in antibody-dependent enhancement of infection.
Example 8 (FIG. 8) Reaction of Convalescent Dengue or Zika Serum with Immobilized Zika and Dengue Wild-Type (WT) and Hyperglycosylated (HX) Exodomain Proteins
(108) The ELISA reactivity of antibodies in a dengue convalescent serum with immobilized Zika and dengue wild-type (WT) and hyperglycosylated (HX) exodomain proteins oriented on the solid phase by capture with a rabbit anti-His-tag monoclonal antibody (
(109) The ELISA reactivity of antibodies in a Zika convalescent serum with immobilized Zika and Dengue wild-type (WT) and hyperglycosylated (HX) exodomain proteins (
(110) The results show that:
(111) 1) the HX Zika antigen of the invention is not susceptible to the off-target recognition of WT Zika exodomain by the convalescent dengue serum.
(112) 2) The off-target recognition of WT Zika exodomain (Aalto) by dengue serum is a fusion-loop directed phenomenon because it is abolished by 4G2 (anti-fusion loop monoclonal antibody) in solution phase at a concentration that causes 80% inhibition against VLPs (10 micrograms per ml). (The antigen on the solid phase in this instance is exodomain rather than VLP).
(113) 3) The ‘Zika’ convalescent serum does not recognize any of three Zika exodomains, but it strongly recognizes WT dengue 2 and WT dengue 4. In the Example 6 the HX Zika antigen of the invention and Aalto's Zika exodomains exhibit reaction with conformation-dependent anti-Zika neutralising antibodies). This demonstrates that this particular Zika serum (positive for Zika plaque neutralisation and Zika NS1 antibodies) is from a subject also exposed to another flavivirus. Because the Zika convalescent serum (unlike the dengue convalescent serum) does not recognize the fusion-loop-cloaked exodomains, it can be concluded that this other flavivirus is not dengue.
(114) 4) The off-target recognition of WT dengue-2 and dengue-4 exodomains by the human Zika convalescent serum is not seen with the HX-cloaked dengue exodomains of the invention. This suggests that it is fusion loop directed and would show false positive in other flavivirus diagnostic tests that do not use glycan-cloaked proteins in accordance with the invention.
(115) 5) The off-target recognition of WT dengue-2 and dengue-4 exodomains by the human Zika convalescent serum is blocked completely by 4G2 showing that it is a fusion loop directed phenomenon.
(116) 6) The dengue convalescent serum recognizes WT 2 & 4 indiscriminately, but clearly prefers the d2 exodomain out of the set of 4. This demonstrates that the fusion loop antigens of the invention have superior selectivity (compared to their wild type equivalent forms) to discriminate between dengue serotypes, due to the glycan cloaking of the fusion loop.
Examples of Diagnostic Use
(117) Various formats of lateral flow test for the detection of antibodies are possible. A widely-used design for the detection of antibodies against viral antigens is to place a line (or spot) of antigen ‘directly’ on a strip of nitrocellulose, and then allow blood/serum/plasma or oral fluid to seep up the strip by capillary action, hydrating a dried nanoparticulate gold reagent, wherein the particles are coated with an antibody (e.g. anti-IgG, anti-IgM), typically followed by a wash buffer. As the reagents proceed along the nitrocellulose strip, specific antibodies are arrested by the antigen, and some of the gold particles are likewise arrested by binding to specific antibodies immobilised on the antigen, excess particles proceeding beyond the observation window—resulting in a brown or pink line in the observation window of the test cassette indicating the presence of specific antibody. In some embodiments of this format, a two-port system is used, one port for the sample application (proximal to the antigen line), and a second port, more distal, for the application of the diluent. Additionally, a control line (or spot) may be used to verify the fluidic performance the test and the functionality of the gold conjugate. This may be formed of a line of purified antibody or of human serum containing the antibody type appropriate to the anti-Ig conjugated nanoparticulate gold reagent. In preliminary studies with this format, we were successful in demonstrating the diagnostic utility of wild type, and the superior specificity of HX versions of the four dengue envelope proteins and Zika. In addition, aware of the phenomenon of surface denaturation of proteins (Sen, Yamaguchi, & Tahara, 2008) which is more common in our experience with nitrocellulose (viz. lateral flow) as a solid phase compared to polystyrene (viz. ELISA) we were influenced in the current lateral flow design by our earlier ELISA studies (see above), to employ indirect immobilisation of the HX envelope proteins. Indirect immobilisation had proven more sensitive in ELISA studies than direct immobilisation of the antigen on the solid phase. To this end we explored the use of anti-His-tag antibodies recognizing the C-terminal hexahistidine tag on the HX proteins described herein as an indirect means to attach the antigens to the solid phase (Example 9).
Example 9: Alternative Formats for Lateral Flow Detection of Antiviral Antibodies
(118) Test Composition (Common to Both Formats A and B of this Example 9)
(119) Absorbent pad—Whatman CF5 (22 mm)
(120) Nitrocellulose—MDI 15μ SS12 (25 mm)
(121) Sample pad—Ahlstrom 8964 (20 mm)—Sample pad can be treated with a red blood cell arresting agent. This can be anti-glycophorin A (AGA) at a suitable concentration (suitable concentrations are known in the art). Pad is soaked with anti-glycophorin A and dried for 24 hrs at 25° C. in low humidity.
(122) Conjugate pad—Ahlstrom 8815 (Format (A) 20 mm, Format (B) 45 mm)—Conjugate pad is soaked with 0.1M Tris-HCl pH8.0 containing 0.25% Tween 20 and 0.5% bovine serum albumin (BSA) and dried overnight at 37° C. followed by 24 hrs at 25° C. in low humidity
(123) Cassette—Kanani Biologicals, India
(124) Gold conjugate (detector) 40 nm colloidal gold (BBI Solutions of Cardiff, Wales, UK) conjugated to anti-human-IgG at 100 units per test on the conjugate pad, dried for 24 hrs at 25° C. in low humidity. Advantageously the conjugate is made from affinity-purified IgG (e.g., from goat or rabbit) or a suitable monoclonal antibody such as a pan-subclass-reactive IgG antibody or a monoclonal anti-IgM antibody. Such antibodies are known in the art and are available from Sigma, AbCAM or Merck-Millipore. Gold conjugates (where applicable) were applied in 2 mM tetrasodium borate, 10% sucrose, 0.95% sodium azide pH 9.0.
(125) Running Buffer was prepared from Oxoid PBS pH 7.3 tablets using deionised water (NaCl 0.137M, KCl 0.003M, disodium hydrogen phosphate 0.008M, potassium dihydrogen phosphate 0.0015M), containing additionally 0.5% Tween-20 and (where indicated) 0.5% PEG 20K (average Mn 20,000) CAS Number 25322-68-3.
(126) Antigens
(127) The antigens used were as follows: (in ‘direct’ formats, antigen was applied directly to the nitrocellulose strip in Oxoid-PBS without additives and dried as above; in ‘indirect’ formats, where antigen was applied to the sample application pad, antigens were applied in Oxoid-PBS without additives and dried as above).
(128) Dengue HX antigen D1: Hyperglycosylated exodomain D1 (from pCRO21) (SEQ ID NO: 24)
(129) Dengue HX antigen D2: Hyperglycosylated exodomain D2 (from pCRO22) (SEQ ID NO: 25)
(130) Dengue HX antigen D3: Hyperglycosylated exodomain D3 (from pCRO23) (SEQ ID NO: 26)
(131) Dengue HX antigen D4 Hyperglycosylated exodomain D4 (from pCRO24) (SEQ ID NO: 27)
(132) Zika HX antigen: Hyperglycosylated exodomain Zika (from pCRO28) (SEQ ID NO: 28)
(133) The alignment of the E-protein fusion loop amino acids 98-110 of a group of wild-type sequences of flaviviruses and recombinant analogue sequences of the invention is shown in Table 1, for each of the Dengue HX antigens D1-D4 the hyperglycosylated exodomain fusion loop sequence was DRGNGSGCGLNGS (SEQ ID NO: 2) for the Zika HX antigen the hyperglycosylated exodomain fusion loop sequence was RNHTNGCGLFGK (SEQ ID NO: 5) Dengue HX antigens D1, D2, D3 and D4 at 100 ng each antigen per test on the sample pad, dried for 24 hrs at 25° C. in low humidity.
(134) Zika HX antigen at 50 ng per test on the sample pad, dried for 24 hrs at 25° C. in low humidity.
(135) Nitrocellulose
(136) Test Line—Mouse mab anti-6×His at 0.3 mg/mL (RT0266 Bioxcell)
(137) Control Line—Human IgG at 0.75 mg/mL (Sigma-Aldrich-Merck 18640—IgG from human serum)
(138) A: Single Port Format for Lateral Flow Detection of Antiviral Antibodies
(139) As shown in
(140) B: Two-Port Format for the Lateral-Flow Detection of Antiviral Antibodies
(141) As shown in
Example 10: Indirect Immobilisation of Mobile Dengue HX Exodomain Antigens Via Anti-His-Tag Monoclonal Antibody Allows Detection of Neutralising Antibodies in Lateral Flow
(142) A: Using the test format of Example 9A (two ports used) 15 ul of normal human plasma (spiked with a two-fold serial dilution of human dengue monoclonal neutralising antibodies DV78 and DV18 (Absolute Antibody Ltd., Oxford UK) in equal amounts, from 1 to 0.0078 ug) was added to the square sample pad port, and allowed to incubate at room temperature for 3 minutes. Next, 45 ul of running buffer was added to the sample port, and the device was run for a further 17 minutes. 90 ul of running buffer was then added to the round port to release the gold anti-human-IgG conjugate. The test was read after a total of run time of 40 minutes. This demonstration illustrates a sensitive capability of this test format to detect neutralising anti-dengue antibodies (DV78 and DV18 recognise epitopes in the DI and DII of dengue virus), 7.8 ng of specific antibody was detectable. In this particular example HX versions of dengue-2 and dengue-4 exodomains were used.
(143) B: Results obtained in this format with control serum NM (not exposed to flavivirus infection or vaccination) and a dengue positive serum ‘2965’ are shown in
(144) Example 10 demonstrates the utility of the HX dengue exodomains 2 and 4, in serodiagnosis of dengue in lateral flow format. It also demonstrates the advantage and utility of capturing the antigen (initially mobile) using an anti-tag antibody. A monoclonal anti-His-tag antibody was used, although there are other tag-antibody pairs that could be used (such as FLAGtag and StrepTag-II). (Such additional pairs can be used to multiplex the assay, whereby a second line of anti-tag would recognize a second tag on a second antigen involved in the test, a third a third etc.). By using a tag antibody for capture of the mobile antigen (and antigen-antibody-complexes and antigen-antibody-colloidal gold conjugate materials), a prozone effect is avoided. This contrasts to an alternative scenario where one might have used an antigen-specific (e.g. murine) monoclonal antibody for the purpose of arrest at the test line or spot. In this latter alternative scenario, the test is defeated by the presence (if present) of an antibody in the test sample against the same or overlapping epitope as is recognized by the antigen-specific capture antibody. (Such adverse competition for antigen binding can be mitigated by the use of multiple monoclonal or a polyclonal affinity purified antigen specific antibody, but cannot be eliminated by this means). In the Example 10 and elsewhere in other examples we have detected IgG antibodies by virtue of a colloidal gold conjugate with surface bound anti-human-IgG. However, it should be apparent that other antibody classes and subclasses can be detected in this way (e.g. IgM, IgA and subclasses of IgG) by use of appropriately specific antibodies on the colloidal gold reagent, and likewise by use of corresponding pure antibodies comprising the control line (i.e. IgM for an IgM test, IgA for an IgA test, IgG for an IgG subclass-1 for an IgG subclass-1 test etc.). For tests designed to measure IgG, when using a monoclonal or affinity-purified anti-IgG colloidal gold conjugate, it is important to choose one that has a balanced reactivity across the IgG subclasses, in order to give a representative signal in the lateral flow tests. Such antibodies are known in the art, and are commercially available.
(145) The antigens used in serodiagnostic tests are sometimes more expensive than antibodies when antibodies are used in such tests. This is particularly important in lateral flow tests targeting mass markets. (This is a feature of improvements in recent years in the facile production of monoclonal antibodies in particular, e.g. by transient expression in HEK cells, where the antibodies can be produced at high yield, e.g. several grams per litre of culture). Therefore, an additional advantage of exploiting anti-tag antibodies for indirect antigen-capture in lateral flow tests is that the test is made more economic with respect to antigen costs in manufacturing. The economic advantage of this approach is further enhanced by the stronger recognition of indirectly-immobilised antigen (via anti-tag antibody), compared to that deposited directly on the lateral flow strip (e.g. a nitrocellulose strip).
(146) A further feature of Example 10 is demonstration that the HX class of flavivirus exodomain antigens (having glycans in the fusion loop) have intact neutralising epitopes throughout the molecule (except, as noted above, for the fusion loop—in which the epitope is glycan-cloaked). Thus as described above it was demonstrated for Zika HX exodomain that immobilisation via its His-tag allowed functional display of two neutralising epitopes, namely those recognised by murine monoclonal antibodies ZV48 and ZV67 (human antibodies from Absolute Antibody Ltd., Oxford). Example 10 demonstrates that HX versions of dengue-2 and dengue-4 exodomains functionally display epitopes recognized by DV78 and DV18, which are located in domains [I and II]. These data and arguments strongly support the use of the HX antigens in serodiagnostic tests, in particular point-of-care serodiagnostic tests, for the detection and measurement of neutralising antibodies. Antibodies against the fusion loop, which dominate the antibody response, are poorly neutralising and rapidly decline to concentrations that enhance infection. The point-of-care diagnostics described here for dengue and Zika can therefore be used to measure vaccine performance, e.g. to determine whether an additional dose may be needed, or how well a vaccine is performing in a subject already exposed to another flavivirus by reason of infection or vaccination (e.g. with yellow fever or dengue vaccines). They may also be combined (dengue and Zika, by using different tag/anti-tag pairs, e.g. His-tagged dengue antigen being captured by an anti-His-tag line, and FLAG-tagged Zika antigen being captured by an anti-FLAG antibody).
Example 11: Direct Spotting/Immobilisation of Antigen on Nitrocellulose (as Distinct from Capture of Mobile Phase Antigen Via Anti-Tag)
(147)
(148) Results: 1&2 verify the performance of the conjugate in expected rank of intensity; 3 showed a very faint signal (not visible in the photograph), likewise 4; 5&6, controls for non-specific binding of reactants, were negative; 7&8 (negative and positive test sera) gave the desired result, i.e. negative in 7, positive in 8. These findings demonstrate (in the case of directly applied immobile antigen) the value of an intervening wash, because signal was weak (not evident on photograph) when no wash was used. Moreover, relatively large amounts of antigen were required, even of the strongly reactive fusion-loop-intact forms, in this test configuration. (Where reagents or solutions are not defined in this Example, they are the same as in Example 9).
(149) Example 11 demonstrates the utility of direct immobilisation by spotting of exodomain antigens on nitrocellulose, as distinct from having them in the mobile phase of the test (which is the format in all other Examples in this application). Direct spotting/immobilisation of antigen on nitrocellulose (as distinct from capture of mobile phase antigen via anti-tag) was effective, but less sensitive than indirect immobilisation of mobile antigen via anti-tag antibody.
(150) In Example 11 wild type antigens are used (dengue-2 and dengue-4) but it is clear that HX versions of flavivirus antigens could be used in this format. In the format of Example 11 it would be possible to improve sensitivity by use of luminescent detection, as described (Laing, 1986), or using fluorophore-labelled anti-human-Ig, with a fluorophore such as a quantum dot or a lanthanide which have high quantum efficiency and low photobleaching, as well as a favourable Stokes shift (particularly in the case of lanthanides such as Europium chelates). Europium chelates are also amenable to time resolved fluorescence which is more sensitive because the fluorescence measurement is made once the light source is turned off, and does not contribute noise to the signal. Clearly these sensitive luminescent and fluorescent methods could be applied to other Examples given in this patent application, if desirable or necessary.
Example 12: Avoidance of Off-Target Recognition of Dengue Envelope Antigens by Zika Macaque Convalescent Serum IgG Antibodies by Using Dengue-HX Antigens
(151) As shown in
(152) These results show a strong off-target recognition of wild type dengue antigens by the Zika convalescent serum which is not exhibited by the tests conducted with the HX versions of the same antigens, which nevertheless correctly identify the dengue positive human serum. The macaque ‘G16’ was bred in captivity and cannot have been exposed to dengue. The strong ‘quasi specific’ recognition of the WT dengue antigens by the macaque serum is a true antigen-specific reaction, explained by reaction of anti-Zika antibodies in the macaque serum with the dengue fusion loop, which is effectively cloaked (masked) by glycosylation in the case of the HX antigens. Results by test are 1 negative; 2 negative; 3 positive; 4 weakly positive (much less than false positive in 3); 5, positive; 6 positive; 7 weak positive; 8 negative. 100 ng of each antigen (200 ng total) was used in each test. ‘8’, using HX versions of the dengue antigens, was negative as expected (desirable result) whereas ‘7’ gave rise to a weak non-specific binding reaction of this non-flavivirus exposed subject (VM) to the wild-type version of the antigen (undesirable result).
(153) Example 12 demonstrates the superior performance of dengue-HX antigens (combined dengue-2 and dengue-4 HX antigens) compared to their wild type equivalents, the wild type versions showing strong off-target recognition by anti-Zika antibodies. In this example a captive-bred macaque was used. This obviates the uncertainty, in the case of human sera, that a person may also have been exposed to dengue as well as Zika, because there is high co-endemicity of Zika and dengue such that the majority of Zika-exposed human subjects will also have been exposed to dengue.
Example 13: Equivalent Performance of Mammalian and Insect-Expressed (Tni Cell) Dengue 1, 2, 3, 4 HX Antigens, and Avoidance of False Positivity with Yellow Fever Vaccinated Subjects
(154) HX versions of the four dengue proteins were made in either human embryonic kidney cells or in insect (Tni) cells using a secretory-type baculovirus vector expression system (Flash-Bac Ultra) according to manufacturer's instructions.
Example 14: Utility and Specificity of HX Zika and Dengue Antigens in Lateral Flow Testing Compared to Wild Type Zika Antigen
(155) In this example the reactivity of HX versions of the complete set of dengue antigens 1, 2, 3 &4 (as a mixture) and of the HX Zika antigen are compared for a dengue positive serum +D=serum 2965, and a Zika positive serum +Z (G16 macacque Zika convalescent serum, and a control human serum positive for neither.
(156)
(157)
(158)
(159) Example 14 demonstrates utility and specificity of HX Zika and dengue antigens in lateral flow testing compared to wild type Zika antigen. In this example insect-cell expressed dengue and Zika antigens were used. The extremely strong reaction seen with the HX version of the insect-expressed Zika antigen can be traded off with respect to specificity, by using less antigen in the test or less anti-His-tag antibody in the test line, in order to render the weak off-target recognition of dengue HX antigens undetectable (i.e. invisible).
Example 15: Performance of the HX Antigens in Lateral Flow Testing of Human Whole Blood
(160) Example 15 demonstrates the performance of various configurations of the test with whole blood, all based on the format of Example 9A (using a single port). For a point-of-care test it is most useful and convenient if it can be conducted using blood without the need for plasma or serum separation. First it was investigated whether it was advantageous to use an anti-glycophorin antibody in the sample pad to absorb-out red blood cells preventing them from running on the nitrocellulose and complicating the reading of the device. It was found that the anti-glycophorin antibody reduced the signal of the test to an unacceptable degree. However, it is noted that it may nevertheless be advantageous to include a lesser amount of such an antibody in the sample pad, in order to absorb out heterophile antibodies (human anti-mouse-IgG antibodies) that occasionally give false positives in tests using mouse monoclonal antibodies. In the format of Example 15, without anti-glycophorin antibody, specific reactivity of dengue positive samples was demonstrated without problems of false positivity due to yellow fever vaccination in five subjects. Also in this format, there was sensitive detection of dengue neutralising antibodies D18 and D78.
(161) A: The test requirement for anti-glycophorin A (AGA) in the dengue test was investigated. Tests were run according to Example 9A, but also including 0.5% PEG20K in the running buffer. Samples were made from serum mixed with an equal volume of centrifuge-sedimented red cell fraction from whole normal blood. 10 ul samples of blood were used. 100 ng of each HX dengue antigen was used and 100 units of anti-human-IgG gold conjugate.
(162) B: Tests were run as in 15A above including testing of serum JF, a yellow-fever vaccine. No AGA was used. There was minimal breakthrough of red blood cells into the test window (
(163) C: Tests were run as above in 15A and 15B, with 5 ul of negative sample which had been spiked with various levels of D18/D78 antibodies (Absolute Antibody Ltd., Oxford) in equal mixture. 100 ul of running buffer was used as chase. All tests used 100 ng of each dengue insect derived HX antigen and 100 units of anti-human-IgG gold conjugate. The results are shown in
(164) D: Blood samples were made up by replacing the plasma of a normal blood sample from which red blood cells were sedimented, with various plasmas (negative, positive and yellow-fever vaccinated) and resuspended. 100 ul of running buffer was used as chase. 100 ng of each of the four insect-derived dengue HX antigens were used, plus 100 units of anti-hu-IgG gold conjugate. “+” is the positive control used in the various previous test examples above. A very favourable profile of positivity was observed—all positive samples gave a positive test line, none of the yellow fever vaccinated subjects or the negative control subject, gave a positive test line (
Example 16: Production of all Seven HX Proteins in Insect-Cells (Tni) and Comparison of Insect-Expressed (Tni) Proteins to Human Expressed (HEK) HX Proteins
(165) HX proteins were initially produced in HEK cells, however it was difficult to achieve efficient expression of some of the proteins. To seek to improve protein expression, HX proteins were produced in Tni insect cells in the ‘Flash-Bac-Ultra’ baculovirus system of Oxford Expression Technologies Ltd. According to the manufacturer's instructions. Protein purification was performed using Strep-TactinXT® Superflow® system (IBA Lifesciences), according to the manufacturer's instructions.
(166) The results of expression of Zika HX-Strep-tag-II from insect (Tni) cells are shown in
(167) In Tni cells, an approximately 10-fold improvement in productivity (relative to expression in HEK or CHO) was achieved for the ‘difficult-to produce’ dengue-1-HX and Zika-HX antigens, which now expressed at levels approaching those of the most abundantly-expressed antigens such as dengue-2-HX. High levels of expression have been achieved subsequently in CHO cells, by optimising signal sequence and by cell-line development.
(168) As it was possible that the HEK-expressed antigens might differ from the baculovirus-expressed forms (notably these cell lines produce different glycan structures), bridging studies were performed with the lateral flow tests to determine if these differences influenced the performance of the test with human sera, or whether there were differences in reactivity in ELISA format with panels of monoclonal antibodies.
(169) The antigenic integrity of insect (Tni) expressed HX forms of the dengue and Zika proteins (having one or more glycans in the fusion loop) was probed with a panel of murine anti-Zika monoclonal antibodies (some of which had known specificity and properties), and with 4G2—a flavivirus cross-reactive antibody which was raised against dengue-2 wild type and which recognizes the conserved fusion loop (Table A). This was done in order to ensure that the data generated earlier on the HEK-expressed proteins would translate effectively when the insect (Tni) expressed antigens were used in the LF tests, in place of the HEK-expressed proteins. These studies confirmed that the baculovirus-produced antigens were equivalent to the HEK-expressed forms in their recognition by well-characterised murine monoclonal antibodies. Also, glycan analyses were conducted on the baculovirus-expressed proteins which confirmed that the fusion-loop planting efficiency of the glycans was the same for the baculovirus-expressed as was the case for the HEK-expressed proteins, although the glycans were, as expected, different between insect and HEK expression.
(170) TABLE-US-00011 TABLE 10 Recognition (absorbance values) of baculovirus-expressed and HEK- expressed HX and wt dengue and Zika antigens by murine MCABs Ab type enhancing anti-D2 Neutralising Neutralising Enhancing neut. &/enh. Unknown Fusion-loop Aba ab name Aalto a Z ZV48 ZV67 ZKA78 ZKA64 ab 4G2 insect D1HX 0.0 0.1 0.0 0.2 0.2 0.1 insect D2HX 0.0 0.1 0.0 0.2 0.1 0.1 insect D3HX 0.0 0.1 0.0 0.2 0.3 0.1 insect D4HX 0.0 0.1 0.0 0.1 0.1 0.1 insect ZHX 1.1 1.5 0.0 0.7 2.0 0.1 insect Z-wt 1.1 1.2 1.4 0.8 1.0 0.9 HEK D1HX 0.0 0.1 0.0 0.1 0.1 0.1 HEK D2HX 0.0 0.0 0.0 0.1 0.1 0.1 HEK D3HX 0.0 0.0 0.0 0.1 0.1 0.1 HEK D4HX 0.0 0.0 0.0 0.1 0.1 0.1 HEK ZHX 1.2 1.1 0.0 0.9 3.1 0.1 Antigenic ‘audit’ of HEK vs Tni-expressed HX proteins, vs Zika-wildtype. Tni-expressed antigen had an equivalent recognition profile to the equivalent HEK-expressed material with murine MCABs, were recognized by neutralizing antibody but not by cross-reactive enhancing antibody and were able to detect neutralising antibody, thus had the capability to be used to assess exposure to flavivirus and inform vaccine use. There was a very similar profile of reactivity of our Zika antigen secreted from insect (Tni) cells in the baculovirus system, compared to the equivalent antigen secreted from HEK cells. Notably, in both cases, the ‘HX’ forms (into which glycosylation sites have been introduced and which have been modified post-translationally by glycosylation) were recognized appropriately by four conformation-sensitive anti-Zika E protein antibodies, but not by dengue-cross-reactive antibodies ZKA78 and 4G2 (which strongly recognize wild-type Zika).
Example 17: Comparison of Excivion Dengue-LF to SD Dengue Ag+Ab Duo (Field Study Done in Pune, India)
(171) Comparison of Excivion dengue-LF prototype test of the invention to the SD BIOLINE Dengue DUO® (SDB DD) NS1 Ag and IgG/IgM ICT (SD Dengue Ag+Ab Duo) test was performed in a field study carried out in Pune, India.
(172) The manufacturer's instructions for the use of SD BIOLINE Dengue DUO® (SDB DD) NS1 Ag and IgG/IgM ICT (Standard Diagnostics, Inc., Korea) were followed, and are described previously (Wang S M, Sekaran S D. Early diagnosis of dengue infection using a commercial Dengue Duo rapid test kit for the detection of NS1, IgM, and IgG. Am J Trop Med Hyg. 2010; 83(3):690-5).
(173) The Excivion dengue-LF prototype test (without preabsorption).
(174) The Excivion dengue-LF test was used to perform 47 tests on the sera tested previously with SD Dengue LF test (
(175) Excivion −Omega dengue-LF test had “increased sensitivity for IgG”. (statistical analysis: Wilcoxon Matched Pairs Test N 47 T 0. Z 5.23162 p-level 1.6803E-7).
Example 18: Test Performance of Early Test Prototypes
(176) The performance of early test prototypes (no preabsorption) was assessed first with panels of sera available from commercial and academic sources. Tests were performed using dengue and Zika serum and plasma panels collected in endemic territories or from returning traveller populations including Thailand, the Dominican Republic, USA and UK-Trinidad, collected before and ‘after’ the advent of Zika as a pandemic disease, although it became apparent that Zika had arrived in Brazil in 2013 (earlier than previously thought). The test was also demonstrated to work with whole blood, its intended use.
(177) Tests were also performed on NIBSC sera, noted above, from non-human primates that had been exposed only to Zika. The specificity seen in earlier ELISA tests with the NIBSC primate sera, was replicated in the LF format Zika test. These primate Zika-convalescent sera were positive in the Zika LF test, but negative in the dengue LF test, employing our proprietary ‘HX’ antigens. In contrast, the wild type Zika antigen (i.e. without cloaking of the fusion loop) gave rise to false-positives with dengue sera when used in LF-tests. These findings confirmed the superior diagnostic accuracy of our HX antigens over the wild-type (natural) equivalent proteins, in a lateral-flow (as well as ELISA) context.
(178)
(179) When human sera were used in the LF tests, we found that the tests had excellent sensitivity (judged initially by spiking normal human sera with monoclonal anti-Zika and anti-dengue antibodies). However, we found that there was a significant false-positivity rate in both the Zika and dengue LF tests, employing our HX antigens, when endemic panels of sera were tested. We attributed these false positives to ‘mosaic’ epitopes representing short segments of identity in amino-acid sequence common between dengue and Zika viruses (and to a lesser extent with other human flaviviruses), less immune-dominant than the fusion-loop. We were able to obviate false signals due to yellow fever vaccination by increasing the ‘stringency’ of the test (via manipulating running-buffer composition), however this was not sufficient to abolish all false signals originating from (putative) dengue-only and Zika-only sera. We then embarked on a modified strategy of test design, which incorporated ‘off-target-pre-absorption’ to deal with remaining cross-reactions.
Example 19 Characterisation of Off-Target Pre-Absorption
(180) Preabsorption is a well-established technique which is sometimes used in serological analyses to prevent signals being generated by cross-reactive antibodies. In the case of flaviviruses this is difficult to achieve, but our advent of cloaking the fusion loop and obviating signals from the major cross-reactive site of this family of viruses makes it possible to exploit preabsorption to minimize signals from cross-reactive antibodies in LF tests for Zika and dengue. In the Zika test we have described Zika as the target antigen, and preabsorbing dengue antigen as the ‘off-target’ antigen. In the dengue test we have described (below) dengue 1,2,3,4 as the target antigen and Zika as the off-target antigen. Because the mosaic epitopes (defined above) were present in sequence alignments across Zika and all four strains of dengue, we used dengue-2-HX for preabsorption instead of all four dengue serotypes. In configuring tests with preabsorbing antigens it was necessary to construct versions of dengue-2-HX (SEQ ID NO: 2) and Zika-HX (SEQ ID NO: 5) that had either no tag or a different tag than a His tag, otherwise they would contribute signal in the test from cross-reactive antibodies. For this reason we constructed Strep-tag-II versions of the dengue-2-HX and Zika-HX antigens. HX versions were used for preabsorption because they were more productive than the wild type versions in recombinant expression. By configuring the tests in this way we were able to engineer the capture and blockade of antigen combining sites of cross-reactive antibodies with Strep-tag-II versions of the off-target antigens, whereby such antigen-antibody complexes were not captured by the anti-His-tag line and ran off the end of the observation window of the test without contributing signal.
(181) TABLE-US-00012 TABLE 11 Comparison of LF tests with and without off-target-preabsorption, vs. commercial ELISAs Concur Concur Commercial ELISA Tests Den LF Z LF D Z Z D Chik WNV D D + Z Z Z + D (with (with Sample M G M G M G M G G G G G abs'n) abs'n) 55 + + − + + + − + +++ +++ +++ +++ Green Green 56 + + − − − − − − − − + + Green Green 57 + + − − − − − − − − ++ + Green Green 58 + + − + − − − + +++ +++ +++ +++ Green Green 59 + − + + − − − − + + − − Green Green 60 + − − − − − − − − − − − Green Green 63 − − − + − − − − ++ ++ + − Green Green 64 − − + − − − − − + + − − Green Green 65 − − − + − − − − + − + − Red Green 66 − − − + + + − − ++ + − − Green Green 67 − − − + + + − ? ++ + ++ − Green Green 68 − + − + + + − ? +++ ++ +++ − Green Red 69 − − − + + + − − ++ + +++ − Green Green 70 − − − + + + − + + + ++ − Green Green 71 − − − − − − + − − − − − Green Green 72 − − ? + − − + + − − − − Red Green 73 − − − − − − + ? − − − − Green Green 74 − − − − − − + − − − − − Green Green 75 − − − + − − + + + - +++ ++ Red Red 76 − − − + − − − + − − − − Red Green Comparative studies conducted with commercial ELISA tests on endemic sera positive for various flaviviruses: The performance of the Zika and dengue LF tests, with and without ‘off-target’ preabsorption’ was compared to that of commercial ELISA tests. In the dengue LF, Zika-HX was used for absorption; in the Zika LF, dengue-2HX was used for off-target preabsorption. Under these circumstances there was good agreement (depicted by light shading (green) in concur D, and concur Z columns) with the commercial ELISA tests.
(182) As multiple-exposure (and cross-reactivity of antibodies) was expected to be commonplace in endemic areas, it was desirable to employ tests with off-target pre-absorption. Thus we set about determining what amounts of off-target antigen would be needed in each of the pair of LF devices (one Zika and one Dengue test making up the pair), and found that 1 ug per device was adequate for the dengue test, and 5 ug per device for the Zika test (these larger amounts of antigen compare to 200 ng amounts of the test antigen in each device). In the case of the Zika test, off-target preabsorption using all four dengue antigens in Strep-tag form was considered, but after comparison of the sequences and X-ray structures of the wild-type Zika and dengue antigens, it was concluded that use of dengue-2-HX-Strep-tag alone would be sufficient for off-target pre-absorption, because the ‘islands of identity’ in the dengue and Zika HX sequences (comprising residual cross-reactive ‘mosaic’ epitopes) were essentially the same across all four dengue serotypes, additionally the dengue-2-HX was easiest to produce. Pre-absorption with the panel sera with dengue-2-HX was successful and provided LF Zika devices, equipped with off-target pre-absorption built into the LF device for the field testing in Brazil (which were tested alongside the analogously configured dengue LF test devices).
(183)
Example 20 Modified LF Test Design
(184) In a particular embodiment, the Zika test contained non-His-tagged (Strep-tagged) dengue antigen, and (conversely) the dengue test contained Strep-tagged Zika antigen, the off-target antigens were included to bind to cross-reactive antibodies, but not result in gold-particle-arrest (visually detectable signal) on the test line (which was effected by an anti-His-tag antibody). Because the HX antigens were much easier to produce, the off-target antigens were made as HX (rather than wild-type) forms.
(185) In conventional LF tests for this purpose, test line has an anti-Ig (anti-IgM or anti-IgG) and the gold conjugate is labelled with antigen, giving rise to arrest of gold particles by human antibodies that bridge the solid-phase anti-Ig of the test line with gold particles. In our preferred configuration, the test-line is monoclonal-anti-His-tag. Off-target antigens (for pre-absorption) are Strep-tagged (using StrepTag-II), allowing them to flow past the test line. Of a number of architectures tested, we found this configuration to be most sensitive, while simple and easy to manufacture. By use of monoclonal and recombinant reagents exclusively, it is ‘infinitely’ scalable.
(186) A depiction of embodiments of the LF test devices is given in
Example 21 Clinical Field Studies in Rio (Flavivirus Reference Laboratory, Oswaldo Cruz)
(187) In the devices used in the clinical field studies in Rio, the LF tests used 1 ug of Zika-HX and 5 ug of den-2-HX for off-target-pre-absorption of cross-reactive antibodies.
(188) In field testing conducted in Rio de Janiero, the Zika test was found to be 100% sensitive (positive in 50 samples out of 50) in detecting cases of Zika confirmed by PCR (in samples from 2016) that were found to be positive in the blockade of binding assay ‘BOB’, a recently developed ‘second generation’ Zika ELISA assay based on blockade of NS1 monoclonal antibody binding, regarded as the most reliable laboratory-based test for Zika antibodies (Balmaseda A et al Proc Natl Acad Sci USA. 2017 Aug. 1; 114(31):8384-8389. doi: 10.1073/pnas.1704984114. Epub 2017 Jul. 17).
(189) Using our tests all of these samples were strongly and unambiguously (++++) positive (whereas in the BOB test they exhibited a range of positivity). This result tallied with our own observations with panel sera in which our LF tests were found (in the case of dengue) to be very significantly more sensitive than Standard Diagnostics' LF test with respect to IgG detection. Our Zika test was also very good at detecting seroconversion (elevation of antibodies indicative of recent infection) in serial samples (p<0.01)
(190) In the Rio field testing, the earliest sera we were able to obtain were from 2014, which was believed to be before significant Zika circulation had occurred in Rio, although it has recently been recognized that Zika was, in fact, circulating in Brazil in 2013, meaning that there could be some Zika cases in this sample set collected in 2014. We found a 4% positivity rate for Zika (calling any result >3 as positive) with our LF test in this group. However, two tests for Zika NS1 antigen put this figure even higher—at 50%. This result indicated that our Zika test is more specific than the commercial NS1-based Zika ELISA tests, which are false positive in 50% of cases. With respect to our dengue LF test, we found a positivity of 92% (46/50) in the 2014 samples, which tallies with an expected 90-95% dengue seropositivity in the general (Rio) population from various literature studies. The results of testing are shown in
CONCLUSION
(191) We have developed a pair of cheap, simple point-of-care devices for detecting and distinguishing prior dengue or Zika infection, the devices are convenient, do not require a laboratory environment for their performance. The performance of the tests is better than previous commercial LFs and ELISAs and they will find many uses in the diagnosis and monitoring of these infections, including their demonstrated use as a ‘companion diagnostic’ with potential to increase the safety of use of vaccines.
(192) TABLE-US-00013 Sequence Listing Free Text SEQ ID NO: 1 DRGWGNGCGLFGK SEQ ID NO: 2 DRGNGSGCGLNGS, SEQ ID NO: 3 DRGNGSGCGLFGK SEQ ID NO: 4 DRGWGNGCGLNGS SEQ ID NO: 5 DRNHTNGCGLFGK. SEQ ID NO: 6 DRGWGNGCGNHTK SEQ ID NO: 7 pCR025 fragment CKRTLVDRGNGSGCGLNGSGSLVTCAKFA SEQ ID NO: 8 pCR029 fragment CKRTLVDRGWGNGCGNHTKGSLVTCAKFA SEQ ID NO: 9 pCR030 fragment CKRTLVDRGNGSGCGLFGKGSLVTCAKFA SEQ ID NO: 10 pCR031 fragment CKRTLVDRGWGNGCGLNGSGSLVTCAKFA SEQ ID NO: 11 DRGWGNNCTLFGK SEQ ID NO: 12 DRGWGNNCSLFGK pCR021 (SEQ ID NO: 13) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgcca tgcggtgcgt ggggatcggc aatcgcgatt ttgtagaagg 961 actatctggt gccacgtggg tcgatgtggt tcttgaacac gggtcatgcg tgaccacgat 1021 ggctaaggat aagccgacct tggacatcga actactgaaa accgaggtca caaaccctgc 1081 tgtgctccgc aagctgtgca tcgaggctaa gatttccaac acaactactg atagccgctg 1141 ccccacccaa ggcgaggcga ccctcgttga agagcaggac agcaacttcg tgtgtcgccg 1201 gactttcgtg gaccgcggta atgggtccgg atgcggactt aacggatctg gttccttact 1261 gacttgcgcc aaatttaagt gcgtgactaa gttagagggg aaaatcgttc agtatgagaa 1321 cttaaaatac tcggtgatag ttaccgtgca cacaggcgac cagcatcaag ttgggaacga 1381 aacgacagag cacgggacaa tagcgaccat taccccacag gctccaacga gcgaaattca 1441 gctgacagac tacggtgcac tcaccctgga ctgtagccca cggaccgggc tagactttaa 1501 cgagatggtg ctcctgacta tgaaggaaaa gtcatggttg gtgcacaagc agtggttcct 1561 tgatcttcca ttgccctgga cctctggcgc ttcgacctca caagagactt ggaacaggca 1621 ggacttgctc gtgacattca aaacggctca cgctaaaaag caagaggtcg tggttctggg 1681 gagtcaggaa ggcgctatgc ataccgcgtt aacaggggct acagagatcc agaccagtgg 1741 aacaaccact attttcgccg ggcatcttaa gtgtaggctg aagatggata agttgaccct 1801 gaaaggtatg tcatatgtga tgtgcaccgg tagtttcaaa ctggagaaag aagtggccga 1861 aacccagcat ggaacagtac tggtgcaagt caaatatgag ggcaccgatg caccatgtaa 1921 aatacccttc agcgcacaag acgagaaggg agttacccag aacggtaggc tgataacagc 1981 caatccaatc gtcaccgata aggagaaacc agtaaacatc gaaaccgagc cacccttcgg 2041 cgaaagctac atcgtggtcg gcgctggcga gaaagcactt aagctgagct ggtttaagaa 2101 aggtagcacg ggcggcggca gccatcatca ccatcatcac tgagctagCT TGACTGACTG 2161 AGATACAGCG TACCTTCAGC TCACAGACAT GATAAGATAC ATTGATGAGT TTGGACAAAC 2221 CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA ATTTGTGATG CTATTGCTTT 2281 ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA TTCATTTTAT 2341 GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTAAAGC AAGTAAAACC TCTACAAATG 2401 TGGTATTGGC CCATCTCTAT CGGTATCGTA GCATAACCCC TTGGGGCCTC TAAACGGGTC 2461 TTGAGGGGTT TTTTGTGCCC CTCGGGCCGG ATTGCTATCT ACCGGCATTG GCGCAGAAAA 2521 AAATGCCTGA TGCGACGCTG CGCGTCTTAT ACTCCCACAT ATGCCAGATT CAGCAACGGA 2581 TACGGCTTCC CCAACTTGCC CACTTCCATA CGTGTCCTCC TTACCAGAAA TTTATCCTTA 2641 AGGTCGTCAG CTATCCTGCA GGCGATCTCT CGATTTCGAT CAAGACATTC CTTTAATGGT 2701 CTTTTCTGGA CACCACTAGG GGTCAGAAGT AGTTCATCAA ACTTTCTTCC CTCCCTAATC 2761 TCATTGGTTA CCTTGGGCTA TCGAAACTTA ATTAACCAGT CAAGTCAGCT ACTTGGCGAG 2821 ATCGACTTGT CTGGGTTTCG ACTACGCTCA GAATTGCGTC AGTCAAGTTC GATCTGGTCC 2881 TTGCTATTGC ACCCGTTCTC CGATTACGAG TTTCATTTAA ATCATGTGAG CAAAAGGCCA 2941 GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC 3001 CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT 3061 ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT 3121 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG 3181 CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA 3241 CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA 3301 CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC 3361 GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG 3421 AAGAACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG 3481 TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA 3541 GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC 3601 TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG 3661 GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA 3721 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT 3781 CTGTCTATTT CGTTCATCCA TAGTTGCATT TAAATTTCCG AACTCTCCAA GGCCCTCGTC 3841 GGAAAATCTT CAAACCTTTC GTCCGATCCA TCTTGCAGGC TACCTCTCGA ACGAACTATC 3901 GCAAGTCTCT TGGCCGGCCT TGCGCCTTGG CTATTGCTTG GCAGCGCCTA TCGCCAGGTA 3961 TTACTCCAAT CCCGAATATC CGAGATCGGG ATCACCCGAG AGAAGTTCAA CCTACATCCT 4021 CAATCCCGAT CTATCCGAGA TCCGAGGAAT ATCGAAATCG GGGCGCGCCT GGTGTACCGA 4081 GAACGATCCT CTCAGTGCGA GTCTCGACGA TCCATATCGT TGCTTGGCAG TCAGCCAGTC 4141 GGAATCCAGC TTGGGACCCA GGAAGTCCAA TCGTCAGATA TTGTACTCAA GCCTGGTCAC 4201 GGCAGCGTAC CGATCTGTTT AAACCTAGAT ATTGATAGTC TGATCGGTCA ACGTATAATC 4261 GAGTCCTAGC TTTTGCAAAC ATCTATCAAG AGACAGGATC AGCAGGAGGC TTTCGCATGA 4321 GTATTCAACA TTTCCGTGTC GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT 4381 TTGCTCACCC AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCGCGAG 4441 TGGGTTACAT CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG 4501 AACGCTTTCC AATGATGAGC ACTTTTAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTA 4561 TTGACGCCGG GCAAGAGCAA CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG 4621 AGTATTCACC AGTCACAGAA AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA 4681 GTGCTGCCAT AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATTGGAG 4741 GACCGAAGGA GCTAACCGCT TTTTTGCACA ACATGGGGGA TCATGTAACT CGCCTTGATC 4801 GTTGGGAACC GGAGCTGAAT GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG 4861 TAGCAATGGC AACAACCTTG CGTAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC 4921 GGCAACAGTT GATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG 4981 CCCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG 5041 GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA 5101 CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC 5161 TGATTAAGCA TTGGTAACCG ATTCTAGGTG CATTGGCGCA GAAAAAAATG CCTGATGCGA 5221 CGCTGCGCGT CTTATACTCC CACATATGCC AGATTCAGCA ACGGATACGG CTTCCCCAAC 5281 TTGCCCACTT CCATACGTGT CCTCCTTACC AGAAATTTAT CCTTAAGATC CCGAATCGTT 5341 TAAACTCGAC TCTGGCTCTA TCGAATCTCC GTCGTTTCGA GCTTACGCGA ACAGCCGTGG 5401 CGCTCATTTG CTCGTCGGGC ATCGAATCTC GTCAGCTATC GTCAGCTTAC CTTTTTGGCA 5461 pCR022 (SEQ ID NO: 14) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgcca tgcgctgcat cgggatcagc aatcgcgact ttgtggaagg 961 agtcagcggc ggatcatggg tggacatcgt gcttgagcac ggcagctgcg tgaccactat 1021 ggcaaagaat aagccgactc tggattttga actcattaaa accgaggcga agcagcccgc 1081 aactctgagg aagtactgca tcgaggccaa actgactaac actaccaccg aatcacggtg 1141 cccgacccaa ggcgaaccga gcctgaacga agagcaggat aagagatttg tctgcaagca 1201 ctcaatggtg gaccggggga atggatccgg ctgcggactg aacggatctg ggggcattgt 1261 gacttgcgca atgttcacct gtaaaaagaa catggagggc aaggtcgtgc agccagagaa 1321 cctggaatac accattgtca ttactccaca ttccggagag gaacacgccg tcggcaacga 1381 cactggaaaa catgggaagg aaattaagat caccccgcag tcgtcaatta ccgaggcaga 1441 actcaccggg tacggcactg tcactatgga gtgctcaccg agaactgggt tggatttcaa 1501 tgagatggtg ctcctacaga tggagaacaa ggcatggctc gtgcaccggc aatggtttct 1561 cgacctgccg ctgccttggc tccctggggc cgacactcaa ggctcgaatt ggattcagaa 1621 ggaaacgctg gtcacgttca agaaccccca tgccaagaag caagacgtgg tggtcctggg 1681 ctcgcaagaa ggagctatgc acaccgctct gaccggcgcg accgaaatcc aaatgtcatc 1741 aggcaacctc ctgttcactg gccacctcaa atgccggctg agaatggata agctgcaact 1801 gaaaggtatg tcctactcga tgtgcaccgg taaatttaaa gtggtgaaag agatcgctga 1861 aactcagcac ggtaccatcg tcatcagggt gcagtacgag ggagacggct caccctgcaa 1921 aatccccttc gaaatcatgg acctcgaaaa gagacacgtg ctgggccgcc tgatcaccgt 1981 taacccgatc gtgaccgaga aagacagccc ggtgaatatt gaagcggaac ctccgttcgg 2041 cgacagctac atcattatcg gcgtggaacc gggccagctg aagcttaatt ggttcaaaaa 2101 ggggtccagc ggcggcggca gccatcatca ccatcatcac tgagctagCT TGACTGACTG 2161 AGATACAGCG TACCTTCAGC TCACAGACAT GATAAGATAC ATTGATGAGT TTGGACAAAC 2221 CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA ATTTGTGATG CTATTGCTTT 2281 ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA TTCATTTTAT 2341 GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTAAAGC AAGTAAAACC TCTACAAATG 2401 TGGTATTGGC CCATCTCTAT CGGTATCGTA GCATAACCCC TTGGGGCCTC TAAACGGGTC 2461 TTGAGGGGTT TTTTGTGCCC CTCGGGCCGG ATTGCTATCT ACCGGCATTG GCGCAGAAAA 2521 AAATGCCTGA TGCGACGCTG CGCGTCTTAT ACTCCCACAT ATGCCAGATT CAGCAACGGA 2581 TACGGCTTCC CCAACTTGCC CACTTCCATA CGTGTCCTCC TTACCAGAAA TTTATCCTTA 2641 AGGTCGTCAG CTATCCTGCA GGCGATCTCT CGATTTCGAT CAAGACATTC CTTTAATGGT 2701 CTTTTCTGGA CACCACTAGG GGTCAGAAGT AGTTCATCAA ACTTTCTTCC CTCCCTAATC 2761 TCATTGGTTA CCTTGGGCTA TCGAAACTTA ATTAACCAGT CAAGTCAGCT ACTTGGCGAG 2821 ATCGACTTGT CTGGGTTTCG ACTACGCTCA GAATTGCGTC AGTCAAGTTC GATCTGGTCC 2881 TTGCTATTGC ACCCGTTCTC CGATTACGAG TTTCATTTAA ATCATGTGAG CAAAAGGCCA 2941 GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC 3001 CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT 3061 ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT 3121 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG 3181 CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA 3241 CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA 3301 CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC 3361 GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG 3421 AAGAACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG 3481 TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA 3541 GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC 3601 TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG 3661 GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA 3721 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT 3781 CTGTCTATTT CGTTCATCCA TAGTTGCATT TAAATTTCCG AACTCTCCAA GGCCCTCGTC 3841 GGAAAATCTT CAAACCTTTC GTCCGATCCA TCTTGCAGGC TACCTCTCGA ACGAACTATC 3901 GCAAGTCTCT TGGCCGGCCT TGCGCCTTGG CTATTGCTTG GCAGCGCCTA TCGCCAGGTA 3961 TTACTCCAAT CCCGAATATC CGAGATCGGG ATCACCCGAG AGAAGTTCAA CCTACATCCT 4021 CAATCCCGAT CTATCCGAGA TCCGAGGAAT ATCGAAATCG GGGCGCGCCT GGTGTACCGA 4081 GAACGATCCT CTCAGTGCGA GTCTCGACGA TCCATATCGT TGCTTGGCAG TCAGCCAGTC 4141 GGAATCCAGC TTGGGACCCA GGAAGTCCAA TCGTCAGATA TTGTACTCAA GCCTGGTCAC 4201 GGCAGCGTAC CGATCTGTTT AAACCTAGAT ATTGATAGTC TGATCGGTCA ACGTATAATC 4261 GAGTCCTAGC TTTTGCAAAC ATCTATCAAG AGACAGGATC AGCAGGAGGC TTTCGCATGA 4321 GTATTCAACA TTTCCGTGTC GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT 4381 TTGCTCACCC AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCGCGAG 4441 TGGGTTACAT CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG 4501 AACGCTTTCC AATGATGAGC ACTTTTAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTA 4561 TTGACGCCGG GCAAGAGCAA CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG 4621 AGTATTCACC AGTCACAGAA AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA 4681 GTGCTGCCAT AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATTGGAG 4741 GACCGAAGGA GCTAACCGCT TTTTTGCACA ACATGGGGGA TCATGTAACT CGCCTTGATC 4801 GTTGGGAACC GGAGCTGAAT GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG 4861 TAGCAATGGC AACAACCTTG CGTAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC 4921 GGCAACAGTT GATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG 4981 CCCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG 5041 GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA 5101 CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC 5161 TGATTAAGCA TTGGTAACCG ATTCTAGGTG CATTGGCGCA GAAAAAAATG CCTGATGCGA 5221 CGCTGCGCGT CTTATACTCC CACATATGCC AGATTCAGCA ACGGATACGG CTTCCCCAAC 5281 TTGCCCACTT CCATACGTGT CCTCCTTACC AGAAATTTAT CCTTAAGATC CCGAATCGTT 5341 TAAACTCGAC TCTGGCTCTA TCGAATCTCC GTCGTTTCGA GCTTACGCGA ACAGCCGTGG 5401 CGCTCATTTG CTCGTCGGGC ATCGAATCTC GTCAGCTATC GTCAGCTTAC CTTTTTGGCA 5461 pCR023 (SEQ ID NO: 15) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgcca tgagatgtgt gggcgtgggg aaccgcgact ttgtcgaagg 961 attaagtggc gcgacctggg tagacgtcgt gctggagcac ggagggtgcg tcacaaccat 1021 ggccaagaac aagcccaccc ttgacattga acttcaaaag acagaagcta ctcagctggc 1081 tacactgcgc aagctgtgca tagagggaaa aatcaccaac ataactacgg actcgaggtg 1141 tcccacacag ggtgaagcgg tcttgcctga agaacaggat cagaattatg tttgtaaaca 1201 tacttatgta gacaggggga atggatccgg gtgcggtctg aacggatctg gttccctagt 1261 cacatgcgct aagttccagt gcctcgagcc tatcgaaggt aaagtggtcc agtacgagaa 1321 tcttaagtac accgtgatca tcacggtcca tacaggagat caacaccagg ttggaaacga 1381 gacccaagga gtcactgccg aaatcacacc gcaggccagc acgacggagg ctattttgcc 1441 ggagtatggg acactgggac tggaatgctc ccctaggacg ggactagatt ttaatgagat 1501 gattctgctg acaatgaaga acaaggcttg gatggtgcat cgtcaatggt tctttgatct 1561 gccactgccg tgggccagcg gcgccacgac agagacccca acctggaatc gaaaagagct 1621 gctggtcaca ttcaaaaacg cacacgccaa aaagcaagaa gtggtagtgc ttggctccca 1681 ggaaggtgcc atgcacactg cactcacagg ggctactgaa attcagaatt caggaggcac 1741 ttctattttc gccggccacc tcaaatgccg gttaaagatg gacaagctgg aactgaaagg 1801 tatgtcgtac gcaatgtgca ctaatacatt tgtgctaaag aaggaagtct ccgagactca 1861 gcacgggaca atactgatta aggtggaata caaaggtgag gatgctccct gtaagatccc 1921 cttctctact gaggatggtc agggcaaagc tcataatggt cggttgatca cagcgaatcc 1981 agtggttaca aagaaggagg agccagtgaa tatcgaagca gaacctccct tcggtgagtc 2041 aaacattgtc atcggtatcg gagataacgc tcttaagata aactggtaca aaaagggatc 2101 tagcggcggc ggcagccatc atcaccatca tcactgagct agCTTGACTG ACTGAGATAC 2161 AGCGTACCTT CAGCTCACAG ACATGATAAG ATACATTGAT GAGTTTGGAC AAACCACAAC 2221 TAGAATGCAG TGAAAAAAAT GCTTTATTTG TGAAATTTGT GATGCTATTG CTTTATTTGT 2281 AACCATTATA AGCTGCAATA AACAAGTTAA CAACAACAAT TGCATTCATT TTATGTTTCA 2341 GGTTCAGGGG GAGGTGTGGG AGGTTTTTTA AAGCAAGTAA AACCTCTACA AATGTGGTAT 2401 TGGCCCATCT CTATCGGTAT CGTAGCATAA CCCCTTGGGG CCTCTAAACG GGTCTTGAGG 2461 GGTTTTTTGT GCCCCTCGGG CCGGATTGCT ATCTACCGGC ATTGGCGCAG AAAAAAATGC 2521 CTGATGCGAC GCTGCGCGTC TTATACTCCC ACATATGCCA GATTCAGCAA CGGATACGGC 2581 TTCCCCAACT TGCCCACTTC CATACGTGTC CTCCTTACCA GAAATTTATC CTTAAGGTCG 2641 TCAGCTATCC TGCAGGCGAT CTCTCGATTT CGATCAAGAC ATTCCTTTAA TGGTCTTTTC 2701 TGGACACCAC TAGGGGTCAG AAGTAGTTCA TCAAACTTTC TTCCCTCCCT AATCTCATTG 2761 GTTACCTTGG GCTATCGAAA CTTAATTAAC CAGTCAAGTC AGCTACTTGG CGAGATCGAC 2821 TTGTCTGGGT TTCGACTACG CTCAGAATTG CGTCAGTCAA GTTCGATCTG GTCCTTGCTA 2881 TTGCACCCGT TCTCCGATTA CGAGTTTCAT TTAAATCATG TGAGCAAAAG GCCAGCAAAA 2941 GGCCAGGAAC CGTAAAAAGG CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA 3001 CGAGCATCAC AAAAATCGAC GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG 3061 ATACCAGGCG TTTCCCCCTG GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT 3121 TACCGGATAC CTGTCCGCCT TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC ATAGCTCACG 3181 CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC 3241 CCCCGTTCAG CCCGACCGCT GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT 3301 AAGACACGAC TTATCGCCAC TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA 3361 TGTAGGCGGT GCTACAGAGT TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGAAC 3421 AGTATTTGGT ATCTGCGCTC TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC 3481 TTGATCCGGC AAACAAACCA CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT 3541 TACGCGCAGA AAAAAAGGAT CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC 3601 TCAGTGGAAC GAAAACTCAC GTTAAGGGAT TTTGGTCATG AGATTATCAA AAAGGATCTT 3661 CACCTAGATC CTTTTAAATT AAAAATGAAG TTTTAAATCA ATCTAAAGTA TATATGAGTA 3721 AACTTGGTCT GACAGTTACC AATGCTTAAT CAGTGAGGCA CCTATCTCAG CGATCTGTCT 3781 ATTTCGTTCA TCCATAGTTG CATTTAAATT TCCGAACTCT CCAAGGCCCT CGTCGGAAAA 3841 TCTTCAAACC TTTCGTCCGA TCCATCTTGC AGGCTACCTC TCGAACGAAC TATCGCAAGT 3901 CTCTTGGCCG GCCTTGCGCC TTGGCTATTG CTTGGCAGCG CCTATCGCCA GGTATTACTC 3961 CAATCCCGAA TATCCGAGAT CGGGATCACC CGAGAGAAGT TCAACCTACA TCCTCAATCC 4021 CGATCTATCC GAGATCCGAG GAATATCGAA ATCGGGGCGC GCCTGGTGTA CCGAGAACGA 4081 TCCTCTCAGT GCGAGTCTCG ACGATCCATA TCGTTGCTTG GCAGTCAGCC AGTCGGAATC 4141 CAGCTTGGGA CCCAGGAAGT CCAATCGTCA GATATTGTAC TCAAGCCTGG TCACGGCAGC 4201 GTACCGATCT GTTTAAACCT AGATATTGAT AGTCTGATCG GTCAACGTAT AATCGAGTCC 4261 TAGCTTTTGC AAACATCTAT CAAGAGACAG GATCAGCAGG AGGCTTTCGC ATGAGTATTC 4321 AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT GTTTTTGCTC 4381 ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCG CGAGTGGGTT 4441 ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC GAAGAACGCT 4501 TTCCAATGAT GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC CGTATTGACG 4561 CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG GTTGAGTATT 4621 CACCAGTCAC AGAAAAGCAT CTTACGGATG GCATGACAGT AAGAGAATTA TGCAGTGCTG 4681 CCATAACCAT GAGTGATAAC ACTGCGGCCA ACTTACTTCT GACAACGATT GGAGGACCGA 4741 AGGAGCTAAC CGCTTTTTTG CACAACATGG GGGATCATGT AACTCGCCTT GATCGTTGGG 4801 AACCGGAGCT GAATGAAGCC ATACCAAACG ACGAGCGTGA CACCACGATG CCTGTAGCAA 4861 TGGCAACAAC CTTGCGTAAA CTATTAACTG GCGAACTACT TACTCTAGCT TCCCGGCAAC 4921 AGTTGATAGA CTGGATGGAG GCGGATAAAG TTGCAGGACC ACTTCTGCGC TCGGCCCTTC 4981 CGGCTGGCTG GTTTATTGCT GATAAATCTG GAGCCGGTGA GCGTGGGTCT CGCGGTATCA 5041 TTGCAGCACT GGGGCCAGAT GGTAAGCCCT CCCGTATCGT AGTTATCTAC ACGACGGGGA 5101 GTCAGGCAAC TATGGATGAA CGAAATAGAC AGATCGCTGA GATAGGTGCC TCACTGATTA 5161 AGCATTGGTA ACCGATTCTA GGTGCATTGG CGCAGAAAAA AATGCCTGAT GCGACGCTGC 5221 GCGTCTTATA CTCCCACATA TGCCAGATTC AGCAACGGAT ACGGCTTCCC CAACTTGCCC 5281 ACTTCCATAC GTGTCCTCCT TACCAGAAAT TTATCCTTAA GATCCCGAAT CGTTTAAACT 5341 CGACTCTGGC TCTATCGAAT CTCCGTCGTT TCGAGCTTAC GCGAACAGCC GTGGCGCTCA 5401 TTTGCTCGTC GGGCATCGAA TCTCGTCAGC TATCGTCAGC TTACCTTTTT GGCA // pCR024 (SEQ ID NO: 16) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgcca tgcgatgcgt gggggtgggc aatagagatt tcgtggaagg 961 ggtgtctgga ggggcatggg tggatctggt gctggagcac ggcggatgtg tcacaactat 1021 ggcccagggg aagccaaccc tggatttcga gctaactaag accacagcta aggaggtagc 1081 cctgcttcgg acttactgta ttgaggcatc catctctaac atcaccaccg ccacgagatg 1141 cccgacacag ggcgaaccct acttgaagga agaacaggat cagcagtaca tttgccggcg 1201 cgatgttgtt gatagaggca atggctccgg gtgtggcctc aacggctctg gtggggtggt 1261 cacctgtgcc aagttcagct gttctggcaa gatcacggga aatctggtgc aaattgaaaa 1321 tttggaatat acggtcgttg tgactgtcca caatggcgat acacatgctg tgggcaacga 1381 taccagtaac cacggcgtca ccgcgatgat aactccccgg agcccatctg ttgaagttaa 1441 actgcccgat tacggagagt tgacactcga ctgcgaaccg aggtctggaa tagatttcaa 1501 cgagatgata cttatgaaaa tgaagaaaaa gacctggctc gtacacaagc agtggttttt 1561 ggatttgccc ctcccttgga ccgcaggggc cgataccagc gaggtgcatt ggaattacaa 1621 agagcgcatg gtgactttca aagtgcccca cgcaaagcgg caagatgtga ctgtattagg 1681 atcacaggaa ggcgctatgc attccgccct ggctggtgcc acggaggtgg attcaggaga 1741 cggtaaccat atgtttgctg gccacctcaa atgtaaggtc cgcatggaaa aacttcgcat 1801 taaaggaatg tcctacacga tgtgctcagg aaagttctct atcgacaagg aaatggccga 1861 gactcagcat ggaacgactg tagtcaaggt gaaatatgaa ggtgccgggg cgccttgcaa 1921 ggtgccaatc gaaatccgag acgttaacaa ggagaaggtg gttgggagga ttataagtag 1981 cactccgctc gcagagaaca ccaatagcgt gactaacata gaactggagc ccccttttgg 2041 ggatagctac attgtgattg gagtagggaa tagtgcacta acattgcact ggttcagaaa 2101 agggtcttca ggcggcggca gccatcatca ccatcatcac tgagctagCT TGACTGACTG 2161 AGATACAGCG TACCTTCAGC TCACAGACAT GATAAGATAC ATTGATGAGT TTGGACAAAC 2221 CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA ATTTGTGATG CTATTGCTTT 2281 ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA TTCATTTTAT 2341 GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTAAAGC AAGTAAAACC TCTACAAATG 2401 TGGTATTGGC CCATCTCTAT CGGTATCGTA GCATAACCCC TTGGGGCCTC TAAACGGGTC 2461 TTGAGGGGTT TTTTGTGCCC CTCGGGCCGG ATTGCTATCT ACCGGCATTG GCGCAGAAAA 2521 AAATGCCTGA TGCGACGCTG CGCGTCTTAT ACTCCCACAT ATGCCAGATT CAGCAACGGA 2581 TACGGCTTCC CCAACTTGCC CACTTCCATA CGTGTCCTCC TTACCAGAAA TTTATCCTTA 2641 AGGTCGTCAG CTATCCTGCA GGCGATCTCT CGATTTCGAT CAAGACATTC CTTTAATGGT 2701 CTTTTCTGGA CACCACTAGG GGTCAGAAGT AGTTCATCAA ACTTTCTTCC CTCCCTAATC 2761 TCATTGGTTA CCTTGGGCTA TCGAAACTTA ATTAACCAGT CAAGTCAGCT ACTTGGCGAG 2821 ATCGACTTGT CTGGGTTTCG ACTACGCTCA GAATTGCGTC AGTCAAGTTC GATCTGGTCC 2881 TTGCTATTGC ACCCGTTCTC CGATTACGAG TTTCATTTAA ATCATGTGAG CAAAAGGCCA 2941 GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC 3001 CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT 3061 ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT 3121 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG 3181 CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA 3241 CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA 3301 CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC 3361 GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG 3421 AAGAACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG 3481 TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA 3541 GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC 3601 TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG 3661 GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA 3721 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT 3781 CTGTCTATTT CGTTCATCCA TAGTTGCATT TAAATTTCCG AACTCTCCAA GGCCCTCGTC 3841 GGAAAATCTT CAAACCTTTC GTCCGATCCA TCTTGCAGGC TACCTCTCGA ACGAACTATC 3901 GCAAGTCTCT TGGCCGGCCT TGCGCCTTGG CTATTGCTTG GCAGCGCCTA TCGCCAGGTA 3961 TTACTCCAAT CCCGAATATC CGAGATCGGG ATCACCCGAG AGAAGTTCAA CCTACATCCT 4021 CAATCCCGAT CTATCCGAGA TCCGAGGAAT ATCGAAATCG GGGCGCGCCT GGTGTACCGA 4081 GAACGATCCT CTCAGTGCGA GTCTCGACGA TCCATATCGT TGCTTGGCAG TCAGCCAGTC 4141 GGAATCCAGC TTGGGACCCA GGAAGTCCAA TCGTCAGATA TTGTACTCAA GCCTGGTCAC 4201 GGCAGCGTAC CGATCTGTTT AAACCTAGAT ATTGATAGTC TGATCGGTCA ACGTATAATC 4261 GAGTCCTAGC TTTTGCAAAC ATCTATCAAG AGACAGGATC AGCAGGAGGC TTTCGCATGA 4321 GTATTCAACA TTTCCGTGTC GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT 4381 TTGCTCACCC AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCGCGAG 4441 TGGGTTACAT CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG 4501 AACGCTTTCC AATGATGAGC ACTTTTAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTA 4561 TTGACGCCGG GCAAGAGCAA CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG 4621 AGTATTCACC AGTCACAGAA AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA 4681 GTGCTGCCAT AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATTGGAG 4741 GACCGAAGGA GCTAACCGCT TTTTTGCACA ACATGGGGGA TCATGTAACT CGCCTTGATC 4801 GTTGGGAACC GGAGCTGAAT GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG 4861 TAGCAATGGC AACAACCTTG CGTAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC 4921 GGCAACAGTT GATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG 4981 CCCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG 5041 GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA 5101 CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC 5161 TGATTAAGCA TTGGTAACCG ATTCTAGGTG CATTGGCGCA GAAAAAAATG CCTGATGCGA 5221 CGCTGCGCGT CTTATACTCC CACATATGCC AGATTCAGCA ACGGATACGG CTTCCCCAAC 5281 TTGCCCACTT CCATACGTGT CCTCCTTACC AGAAATTTAT CCTTAAGATC CCGAATCGTT 5341 TAAACTCGAC TCTGGCTCTA TCGAATCTCC GTCGTTTCGA GCTTACGCGA ACAGCCGTGG 5401 CGCTCATTTG CTCGTCGGGC ATCGAATCTC GTCAGCTATC GTCAGCTTAC CTTTTTGGCA 5461 // pCR028 (SEQ ID NO: 17) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgccA TCAGGTGCAT TGGAGTCAGC AACAGGGACT TCGTCGAAGG 961 CATGTCCGGC GGCACCTGGG TGGATGTGGT GCTCGAACAC GGCGGATGCG TGACCGTCAT 1021 GGCCCAGGAC AAGCCTACCG TCGATATTGA GCTGGTGACC ACCACAGTGA GCAACATGGC 1081 CGAAGTGAGA AGCTACTGCT ATGAGGCCTC CATCAGCGAT ATGGCTTCCG ATTCCAGATG 1141 CCCCACACAG GGAGAGGCTT ATCTGGACAA ACAGTCCGAC ACCCAGTACG TCTGCAAAAG 1201 AACCCTGGTG GACAGAaacc acaccAACGG ATGCGGCCTG TTCGGCAAAG GCAGCCTCGT 1261 GACATGTGCC AAGTTCGCCT GCAGCAAAAA GATGACCGGC AAGTCCATCC AGCCCGAGAA 1321 CCTGGAATAC AGGATCATGC TGTCCGTGCA TGGATCCCAG CACTCCGGCA TGATCGTCAA 1381 CGATACCGGC CACGAGACCG ACGAGAACAG GGCTAAAGTG GAGATCACCC CCAACAGCCC 1441 TAGAGCCGAA GCTACACTGG GCGGCTTCGG AAGCCTGGGC CTGGATTGCG AACCCAGGAC 1501 CGGCCTGGAT TTCAGCGACC TGTATTACCT GACCATGAAC AATAAGCACT GGCTGGTGCA 1561 CAAGGAATGG TTCCACGACA TCCCCCTGCC TTGGCATGCT GGCGCCGATA CCGGCACACC 1621 TCACTGGAAC AATAAGGAAG CCCTGGTCGA GTTTAAGGAC GCCCACGCCA AAAGACAGAC 1681 CGTGGTGGTG CTGGGAAGCC AGGAGGGAGC TGTCCACACA GCCCTGGCCG GAGCTCTGGA 1741 AGCCGAGATG GATGGCGCCA AGGGCAGGCT GAGCTCCGGC CACCTGAAAT GCAGGCTCAA 1801 GATGGACAAG CTGAGGCTGA AGGGCGTGAG CTACAGCCTG TGCACCGCCG CTTTCACCTT 1861 TACCAAGATC CCTGCCGAGA CACTGCACGG CACCGTCACC GTGGAGGTGC AATACGCCGG 1921 AACCGATGGA CCTTGCAAAG TGCCTGCCCA GATGGCTGTG GATATGCAGA CCCTCACACC 1981 CGTCGGCAGG CTGATCACCG CCAATCCCGT CATTACCGAG TCCACCGAGA ACAGCAAGAT 2041 GATGCTcGAG CTCGATCCCC CCTTTGGCGA CAGCTACATT GTGATCGGCG TGGGCGAGAA 2101 GAAGATCACC CACCATTGGC ACAGAAGCGG CTCCACAggg ggtagcggtg gtagcggagg 2161 tagccatcac caccatcacc actgagctag CTTGACTGAC TGAGATACAG CGTACCTTCA 2221 GCTCACAGAC ATGATAAGAT ACATTGATGA GTTTGGACAA ACCACAACTA GAATGCAGTG 2281 AAAAAAATGC TTTATTTGTG AAATTTGTGA TGCTATTGCT TTATTTGTAA CCATTATAAG 2341 CTGCAATAAA CAAGTTAACA ACAACAATTG CATTCATTTT ATGTTTCAGG TTCAGGGGGA 2401 GGTGTGGGAG GTTTTTTAAA GCAAGTAAAA CCTCTACAAA TGTGGTATTG GCCCATCTCT 2461 ATCGGTATCG TAGCATAACC CCTTGGGGCC TCTAAACGGG TCTTGAGGGG TTTTTTGTGC 2521 CCCTCGGGCC GGATTGCTAT CTACCGGCAT TGGCGCAGAA AAAAATGCCT GATGCGACGC 2581 TGCGCGTCTT ATACTCCCAC ATATGCCAGA TTCAGCAACG GATACGGCTT CCCCAACTTG 2641 CCCACTTCCA TACGTGTCCT CCTTACCAGA AATTTATCCT TAAGGTCGTC AGCTATCCTG 2701 CAGGCGATCT CTCGATTTCG ATCAAGACAT TCCTTTAATG GTCTTTTCTG GACACCACTA 2761 GGGGTCAGAA GTAGTTCATC AAACTTTCTT CCCTCCCTAA TCTCATTGGT TACCTTGGGC 2821 TATCGAAACT TAATTAACCA GTCAAGTCAG CTACTTGGCG AGATCGACTT GTCTGGGTTT 2881 CGACTACGCT CAGAATTGCG TCAGTCAAGT TCGATCTGGT CCTTGCTATT GCACCCGTTC 2941 TCCGATTACG AGTTTCATTT AAATCATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG 3001 TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA 3061 AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT 3121 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT 3181 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT 3241 CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC 3301 CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 3361 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC 3421 TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT 3481 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA 3541 ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 3601 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA 3661 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT 3721 TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA 3781 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 3841 CATAGTTGCA TTTAAATTTC CGAACTCTCC AAGGCCCTCG TCGGAAAATC TTCAAACCTT 3901 TCGTCCGATC CATCTTGCAG GCTACCTCTC GAACGAACTA TCGCAAGTCT CTTGGCCGGC 3961 CTTGCGCCTT GGCTATTGCT TGGCAGCGCC TATCGCCAGG TATTACTCCA ATCCCGAATA 4021 TCCGAGATCG GGATCACCCG AGAGAAGTTC AACCTACATC CTCAATCCCG ATCTATCCGA 4081 GATCCGAGGA ATATCGAAAT CGGGGCGCGC CTGGTGTACC GAGAACGATC CTCTCAGTGC 4141 GAGTCTCGAC GATCCATATC GTTGCTTGGC AGTCAGCCAG TCGGAATCCA GCTTGGGACC 4201 CAGGAAGTCC AATCGTCAGA TATTGTACTC AAGCCTGGTC ACGGCAGCGT ACCGATCTGT 4261 TTAAACCTAG ATATTGATAG TCTGATCGGT CAACGTATAA TCGAGTCCTA GCTTTTGCAA 4321 ACATCTATCA AGAGACAGGA TCAGCAGGAG GCTTTCGCAT GAGTATTCAA CATTTCCGTG 4381 TCGCCCTTAT TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC CCAGAAACGC 4441 TGGTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCGCG AGTGGGTTAC ATCGAACTGG 4501 ATCTCAACAG CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGCTTT CCAATGATGA 4561 GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG TATTGACGCC GGGCAAGAGC 4621 AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT TGAGTATTCA CCAGTCACAG 4681 AAAAGCATCT TACGGATGGC ATGACAGTAA GAGAATTATG CAGTGCTGCC ATAACCATGA 4741 GTGATAACAC TGCGGCCAAC TTACTTCTGA CAACGATTGG AGGACCGAAG GAGCTAACCG 4801 CTTTTTTGCA CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA 4861 ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGTAGCAATG GCAACAACCT 4921 TGCGTAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC CCGGCAACAG TTGATAGACT 4981 GGATGGAGGC GGATAAAGTT GCAGGACCAC TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT 5041 TTATTGCTGA TAAATCTGGA GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG 5101 GGCCAGATGG TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA 5161 TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG CATTGGTAAC 5221 CGATTCTAGG TGCATTGGCG CAGAAAAAAA TGCCTGATGC GACGCTGCGC GTCTTATACT 5281 CCCACATATG CCAGATTCAG CAACGGATAC GGCTTCCCCA ACTTGCCCAC TTCCATACGT 5341 GTCCTCCTTA CCAGAAATTT ATCCTTAAGA TCCCGAATCG TTTAAACTCG ACTCTGGCTC 5401 TATCGAATCT CCGTCGTTTC GAGCTTACGC GAACAGCCGT GGCGCTCATT TGCTCGTCGG 5461 GCATCGAATC TCGTCAGCTA TCGTCAGCTT ACCTTTTTGG CA pCR025 (SEQ ID NO: 18) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgcca tcaggtgcat tggagtcagc aacagggact tcgtcgaagg 961 catgtccggc ggcacctggg tggatgtggt gctcgaacac ggcggatgcg tgaccgtcat 1021 ggcccaggac aagcctaccg tcgatattga gctggtgacc accacagtga gcaacatggc 1081 cgaagtgaga agctactgct atgaggcctc catcagcgat atggcttccg attccagatg 1141 ccccacacag ggagaggctt atctggacaa acagtccgac acccagtacg tctgcaaaag 1201 aaccctggtg gacagaggca atggatccgg atgcggcctg aacggctctg gcagcctcgt 1261 gacatgtgcc aagttcgcct gcagcaaaaa gatgaccggc aagtccatcc agcccgagaa 1321 cctggaatac aggatcatgc tgtccgtgca tggatcccag cactccggca tgatcgtcaa 1381 cgataccggc cacgagaccg acgagaacag ggctaaagtg gagatcaccc ccaacagccc 1441 tagagccgaa gctacactgg gcggcttcgg aagcctgggc ctggattgcg aacccaggac 1501 cggcctggat ttcagcgacc tgtattacct gaccatgaac aataagcact ggctggtgca 1561 caaggaatgg ttccacgaca tccccctgcc ttggcatgct ggcgccgata ccggcacacc 1621 tcactggaac aataaggaag ccctggtcga gtttaaggac gcccacgcca aaagacagac 1681 cgtggtggtg ctgggaagcc aggagggagc tgtccacaca gccctggccg gagctctgga 1741 agccgagatg gatggcgcca agggcaggct gagctccggc cacctgaaat gcaggctcaa 1801 gatggacaag ctgaggctga agggcgtgag ctacagcctg tgcaccgccg ctttcacctt 1861 taccaagatc cctgccgaga cactgcacgg caccgtcacc gtggaggtgc aatacgccgg 1921 aaccgatgga ccttgcaaag tgcctgccca gatggctgtg gatatgcaga ccctcacacc 1981 cgtcggcagg ctgatcaccg ccaatcccgt cattaccgag tccaccgaga acagcaagat 2041 gatgctcgag ctcgatcccc cctttggcga cagctacatt gtgatcggcg tgggcgagaa 2101 gaagatcacc caccattggc acagaagcgg ctccacaggg ggtagcggtg gtagcggagg 2161 tagccatcac caccatcacc actgagctag CTTGACTGAC TGAGATACAG CGTACCTTCA 2221 GCTCACAGAC ATGATAAGAT ACATTGATGA GTTTGGACAA ACCACAACTA GAATGCAGTG 2281 AAAAAAATGC TTTATTTGTG AAATTTGTGA TGCTATTGCT TTATTTGTAA CCATTATAAG 2341 CTGCAATAAA CAAGTTAACA ACAACAATTG CATTCATTTT ATGTTTCAGG TTCAGGGGGA 2401 GGTGTGGGAG GTTTTTTAAA GCAAGTAAAA CCTCTACAAA TGTGGTATTG GCCCATCTCT 2461 ATCGGTATCG TAGCATAACC CCTTGGGGCC TCTAAACGGG TCTTGAGGGG TTTTTTGTGC 2521 CCCTCGGGCC GGATTGCTAT CTACCGGCAT TGGCGCAGAA AAAAATGCCT GATGCGACGC 2581 TGCGCGTCTT ATACTCCCAC ATATGCCAGA TTCAGCAACG GATACGGCTT CCCCAACTTG 2641 CCCACTTCCA TACGTGTCCT CCTTACCAGA AATTTATCCT TAAGGTCGTC AGCTATCCTG 2701 CAGGCGATCT CTCGATTTCG ATCAAGACAT TCCTTTAATG GTCTTTTCTG GACACCACTA 2761 GGGGTCAGAA GTAGTTCATC AAACTTTCTT CCCTCCCTAA TCTCATTGGT TACCTTGGGC 2821 TATCGAAACT TAATTAACCA GTCAAGTCAG CTACTTGGCG AGATCGACTT GTCTGGGTTT 2881 CGACTACGCT CAGAATTGCG TCAGTCAAGT TCGATCTGGT CCTTGCTATT GCACCCGTTC 2941 TCCGATTACG AGTTTCATTT AAATCATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG 3001 TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA 3061 AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT 3121 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT 3181 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT 3241 CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC 3301 CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 3361 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC 3421 TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT 3481 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA 3541 ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 3601 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA 3661 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT 3721 TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA 3781 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 3841 CATAGTTGCA TTTAAATTTC CGAACTCTCC AAGGCCCTCG TCGGAAAATC TTCAAACCTT 3901 TCGTCCGATC CATCTTGCAG GCTACCTCTC GAACGAACTA TCGCAAGTCT CTTGGCCGGC 3961 CTTGCGCCTT GGCTATTGCT TGGCAGCGCC TATCGCCAGG TATTACTCCA ATCCCGAATA 4021 TCCGAGATCG GGATCACCCG AGAGAAGTTC AACCTACATC CTCAATCCCG ATCTATCCGA 4081 GATCCGAGGA ATATCGAAAT CGGGGCGCGC CTGGTGTACC GAGAACGATC CTCTCAGTGC 4141 GAGTCTCGAC GATCCATATC GTTGCTTGGC AGTCAGCCAG TCGGAATCCA GCTTGGGACC 4201 CAGGAAGTCC AATCGTCAGA TATTGTACTC AAGCCTGGTC ACGGCAGCGT ACCGATCTGT 4261 TTAAACCTAG ATATTGATAG TCTGATCGGT CAACGTATAA TCGAGTCCTA GCTTTTGCAA 4321 ACATCTATCA AGAGACAGGA TCAGCAGGAG GCTTTCGCAT GAGTATTCAA CATTTCCGTG 4381 TCGCCCTTAT TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC CCAGAAACGC 4441 TGGTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCGCG AGTGGGTTAC ATCGAACTGG 4501 ATCTCAACAG CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGCTTT CCAATGATGA 4561 GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG TATTGACGCC GGGCAAGAGC 4621 AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT TGAGTATTCA CCAGTCACAG 4681 AAAAGCATCT TACGGATGGC ATGACAGTAA GAGAATTATG CAGTGCTGCC ATAACCATGA 4741 GTGATAACAC TGCGGCCAAC TTACTTCTGA CAACGATTGG AGGACCGAAG GAGCTAACCG 4801 CTTTTTTGCA CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA 4861 ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGTAGCAATG GCAACAACCT 4921 TGCGTAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC CCGGCAACAG TTGATAGACT 4981 GGATGGAGGC GGATAAAGTT GCAGGACCAC TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT 5041 TTATTGCTGA TAAATCTGGA GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG 5101 GGCCAGATGG TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA 5161 TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG CATTGGTAAC 5221 CGATTCTAGG TGCATTGGCG CAGAAAAAAA TGCCTGATGC GACGCTGCGC GTCTTATACT 5281 CCCACATATG CCAGATTCAG CAACGGATAC GGCTTCCCCA ACTTGCCCAC TTCCATACGT 5341 GTCCTCCTTA CCAGAAATTT ATCCTTAAGA TCCCGAATCG TTTAAACTCG ACTCTGGCTC 5401 TATCGAATCT CCGTCGTTTC GAGCTTACGC GAACAGCCGT GGCGCTCATT TGCTCGTCGG 5461 GCATCGAATC TCGTCAGCTA TCGTCAGCTT ACCTTTTTGG CA pCR026 (SEQ ID NO: 19) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgcca tgcggtgcgt ggggatcggc aatcgcgatt ttgtagaagg 961 actatctggt gccacgtggg tcgatgtggt tcttgaacac gggtcatgcg tgaccacgat 1021 ggctaaggat aagccgacct tggacatcga actactgaaa accgaggtca caaaccctgc 1081 tgtgctccgc aagctgtgca tcgaggctaa gatttccaac acaactactg atagccgctg 1141 ccccacccaa ggcgaggcga ccctcgttga agagcaggac agcaacttcg tgtgtcgccg 1201 gactttcgtg gaccgcggta atgggtccgg atgcggactt TTTGGAAAGg gttccttact 1261 gacttgcgcc aaatttaagt gcgtgactaa gttagagggg aaaatcgttc agtatgagaa 1321 cttaaaatac tcggtgatag ttaccgtgca cacaggcgac cagcatcaag ttgggaacga 1381 aacgacagag cacgggacaa tagcgaccat taccccacag gctccaacga gcgaaattca 1441 gctgacagac tacggtgcac tcaccctgga ctgtagccca cggaccgggc tagactttaa 1501 cgagatggtg ctcctgacta tgaaggaaaa gtcatggttg gtgcacaagc agtggttcct 1561 tgatcttcca ttgccctgga cctctggcgc ttcgacctca caagagactt ggaacaggca 1621 ggacttgctc gtgacattca aaacggctca cgctaaaaag caagaggtcg tggttctggg 1681 gagtcaggaa ggcgctatgc ataccgcgtt aacaggggct acagagatcc agaccagtgg 1741 aacaaccact attttcgccg ggcatcttaa gtgtaggctg aagatggata agttgaccct 1801 gaaaggtatg tcatatgtga tgtgcaccgg tagtttcaaa ctggagaaag aagtggccga 1861 aacccagcat ggaacagtac tggtgcaagt caaatatgag ggcaccgatg caccatgtaa 1921 aatacccttc agcgcacaag acgagaaggg agttacccag aacggtaggc tgataacagc 1981 caatccaatc gtcaccgata aggagaaacc agtaaacatc gaaaccgagc cacccttcgg 2041 cgaaagctac atcgtggtcg gcgctggcga gaaagcactt aagctgagct ggtttaagaa 2101 aggtagcacg ggcggcggca gccatcatca ccatcatcac tgagctagCT TGACTGACTG 2161 AGATACAGCG TACCTTCAGC TCACAGACAT GATAAGATAC ATTGATGAGT TTGGACAAAC 2221 CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA ATTTGTGATG CTATTGCTTT 2281 ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA TTCATTTTAT 2341 GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTAAAGC AAGTAAAACC TCTACAAATG 2401 TGGTATTGGC CCATCTCTAT CGGTATCGTA GCATAACCCC TTGGGGCCTC TAAACGGGTC 2461 TTGAGGGGTT TTTTGTGCCC CTCGGGCCGG ATTGCTATCT ACCGGCATTG GCGCAGAAAA 2521 AAATGCCTGA TGCGACGCTG CGCGTCTTAT ACTCCCACAT ATGCCAGATT CAGCAACGGA 2581 TACGGCTTCC CCAACTTGCC CACTTCCATA CGTGTCCTCC TTACCAGAAA TTTATCCTTA 2641 AGGTCGTCAG CTATCCTGCA GGCGATCTCT CGATTTCGAT CAAGACATTC CTTTAATGGT 2701 CTTTTCTGGA CACCACTAGG GGTCAGAAGT AGTTCATCAA ACTTTCTTCC CTCCCTAATC 2761 TCATTGGTTA CCTTGGGCTA TCGAAACTTA ATTAACCAGT CAAGTCAGCT ACTTGGCGAG 2821 ATCGACTTGT CTGGGTTTCG ACTACGCTCA GAATTGCGTC AGTCAAGTTC GATCTGGTCC 2881 TTGCTATTGC ACCCGTTCTC CGATTACGAG TTTCATTTAA ATCATGTGAG CAAAAGGCCA 2941 GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC 3001 CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT 3061 ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT 3121 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG 3181 CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA 3241 CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA 3301 CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC 3361 GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG 3421 AAGAACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG 3481 TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA 3541 GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC 3601 TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG 3661 GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA 3721 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT 3781 CTGTCTATTT CGTTCATCCA TAGTTGCATT TAAATTTCCG AACTCTCCAA GGCCCTCGTC 3841 GGAAAATCTT CAAACCTTTC GTCCGATCCA TCTTGCAGGC TACCTCTCGA ACGAACTATC 3901 GCAAGTCTCT TGGCCGGCCT TGCGCCTTGG CTATTGCTTG GCAGCGCCTA TCGCCAGGTA 3961 TTACTCCAAT CCCGAATATC CGAGATCGGG ATCACCCGAG AGAAGTTCAA CCTACATCCT 4021 CAATCCCGAT CTATCCGAGA TCCGAGGAAT ATCGAAATCG GGGCGCGCCT GGTGTACCGA 4081 GAACGATCCT CTCAGTGCGA GTCTCGACGA TCCATATCGT TGCTTGGCAG TCAGCCAGTC 4141 GGAATCCAGC TTGGGACCCA GGAAGTCCAA TCGTCAGATA TTGTACTCAA GCCTGGTCAC 4201 GGCAGCGTAC CGATCTGTTT AAACCTAGAT ATTGATAGTC TGATCGGTCA ACGTATAATC 4261 GAGTCCTAGC TTTTGCAAAC ATCTATCAAG AGACAGGATC AGCAGGAGGC TTTCGCATGA 4321 GTATTCAACA TTTCCGTGTC GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT 4381 TTGCTCACCC AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCGCGAG 4441 TGGGTTACAT CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG 4501 AACGCTTTCC AATGATGAGC ACTTTTAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTA 4561 TTGACGCCGG GCAAGAGCAA CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG 4621 AGTATTCACC AGTCACAGAA AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA 4681 GTGCTGCCAT AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATTGGAG 4741 GACCGAAGGA GCTAACCGCT TTTTTGCACA ACATGGGGGA TCATGTAACT CGCCTTGATC 4801 GTTGGGAACC GGAGCTGAAT GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG 4861 TAGCAATGGC AACAACCTTG CGTAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC 4921 GGCAACAGTT GATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG 4981 CCCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG 5041 GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA 5101 CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC 5161 TGATTAAGCA TTGGTAACCG ATTCTAGGTG CATTGGCGCA GAAAAAAATG CCTGATGCGA 5221 CGCTGCGCGT CTTATACTCC CACATATGCC AGATTCAGCA ACGGATACGG CTTCCCCAAC 5281 TTGCCCACTT CCATACGTGT CCTCCTTACC AGAAATTTAT CCTTAAGATC CCGAATCGTT 5341 TAAACTCGAC TCTGGCTCTA TCGAATCTCC GTCGTTTCGA GCTTACGCGA ACAGCCGTGG 5401 CGCTCATTTG CTCGTCGGGC ATCGAATCTC GTCAGCTATC GTCAGCTTAC CTTTTTGGCA 5461 pCR027 (SEQ ID NO: 20) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgcca tgcggtgcgt ggggatcggc aatcgcgatt ttgtagaagg 961 actatctggt gccacgtggg tcgatgtggt tcttgaacac gggtcatgcg tgaccacgat 1021 ggctaaggat aagccgacct tggacatcga actactgaaa accgaggtca caaaccctgc 1081 tgtgctccgc aagctgtgca tcgaggctaa gatttccaac acaactactg atagccgctg 1141 ccccacccaa ggcgaggcga ccctcgttga agagcaggac agcaacttcg tgtgtcgccg 1201 gactttcgtg gaccgcggtT GGGGGAATgg atgcggactt aacggatctg gttccttact 1261 gacttgcgcc aaatttaagt gcgtgactaa gttagagggg aaaatcgttc agtatgagaa 1321 cttaaaatac tcggtgatag ttaccgtgca cacaggcgac cagcatcaag ttgggaacga 1381 aacgacagag cacgggacaa tagcgaccat taccccacag gctccaacga gcgaaattca 1441 gctgacagac tacggtgcac tcaccctgga ctgtagccca cggaccgggc tagactttaa 1501 cgagatggtg ctcctgacta tgaaggaaaa gtcatggttg gtgcacaagc agtggttcct 1561 tgatcttcca ttgccctgga cctctggcgc ttcgacctca caagagactt ggaacaggca 1621 ggacttgctc gtgacattca aaacggctca cgctaaaaag caagaggtcg tggttctggg 1681 gagtcaggaa ggcgctatgc ataccgcgtt aacaggggct acagagatcc agaccagtgg 1741 aacaaccact attttcgccg ggcatcttaa gtgtaggctg aagatggata agttgaccct 1801 gaaaggtatg tcatatgtga tgtgcaccgg tagtttcaaa ctggagaaag aagtggccga 1861 aacccagcat ggaacagtac tggtgcaagt caaatatgag ggcaccgatg caccatgtaa 1921 aatacccttc agcgcacaag acgagaaggg agttacccag aacggtaggc tgataacagc 1981 caatccaatc gtcaccgata aggagaaacc agtaaacatc gaaaccgagc cacccttcgg 2041 cgaaagctac atcgtggtcg gcgctggcga gaaagcactt aagctgagct ggtttaagaa 2101 aggtagcacg ggcggcggca gccatcatca ccatcatcac tgagctagCT TGACTGACTG 2161 AGATACAGCG TACCTTCAGC TCACAGACAT GATAAGATAC ATTGATGAGT TTGGACAAAC 2221 CACAACTAGA ATGCAGTGAA AAAAATGCTT TATTTGTGAA ATTTGTGATG CTATTGCTTT 2281 ATTTGTAACC ATTATAAGCT GCAATAAACA AGTTAACAAC AACAATTGCA TTCATTTTAT 2341 GTTTCAGGTT CAGGGGGAGG TGTGGGAGGT TTTTTAAAGC AAGTAAAACC TCTACAAATG 2401 TGGTATTGGC CCATCTCTAT CGGTATCGTA GCATAACCCC TTGGGGCCTC TAAACGGGTC 2461 TTGAGGGGTT TTTTGTGCCC CTCGGGCCGG ATTGCTATCT ACCGGCATTG GCGCAGAAAA 2521 AAATGCCTGA TGCGACGCTG CGCGTCTTAT ACTCCCACAT ATGCCAGATT CAGCAACGGA 2581 TACGGCTTCC CCAACTTGCC CACTTCCATA CGTGTCCTCC TTACCAGAAA TTTATCCTTA 2641 AGGTCGTCAG CTATCCTGCA GGCGATCTCT CGATTTCGAT CAAGACATTC CTTTAATGGT 2701 CTTTTCTGGA CACCACTAGG GGTCAGAAGT AGTTCATCAA ACTTTCTTCC CTCCCTAATC 2761 TCATTGGTTA CCTTGGGCTA TCGAAACTTA ATTAACCAGT CAAGTCAGCT ACTTGGCGAG 2821 ATCGACTTGT CTGGGTTTCG ACTACGCTCA GAATTGCGTC AGTCAAGTTC GATCTGGTCC 2881 TTGCTATTGC ACCCGTTCTC CGATTACGAG TTTCATTTAA ATCATGTGAG CAAAAGGCCA 2941 GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC 3001 CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT 3061 ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT 3121 GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCATAG 3181 CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA 3241 CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA 3301 CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC 3361 GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG 3421 AAGAACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG 3481 TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA 3541 GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC 3601 TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG 3661 GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT AAAGTATATA 3721 TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA TCTCAGCGAT 3781 CTGTCTATTT CGTTCATCCA TAGTTGCATT TAAATTTCCG AACTCTCCAA GGCCCTCGTC 3841 GGAAAATCTT CAAACCTTTC GTCCGATCCA TCTTGCAGGC TACCTCTCGA ACGAACTATC 3901 GCAAGTCTCT TGGCCGGCCT TGCGCCTTGG CTATTGCTTG GCAGCGCCTA TCGCCAGGTA 3961 TTACTCCAAT CCCGAATATC CGAGATCGGG ATCACCCGAG AGAAGTTCAA CCTACATCCT 4021 CAATCCCGAT CTATCCGAGA TCCGAGGAAT ATCGAAATCG GGGCGCGCCT GGTGTACCGA 4081 GAACGATCCT CTCAGTGCGA GTCTCGACGA TCCATATCGT TGCTTGGCAG TCAGCCAGTC 4141 GGAATCCAGC TTGGGACCCA GGAAGTCCAA TCGTCAGATA TTGTACTCAA GCCTGGTCAC 4201 GGCAGCGTAC CGATCTGTTT AAACCTAGAT ATTGATAGTC TGATCGGTCA ACGTATAATC 4261 GAGTCCTAGC TTTTGCAAAC ATCTATCAAG AGACAGGATC AGCAGGAGGC TTTCGCATGA 4321 GTATTCAACA TTTCCGTGTC GCCCTTATTC CCTTTTTTGC GGCATTTTGC CTTCCTGTTT 4381 TTGCTCACCC AGAAACGCTG GTGAAAGTAA AAGATGCTGA AGATCAGTTG GGTGCGCGAG 4441 TGGGTTACAT CGAACTGGAT CTCAACAGCG GTAAGATCCT TGAGAGTTTT CGCCCCGAAG 4501 AACGCTTTCC AATGATGAGC ACTTTTAAAG TTCTGCTATG TGGCGCGGTA TTATCCCGTA 4561 TTGACGCCGG GCAAGAGCAA CTCGGTCGCC GCATACACTA TTCTCAGAAT GACTTGGTTG 4621 AGTATTCACC AGTCACAGAA AAGCATCTTA CGGATGGCAT GACAGTAAGA GAATTATGCA 4681 GTGCTGCCAT AACCATGAGT GATAACACTG CGGCCAACTT ACTTCTGACA ACGATTGGAG 4741 GACCGAAGGA GCTAACCGCT TTTTTGCACA ACATGGGGGA TCATGTAACT CGCCTTGATC 4801 GTTGGGAACC GGAGCTGAAT GAAGCCATAC CAAACGACGA GCGTGACACC ACGATGCCTG 4861 TAGCAATGGC AACAACCTTG CGTAAACTAT TAACTGGCGA ACTACTTACT CTAGCTTCCC 4921 GGCAACAGTT GATAGACTGG ATGGAGGCGG ATAAAGTTGC AGGACCACTT CTGCGCTCGG 4981 CCCTTCCGGC TGGCTGGTTT ATTGCTGATA AATCTGGAGC CGGTGAGCGT GGGTCTCGCG 5041 GTATCATTGC AGCACTGGGG CCAGATGGTA AGCCCTCCCG TATCGTAGTT ATCTACACGA 5101 CGGGGAGTCA GGCAACTATG GATGAACGAA ATAGACAGAT CGCTGAGATA GGTGCCTCAC 5161 TGATTAAGCA TTGGTAACCG ATTCTAGGTG CATTGGCGCA GAAAAAAATG CCTGATGCGA 5221 CGCTGCGCGT CTTATACTCC CACATATGCC AGATTCAGCA ACGGATACGG CTTCCCCAAC 5281 TTGCCCACTT CCATACGTGT CCTCCTTACC AGAAATTTAT CCTTAAGATC CCGAATCGTT 5341 TAAACTCGAC TCTGGCTCTA TCGAATCTCC GTCGTTTCGA GCTTACGCGA ACAGCCGTGG 5401 CGCTCATTTG CTCGTCGGGC ATCGAATCTC GTCAGCTATC GTCAGCTTAC CTTTTTGGCA 5461 // pCR029 (SEQ ID NO: 21) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgccA TCAGGTGCAT TGGAGTCAGC AACAGGGACT TCGTCGAAGG 961 CATGTCCGGC GGCACCTGGG TGGATGTGGT GCTCGAACAC GGCGGATGCG TGACCGTCAT 1021 GGCCCAGGAC AAGCCTACCG TCGATATTGA GCTGGTGACC ACCACAGTGA GCAACATGGC 1081 CGAAGTGAGA AGCTACTGCT ATGAGGCCTC CATCAGCGAT ATGGCTTCCG ATTCCAGATG 1141 CCCCACACAG GGAGAGGCTT ATCTGGACAA ACAGTCCGAC ACCCAGTACG TCTGCAAAAG 1201 AACCCTGGTG GACAGAGGCT GGGGAAACGG ATGCGGCaac cacaccAAAG GCAGCCTCGT 1261 GACATGTGCC AAGTTCGCCT GCAGCAAAAA GATGACCGGC AAGTCCATCC AGCCCGAGAA 1321 CCTGGAATAC AGGATCATGC TGTCCGTGCA TGGATCCCAG CACTCCGGCA TGATCGTCAA 1381 CGATACCGGC CACGAGACCG ACGAGAACAG GGCTAAAGTG GAGATCACCC CCAACAGCCC 1441 TAGAGCCGAA GCTACACTGG GCGGCTTCGG AAGCCTGGGC CTGGATTGCG AACCCAGGAC 1501 CGGCCTGGAT TTCAGCGACC TGTATTACCT GACCATGAAC AATAAGCACT GGCTGGTGCA 1561 CAAGGAATGG TTCCACGACA TCCCCCTGCC TTGGCATGCT GGCGCCGATA CCGGCACACC 1621 TCACTGGAAC AATAAGGAAG CCCTGGTCGA GTTTAAGGAC GCCCACGCCA AAAGACAGAC 1681 CGTGGTGGTG CTGGGAAGCC AGGAGGGAGC TGTCCACACA GCCCTGGCCG GAGCTCTGGA 1741 AGCCGAGATG GATGGCGCCA AGGGCAGGCT GAGCTCCGGC CACCTGAAAT GCAGGCTCAA 1801 GATGGACAAG CTGAGGCTGA AGGGCGTGAG CTACAGCCTG TGCACCGCCG CTTTCACCTT 1861 TACCAAGATC CCTGCCGAGA CACTGCACGG CACCGTCACC GTGGAGGTGC AATACGCCGG 1921 AACCGATGGA CCTTGCAAAG TGCCTGCCCA GATGGCTGTG GATATGCAGA CCCTCACACC 1981 CGTCGGCAGG CTGATCACCG CCAATCCCGT CATTACCGAG TCCACCGAGA ACAGCAAGAT 2041 GATGCTcGAG CTCGATCCCC CCTTTGGCGA CAGCTACATT GTGATCGGCG TGGGCGAGAA 2101 GAAGATCACC CACCATTGGC ACAGAAGCGG CTCCACAggg ggtagcggtg gtagcggagg 2161 tagccatcac caccatcacc actgagctag CTTGACTGAC TGAGATACAG CGTACCTTCA 2221 GCTCACAGAC ATGATAAGAT ACATTGATGA GTTTGGACAA ACCACAACTA GAATGCAGTG 2281 AAAAAAATGC TTTATTTGTG AAATTTGTGA TGCTATTGCT TTATTTGTAA CCATTATAAG 2341 CTGCAATAAA CAAGTTAACA ACAACAATTG CATTCATTTT ATGTTTCAGG TTCAGGGGGA 2401 GGTGTGGGAG GTTTTTTAAA GCAAGTAAAA CCTCTACAAA TGTGGTATTG GCCCATCTCT 2461 ATCGGTATCG TAGCATAACC CCTTGGGGCC TCTAAACGGG TCTTGAGGGG TTTTTTGTGC 2521 CCCTCGGGCC GGATTGCTAT CTACCGGCAT TGGCGCAGAA AAAAATGCCT GATGCGACGC 2581 TGCGCGTCTT ATACTCCCAC ATATGCCAGA TTCAGCAACG GATACGGCTT CCCCAACTTG 2641 CCCACTTCCA TACGTGTCCT CCTTACCAGA AATTTATCCT TAAGGTCGTC AGCTATCCTG 2701 CAGGCGATCT CTCGATTTCG ATCAAGACAT TCCTTTAATG GTCTTTTCTG GACACCACTA 2761 GGGGTCAGAA GTAGTTCATC AAACTTTCTT CCCTCCCTAA TCTCATTGGT TACCTTGGGC 2821 TATCGAAACT TAATTAACCA GTCAAGTCAG CTACTTGGCG AGATCGACTT GTCTGGGTTT 2881 CGACTACGCT CAGAATTGCG TCAGTCAAGT TCGATCTGGT CCTTGCTATT GCACCCGTTC 2941 TCCGATTACG AGTTTCATTT AAATCATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG 3001 TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA 3061 AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT 3121 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT 3181 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT 3241 CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC 3301 CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 3361 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC 3421 TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT 3481 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA 3541 ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 3601 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA 3661 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT 3721 TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA 3781 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 3841 CATAGTTGCA TTTAAATTTC CGAACTCTCC AAGGCCCTCG TCGGAAAATC TTCAAACCTT 3901 TCGTCCGATC CATCTTGCAG GCTACCTCTC GAACGAACTA TCGCAAGTCT CTTGGCCGGC 3961 CTTGCGCCTT GGCTATTGCT TGGCAGCGCC TATCGCCAGG TATTACTCCA ATCCCGAATA 4021 TCCGAGATCG GGATCACCCG AGAGAAGTTC AACCTACATC CTCAATCCCG ATCTATCCGA 4081 GATCCGAGGA ATATCGAAAT CGGGGCGCGC CTGGTGTACC GAGAACGATC CTCTCAGTGC 4141 GAGTCTCGAC GATCCATATC GTTGCTTGGC AGTCAGCCAG TCGGAATCCA GCTTGGGACC 4201 CAGGAAGTCC AATCGTCAGA TATTGTACTC AAGCCTGGTC ACGGCAGCGT ACCGATCTGT 4261 TTAAACCTAG ATATTGATAG TCTGATCGGT CAACGTATAA TCGAGTCCTA GCTTTTGCAA 4321 ACATCTATCA AGAGACAGGA TCAGCAGGAG GCTTTCGCAT GAGTATTCAA CATTTCCGTG 4381 TCGCCCTTAT TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC CCAGAAACGC 4441 TGGTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCGCG AGTGGGTTAC ATCGAACTGG 4501 ATCTCAACAG CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGCTTT CCAATGATGA 4561 GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG TATTGACGCC GGGCAAGAGC 4621 AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT TGAGTATTCA CCAGTCACAG 4681 AAAAGCATCT TACGGATGGC ATGACAGTAA GAGAATTATG CAGTGCTGCC ATAACCATGA 4741 GTGATAACAC TGCGGCCAAC TTACTTCTGA CAACGATTGG AGGACCGAAG GAGCTAACCG 4801 CTTTTTTGCA CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA 4861 ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGTAGCAATG GCAACAACCT 4921 TGCGTAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC CCGGCAACAG TTGATAGACT 4981 GGATGGAGGC GGATAAAGTT GCAGGACCAC TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT 5041 TTATTGCTGA TAAATCTGGA GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG 5101 GGCCAGATGG TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA 5161 TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG CATTGGTAAC 5221 CGATTCTAGG TGCATTGGCG CAGAAAAAAA TGCCTGATGC GACGCTGCGC GTCTTATACT 5281 CCCACATATG CCAGATTCAG CAACGGATAC GGCTTCCCCA ACTTGCCCAC TTCCATACGT 5341 GTCCTCCTTA CCAGAAATTT ATCCTTAAGA TCCCGAATCG TTTAAACTCG ACTCTGGCTC 5401 TATCGAATCT CCGTCGTTTC GAGCTTACGC GAACAGCCGT GGCGCTCATT TGCTCGTCGG 5461 GCATCGAATC TCGTCAGCTA TCGTCAGCTT ACCTTTTTGG // pCR030 (SEQ ID NO: 22) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgccA TCAGGTGCAT TGGAGTCAGC AACAGGGACT TCGTCGAAGG 961 CATGTCCGGC GGCACCTGGG TGGATGTGGT GCTCGAACAC GGCGGATGCG TGACCGTCAT 1021 GGCCCAGGAC AAGCCTACCG TCGATATTGA GCTGGTGACC ACCACAGTGA GCAACATGGC 1081 CGAAGTGAGA AGCTACTGCT ATGAGGCCTC CATCAGCGAT ATGGCTTCCG ATTCCAGATG 1141 CCCCACACAG GGAGAGGCTT ATCTGGACAA ACAGTCCGAC ACCCAGTACG TCTGCAAAAG 1201 AACCCTGGTG GACAGAGGCa acggatccGG ATGCGGCCTG TTCGGCAAAG GCAGCCTCGT 1261 GACATGTGCC AAGTTCGCCT GCAGCAAAAA GATGACCGGC AAGTCCATCC AGCCCGAGAA 1321 CCTGGAATAC AGGATCATGC TGTCCGTGCA TGGATCCCAG CACTCCGGCA TGATCGTCAA 1381 CGATACCGGC CACGAGACCG ACGAGAACAG GGCTAAAGTG GAGATCACCC CCAACAGCCC 1441 TAGAGCCGAA GCTACACTGG GCGGCTTCGG AAGCCTGGGC CTGGATTGCG AACCCAGGAC 1501 CGGCCTGGAT TTCAGCGACC TGTATTACCT GACCATGAAC AATAAGCACT GGCTGGTGCA 1561 CAAGGAATGG TTCCACGACA TCCCCCTGCC TTGGCATGCT GGCGCCGATA CCGGCACACC 1621 TCACTGGAAC AATAAGGAAG CCCTGGTCGA GTTTAAGGAC GCCCACGCCA AAAGACAGAC 1681 CGTGGTGGTG CTGGGAAGCC AGGAGGGAGC TGTCCACACA GCCCTGGCCG GAGCTCTGGA 1741 AGCCGAGATG GATGGCGCCA AGGGCAGGCT GAGCTCCGGC CACCTGAAAT GCAGGCTCAA 1801 GATGGACAAG CTGAGGCTGA AGGGCGTGAG CTACAGCCTG TGCACCGCCG CTTTCACCTT 1861 TACCAAGATC CCTGCCGAGA CACTGCACGG CACCGTCACC GTGGAGGTGC AATACGCCGG 1921 AACCGATGGA CCTTGCAAAG TGCCTGCCCA GATGGCTGTG GATATGCAGA CCCTCACACC 1981 CGTCGGCAGG CTGATCACCG CCAATCCCGT CATTACCGAG TCCACCGAGA ACAGCAAGAT 2041 GATGCTcGAG CTCGATCCCC CCTTTGGCGA CAGCTACATT GTGATCGGCG TGGGCGAGAA 2101 GAAGATCACC CACCATTGGC ACAGAAGCGG CTCCACAggg ggtagcggtg gtagcggagg 2161 tagccatcac caccatcacc actgagctag CTTGACTGAC TGAGATACAG CGTACCTTCA 2221 GCTCACAGAC ATGATAAGAT ACATTGATGA GTTTGGACAA ACCACAACTA GAATGCAGTG 2281 AAAAAAATGC TTTATTTGTG AAATTTGTGA TGCTATTGCT TTATTTGTAA CCATTATAAG 2341 CTGCAATAAA CAAGTTAACA ACAACAATTG CATTCATTTT ATGTTTCAGG TTCAGGGGGA 2401 GGTGTGGGAG GTTTTTTAAA GCAAGTAAAA CCTCTACAAA TGTGGTATTG GCCCATCTCT 2461 ATCGGTATCG TAGCATAACC CCTTGGGGCC TCTAAACGGG TCTTGAGGGG TTTTTTGTGC 2521 CCCTCGGGCC GGATTGCTAT CTACCGGCAT TGGCGCAGAA AAAAATGCCT GATGCGACGC 2581 TGCGCGTCTT ATACTCCCAC ATATGCCAGA TTCAGCAACG GATACGGCTT CCCCAACTTG 2641 CCCACTTCCA TACGTGTCCT CCTTACCAGA AATTTATCCT TAAGGTCGTC AGCTATCCTG 2701 CAGGCGATCT CTCGATTTCG ATCAAGACAT TCCTTTAATG GTCTTTTCTG GACACCACTA 2761 GGGGTCAGAA GTAGTTCATC AAACTTTCTT CCCTCCCTAA TCTCATTGGT TACCTTGGGC 2821 TATCGAAACT TAATTAACCA GTCAAGTCAG CTACTTGGCG AGATCGACTT GTCTGGGTTT 2881 CGACTACGCT CAGAATTGCG TCAGTCAAGT TCGATCTGGT CCTTGCTATT GCACCCGTTC 2941 TCCGATTACG AGTTTCATTT AAATCATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG 3001 TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA 3061 AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT 3121 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT 3181 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT 3241 CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC 3301 CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 3361 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC 3421 TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT 3481 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA 3541 ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 3601 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA 3661 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT 3721 TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA 3781 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 3841 CATAGTTGCA TTTAAATTTC CGAACTCTCC AAGGCCCTCG TCGGAAAATC TTCAAACCTT 3901 TCGTCCGATC CATCTTGCAG GCTACCTCTC GAACGAACTA TCGCAAGTCT CTTGGCCGGC 3961 CTTGCGCCTT GGCTATTGCT TGGCAGCGCC TATCGCCAGG TATTACTCCA ATCCCGAATA 4021 TCCGAGATCG GGATCACCCG AGAGAAGTTC AACCTACATC CTCAATCCCG ATCTATCCGA 4081 GATCCGAGGA ATATCGAAAT CGGGGCGCGC CTGGTGTACC GAGAACGATC CTCTCAGTGC 4141 GAGTCTCGAC GATCCATATC GTTGCTTGGC AGTCAGCCAG TCGGAATCCA GCTTGGGACC 4201 CAGGAAGTCC AATCGTCAGA TATTGTACTC AAGCCTGGTC ACGGCAGCGT ACCGATCTGT 4261 TTAAACCTAG ATATTGATAG TCTGATCGGT CAACGTATAA TCGAGTCCTA GCTTTTGCAA 4321 ACATCTATCA AGAGACAGGA TCAGCAGGAG GCTTTCGCAT GAGTATTCAA CATTTCCGTG 4381 TCGCCCTTAT TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC CCAGAAACGC 4441 TGGTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCGCG AGTGGGTTAC ATCGAACTGG 4501 ATCTCAACAG CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGCTTT CCAATGATGA 4561 GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG TATTGACGCC GGGCAAGAGC 4621 AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT TGAGTATTCA CCAGTCACAG 4681 AAAAGCATCT TACGGATGGC ATGACAGTAA GAGAATTATG CAGTGCTGCC ATAACCATGA 4741 GTGATAACAC TGCGGCCAAC TTACTTCTGA CAACGATTGG AGGACCGAAG GAGCTAACCG 4801 CTTTTTTGCA CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA 4861 ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGTAGCAATG GCAACAACCT 4921 TGCGTAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC CCGGCAACAG TTGATAGACT 4981 GGATGGAGGC GGATAAAGTT GCAGGACCAC TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT 5041 TTATTGCTGA TAAATCTGGA GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG 5101 GGCCAGATGG TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA 5161 TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG CATTGGTAAC 5221 CGATTCTAGG TGCATTGGCG CAGAAAAAAA TGCCTGATGC GACGCTGCGC GTCTTATACT 5281 CCCACATATG CCAGATTCAG CAACGGATAC GGCTTCCCCA ACTTGCCCAC TTCCATACGT 5341 GTCCTCCTTA CCAGAAATTT ATCCTTAAGA TCCCGAATCG TTTAAACTCG ACTCTGGCTC 5401 TATCGAATCT CCGTCGTTTC GAGCTTACGC GAACAGCCGT GGCGCTCATT TGCTCGTCGG 5461 GCATCGAATC TCGTCAGCTA TCGTCAGCTT ACCTTTTTGG CA // pCR031 (SEQ ID NO: 23) ORIGIN 1 GCGATCGCGG CTCCCGACAT CTTGGACCAT TAGCTCCACA GGTATCTTCT TCCCTCTAGT 61 GGTCATAACA GCAGCTTCAG CTACCTCTCA ATTCAAAAAA CCCCTCAAGA CCCGTTTAGA 121 GGCCCCAAGG GGTTATGCTA TCAATCGTTG CGTTACACAC ACAAAAAACC AACACACATC 181 CATCTTCGAT GGATAGCGAT TTTATTATCT AACTGCTGAT CGAGTGTAGC CAGATCTAGT 241 AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT ACATAACTTA 301 CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG TCAATAATGA 361 CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG GTGGAGTATT 421 TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT ACGCCCCCTA 481 TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG ACCTTATGGG 541 ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG CTGATGCGGT 601 TTTGGCAGTA CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC 661 ACCCCATTGA CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT 721 GTCGTAACAA CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT 781 ATATAAGCAG AGCTGGTTTA GTGAACCGTC AGATCAGATC TTTGTCGATC CTACCATCCA 841 CTCGACACAC CCGCCAGCgg ccgccaccat gaaggccaat ctactggtgt tgctgtgtgc 901 ccttgcggcg gcagatgccA TCAGGTGCAT TGGAGTCAGC AACAGGGACT TCGTCGAAGG 961 CATGTCCGGC GGCACCTGGG TGGATGTGGT GCTCGAACAC GGCGGATGCG TGACCGTCAT 1021 GGCCCAGGAC AAGCCTACCG TCGATATTGA GCTGGTGACC ACCACAGTGA GCAACATGGC 1081 CGAAGTGAGA AGCTACTGCT ATGAGGCCTC CATCAGCGAT ATGGCTTCCG ATTCCAGATG 1141 CCCCACACAG GGAGAGGCTT ATCTGGACAA ACAGTCCGAC ACCCAGTACG TCTGCAAAAG 1201 AACCCTGGTG GACAGAGGCT GGGGAAACGG ATGCGGCCTG aacggatccG GCAGCCTCGT 1261 GACATGTGCC AAGTTCGCCT GCAGCAAAAA GATGACCGGC AAGTCCATCC AGCCCGAGAA 1321 CCTGGAATAC AGGATCATGC TGTCCGTGCA TGGATCCCAG CACTCCGGCA TGATCGTCAA 1381 CGATACCGGC CACGAGACCG ACGAGAACAG GGCTAAAGTG GAGATCACCC CCAACAGCCC 1441 TAGAGCCGAA GCTACACTGG GCGGCTTCGG AAGCCTGGGC CTGGATTGCG AACCCAGGAC 1501 CGGCCTGGAT TTCAGCGACC TGTATTACCT GACCATGAAC AATAAGCACT GGCTGGTGCA 1561 CAAGGAATGG TTCCACGACA TCCCCCTGCC TTGGCATGCT GGCGCCGATA CCGGCACACC 1621 TCACTGGAAC AATAAGGAAG CCCTGGTCGA GTTTAAGGAC GCCCACGCCA AAAGACAGAC 1681 CGTGGTGGTG CTGGGAAGCC AGGAGGGAGC TGTCCACACA GCCCTGGCCG GAGCTCTGGA 1741 AGCCGAGATG GATGGCGCCA AGGGCAGGCT GAGCTCCGGC CACCTGAAAT GCAGGCTCAA 1801 GATGGACAAG CTGAGGCTGA AGGGCGTGAG CTACAGCCTG TGCACCGCCG CTTTCACCTT 1861 TACCAAGATC CCTGCCGAGA CACTGCACGG CACCGTCACC GTGGAGGTGC AATACGCCGG 1921 AACCGATGGA CCTTGCAAAG TGCCTGCCCA GATGGCTGTG GATATGCAGA CCCTCACACC 1981 CGTCGGCAGG CTGATCACCG CCAATCCCGT CATTACCGAG TCCACCGAGA ACAGCAAGAT 2041 GATGCTcGAG CTCGATCCCC CCTTTGGCGA CAGCTACATT GTGATCGGCG TGGGCGAGAA 2101 GAAGATCACC CACCATTGGC ACAGAAGCGG CTCCACAggg ggtagcggtg gtagcggagg 2161 tagccatcac caccatcacc actgagctag CTTGACTGAC TGAGATACAG CGTACCTTCA 2221 GCTCACAGAC ATGATAAGAT ACATTGATGA GTTTGGACAA ACCACAACTA GAATGCAGTG 2281 AAAAAAATGC TTTATTTGTG AAATTTGTGA TGCTATTGCT TTATTTGTAA CCATTATAAG 2341 CTGCAATAAA CAAGTTAACA ACAACAATTG CATTCATTTT ATGTTTCAGG TTCAGGGGGA 2401 GGTGTGGGAG GTTTTTTAAA GCAAGTAAAA CCTCTACAAA TGTGGTATTG GCCCATCTCT 2461 ATCGGTATCG TAGCATAACC CCTTGGGGCC TCTAAACGGG TCTTGAGGGG TTTTTTGTGC 2521 CCCTCGGGCC GGATTGCTAT CTACCGGCAT TGGCGCAGAA AAAAATGCCT GATGCGACGC 2581 TGCGCGTCTT ATACTCCCAC ATATGCCAGA TTCAGCAACG GATACGGCTT CCCCAACTTG 2641 CCCACTTCCA TACGTGTCCT CCTTACCAGA AATTTATCCT TAAGGTCGTC AGCTATCCTG 2701 CAGGCGATCT CTCGATTTCG ATCAAGACAT TCCTTTAATG GTCTTTTCTG GACACCACTA 2761 GGGGTCAGAA GTAGTTCATC AAACTTTCTT CCCTCCCTAA TCTCATTGGT TACCTTGGGC 2821 TATCGAAACT TAATTAACCA GTCAAGTCAG CTACTTGGCG AGATCGACTT GTCTGGGTTT 2881 CGACTACGCT CAGAATTGCG TCAGTCAAGT TCGATCTGGT CCTTGCTATT GCACCCGTTC 2941 TCCGATTACG AGTTTCATTT AAATCATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG 3001 TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA 3061 AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT 3121 TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT 3181 GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAT AGCTCACGCT GTAGGTATCT 3241 CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC 3301 CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT 3361 ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC 3421 TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT AGAAGAACAG TATTTGGTAT 3481 CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA 3541 ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA 3601 AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA 3661 AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT 3721 TTTAAATTAA AAATGAAGTT TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA 3781 CAGTTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC 3841 CATAGTTGCA TTTAAATTTC CGAACTCTCC AAGGCCCTCG TCGGAAAATC TTCAAACCTT 3901 TCGTCCGATC CATCTTGCAG GCTACCTCTC GAACGAACTA TCGCAAGTCT CTTGGCCGGC 3961 CTTGCGCCTT GGCTATTGCT TGGCAGCGCC TATCGCCAGG TATTACTCCA ATCCCGAATA 4021 TCCGAGATCG GGATCACCCG AGAGAAGTTC AACCTACATC CTCAATCCCG ATCTATCCGA 4081 GATCCGAGGA ATATCGAAAT CGGGGCGCGC CTGGTGTACC GAGAACGATC CTCTCAGTGC 4141 GAGTCTCGAC GATCCATATC GTTGCTTGGC AGTCAGCCAG TCGGAATCCA GCTTGGGACC 4201 CAGGAAGTCC AATCGTCAGA TATTGTACTC AAGCCTGGTC ACGGCAGCGT ACCGATCTGT 4261 TTAAACCTAG ATATTGATAG TCTGATCGGT CAACGTATAA TCGAGTCCTA GCTTTTGCAA 4321 ACATCTATCA AGAGACAGGA TCAGCAGGAG GCTTTCGCAT GAGTATTCAA CATTTCCGTG 4381 TCGCCCTTAT TCCCTTTTTT GCGGCATTTT GCCTTCCTGT TTTTGCTCAC CCAGAAACGC 4441 TGGTGAAAGT AAAAGATGCT GAAGATCAGT TGGGTGCGCG AGTGGGTTAC ATCGAACTGG 4501 ATCTCAACAG CGGTAAGATC CTTGAGAGTT TTCGCCCCGA AGAACGCTTT CCAATGATGA 4561 GCACTTTTAA AGTTCTGCTA TGTGGCGCGG TATTATCCCG TATTGACGCC GGGCAAGAGC 4621 AACTCGGTCG CCGCATACAC TATTCTCAGA ATGACTTGGT TGAGTATTCA CCAGTCACAG 4681 AAAAGCATCT TACGGATGGC ATGACAGTAA GAGAATTATG CAGTGCTGCC ATAACCATGA 4741 GTGATAACAC TGCGGCCAAC TTACTTCTGA CAACGATTGG AGGACCGAAG GAGCTAACCG 4801 CTTTTTTGCA CAACATGGGG GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA 4861 ATGAAGCCAT ACCAAACGAC GAGCGTGACA CCACGATGCC TGTAGCAATG GCAACAACCT 4921 TGCGTAAACT ATTAACTGGC GAACTACTTA CTCTAGCTTC CCGGCAACAG TTGATAGACT 4981 GGATGGAGGC GGATAAAGTT GCAGGACCAC TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT 5041 TTATTGCTGA TAAATCTGGA GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG 5101 GGCCAGATGG TAAGCCCTCC CGTATCGTAG TTATCTACAC GACGGGGAGT CAGGCAACTA 5161 TGGATGAACG AAATAGACAG ATCGCTGAGA TAGGTGCCTC ACTGATTAAG CATTGGTAAC 5221 CGATTCTAGG TGCATTGGCG CAGAAAAAAA TGCCTGATGC GACGCTGCGC GTCTTATACT 5281 CCCACATATG CCAGATTCAG CAACGGATAC GGCTTCCCCA ACTTGCCCAC TTCCATACGT 5341 GTCCTCCTTA CCAGAAATTT ATCCTTAAGA TCCCGAATCG TTTAAACTCG ACTCTGGCTC 5401 TATCGAATCT CCGTCGTTTC GAGCTTACGC GAACAGCCGT GGCGCTCATT TGCTCGTCGG 5461 GCATCGAATC TCGTCAGCTA TCGTCAGCTT ACCTTTTTGG CA // Hyperglycosylated exodomain D1 (from pCR021) (SEQ ID NO: 24) Hyperglycosylated exodomain D2 (from pCR022) (SEQ ID NO: 25) Hyperglycosylated exodomain D3 (from pCR023) (SEQ ID NO: 26) Hyperglycosylated exodomain D4 (from pCR024) (SEQ ID NO: 27) Hyperglycosylated exodomain Zika(from pCR028) (SEQ ID NO: 28) SEQ ID NO: 24 >DENVl_Eexo = pCR021 MRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCP TQGEATLVEEQDSNFVCRRTFVDRGNGSGCGLNGSGSLLTCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQ VGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMKEKSWLVHKQWFLDLPLPWTSGAS TSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGMSYV MCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSAQDEKGVTQNGRLITANPIVTDKEKPVNIETEPPFGE SYIVVGAGEKALKLSWFKKGSTGGGSHHHHHH SEQ ID NO: 25 >DENV2_Eexo = pCR022 MRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCP TQGEPSLNEEQDKRFVCKHSMVDRGNGSGCGLNGSGGIVTCAMFTCKKNMEGKVVQPENLEYTIVITPHSGEEHA VGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGAD TQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYS MCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGD SYIIIGVEPGQLKLNWFKKGSSGGGSHHHHHH SEQ ID NO: 26 >DENV3_Eexo = pCR023 MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCP TQGEAVLPEEQDQNYVCKHTYVDRGNGSGCGLNGSGSLVTCAKFQCLEPIEGKVVQYENLKYTVIITVHTGDQHQ VGNETQGVTAEITPQASTTEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWASGATTE TPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMC TNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESN IVIGIGDNALKINWYKKGSSGGGSHHHHHH SEQ ID NO: 27 >DENV4_Eexo = pCR024 MRCVGVGNRDEVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASISNITTATRCP TQGEPYLKEEQDQQYICRRDVVDRGNGSGCGLNGSGGVVTCAKFSCSGKITGNLVQIENLEYTVVVTVHNGDTHA VGNDTSNHGVTAMITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGAD TSEVHWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYT MCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGD SYIVIGVGNSALTLHWFRKGSSGGGSHHHHHH SEQ ID NO: 28 >ZIKV_Eexo = pCR025 IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCP TQGEAYLDKQSDTQYVCKRTLVDRGNGSGCGLNGSGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSG MIVNDTGHETDENRAKVEITPNSPRAEATLGGEGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPW HAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRL KGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMM LELDPPFGDSYIVIGVGEKKITHHWHRSGSTGGSGGSGGSHHHHHH SEQ ID NO: 29 >DENVl_Eexo 2.1 (single sequon W101N;N103S) [= insert for pCR026 plasmid] MRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCP TQGEATLVEEQDSNFVCRRTFVDRGNGSGCGLFGKGSLLTCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQ VGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMKEKSWLVHKQWFLDLPLPWTSGAS TSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGMSYV MCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSAQDEKGVTQNGRLITANPIVTDKEKPVNIETEPPFGE SYIVVGAGEKALKLSWFKKGSTGGGSHHHHHH SEQ ID NO: 30 >DENVl_Eexo 2.2 (single sequon F108N;K110S) [= insert for pCR027 plasmid] MRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCP TQGEATLVEEQDSNFVCRRTFVDRGWGNGCGLNGSGSLLTCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQ VGNETTEHGTIATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMKEKSWLVHKQWFLDLPLPWTSGAS TSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLTLKGMSYV MCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSAQDEKGVTQNGRLITANPIVTDKEKPVNIETEPPFGE SYIVVGAGEKALKLSWFKKGSTGGGSHHHHHH SEQ ID NO: 31 >ZIKV_Eexo 2.1 (single sequon G100N;W101H;G102T) [= insert for pCR028 plasmid] IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCP TQGEAYLDKQSDTQYVCKRTLVDRNHTNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSG MIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPW HAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRL KGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMM LELDPPFGDSYIVIGVGEKKITHHWHRSGSTGGSGGSGGSHHHHHH SEQ ID NO: 32 >ZIKV_Eexo 2.2 (single sequon L107N;F108H;G109T) [= insert for pCR029 plasmid] IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCP TQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGNHTKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSG MIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPW HAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRL KGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMM LELDPPFGDSYIVIGVGEKKITHHWHRSGSTGGSGGSGGSHHHHHH SEQ ID NO: 33 >ZIKV_Eexo 2.3 (single sequon W101N;N103S) [= insert for pCR030 plasmid] IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCP TQGEAYLDKQSDTQYVCKRTLVDRGNGSGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSG MIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPW HAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRL KGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMM LELDPPFGDSYIVIGVGEKKITHHWHRSGSTGGSGGSGGSHHHHHH SEQ ID NO: 34 >ZIKV_Eexo 2.4 (single sequon F108N;K110S) [= insert for pCR031 plasmid] IRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCP TQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLNGSGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSG MIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPW HAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRL KGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMM LELDPPFGDSYIVIGVGEKKITHHWHRSGSTGGSGGSGGSHHHHHH
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