Patent application title: ENGINEERED VIRAL VECTOR REDUCES INDUCTION OF INFLAMMATORY AND IMMUNE RESPONSES
Inventors:
Ying Kai Chan (Cambridge, MA, US)
George Church (Brookline, MA, US)
George Church (Brookline, MA, US)
Assignees:
President and Fellows of Harvard College
IPC8 Class: AC12N1586FI
USPC Class:
1 1
Class name:
Publication date: 2019-10-17
Patent application number: 20190316151
Abstract:
Modified viral genomes are able to reduce induction of inflammatory and
immune anti-viral responses. This manifests itself in reduced NF-kB
activity, increased viral transduction rates, and increased expression of
transgenes. Viral genomes are modified by incorporating one or more
oligonucleotide sequences which are able to bind to TLR9 but not induce
activation of it. The oligonucleotide sequences may be synthetic,
bacterial, human, or from any other source.Claims:
1. (canceled)
2. A recombinant viral genome comprising four repeated monomers of TTAGGG (SEQ ID NO: 6).
3. The recombinant viral genome of claim 2, wherein the recombinant viral genome is an adeno-associated virus (AAV) genome.
4. The recombinant viral genome of claim 2, wherein the recombinant viral genome is selected from the group consisting of adenovirus, herpes simplex virus, varicella, variola virus, hepatitis B, cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus, Herpes Zoster, Epstein-Barr virus, human herpes virus 7, Kaposi's sarcoma-associated herpesvirus, and human parvovirus B19.
5. The recombinant viral genome of claim 2, wherein the recombinant viral genome is single stranded.
6. The recombinant viral genome of claim 2, wherein the viral genome is packaged in a virion.
7. The recombinant viral genome of claim 2, wherein the recombinant viral genome comprises a therapeutic gene.
8. The recombinant viral genome of claim 2, wherein the recombinant viral genome is a cytotoxic virus.
9-14. (canceled)
15. The recombinant viral genome of claim 2, wherein the recombinant viral genome comprises a non-human gene.
16. The recombinant viral genome of claim 2, wherein the four repeated monomers of TTAGGG (SEQ ID NO: 6) are inserted downstream of or in a 3' untranslated region of the viral genome.
17. The recombinant viral genome of claim 2, wherein the recombinant viral genome is covalently linked by a phosphodiester bond to a nucleic acid sequence comprising the four repeated monomers of TTAGGG (SEQ ID NO: 6).
18. The recombinant viral genome of claim 2 further comprising a detectable marker.
19. The recombinant viral genome of claim 18, wherein expression of the detectable marker is inducible.
20. The recombinant viral genome of claim 2 comprising the nucleotide sequence of SEQ ID NO: 9.
21-42. (canceled)
43. The recombinant viral genome of claim 2 comprising three repeated monomers of the nucleotide sequence of SEQ ID NO: 9.
44. The recombinant viral genome of claim 2 comprising five repeated monomers of the nucleotide sequence of SEQ ID NO: 9.
45. A method of treating a mammal, comprising: administering to the mammal the recombinant viral genome of claim 2.
46-73. (canceled)
74. A method of producing a recombinant viral genome, comprising: inserting into a viral genome four repeated monomers of TTAGGG (SEQ ID NO: 6) or the nucleotide sequence of SEQ ID NO: 9.
75. A nucleic acid vector comprising the nucleotide sequence of SEQ ID NO: 9.
76-90. (canceled)
91. The recombinant viral genome of claim 2, wherein the recombinant viral genome is self-complementary.
92. A recombinant viral genome comprising a sequence that is at least 95% identical to the sequence of SEQ ID NO: 9.
Description:
RELATED APPLICATION DATA
[0001] This application is continuation of PCT/US2017/036525 filed Jun. 8, 2017, which claims priority to U.S. Provisional Application No. 62/347,302 filed on Jun. 8, 2016, each of which is herein incorporated by reference in its entirety for all purposes.
TECHNICAL FIELD OF THE INVENTION
[0003] This invention is related to the area of viral based therapies. In particular, it relates to recombinant viruses.
BACKGROUND OF THE INVENTION
[0004] Gene therapy has immense potential to prevent, treat, and cure multiple human diseases [1]. In 2012, Glybera.TM. (alipogene tiparvovec) became the first viral gene therapy to be approved for use in the western world [2]. Glybera.TM. utilizes adeno-associated virus (AAV) as a viral vector to deliver the human lipoprotein lipase (LPL) gene to muscle cells in patients with LPL deficiency [3]. Many AAV clinical trials are currently underway or being planned in the U.S. and the EU [4-6].
[0005] AAV is a small, non-enveloped virus that packages a single-stranded linear DNA genome that is approximately 5 kb long, and has been adapted for use as a gene transfer vehicle [4]. The coding regions of AAV are flanked by inverted terminal repeats (ITRs), which act as the origins for DNA replication and serve as the primary packaging signal [7, 8]. Both positive and negative strands are packaged into virions equally well and capable of infection [9-11]. In addition, a small deletion in one of the two ITRs allows packaging of self-complementary vectors, in which the genome self-anneals after viral uncoating. This results in more efficient transduction of cells but reduces the coding capacity by half [12, 13]. While AAV is not associated with any human disease, >70% of humans are seropositive for one or most serotypes [14, 15]. Typical routes of administration for AAV vectors include intravenous, intramuscular, subretinal and intracranal injections. AAV gene therapy is most often used to deliver a wild-type gene to treat monogenic diseases, and AAV vectors have been used to transduce cells in the liver, skeletal and cardiac muscle, retina and central nervous system [16-26]. In addition, there is now growing interest in using AAV to deliver CRISPR-Cas9 gene editing or to deliver broadly neutralizing antibodies against infectious diseases such as human immunodeficiency virus (HIV) and influenza A virus [27-32].
[0006] Despite several advances in gene therapy, a key concern is the inflammatory response elicited by the viral vector [33, 34]. This is best illustrated by a highly publicized case in 1999 when Jesse Gelsinger died four days after gene therapy with adenovirus in a clinical trial due to excessive inflammation [35]. While AAV has been shown to elicit much weaker inflammation in comparison to adenoviruses [36, 37], Glybera.TM. therapy still includes twelve weeks of immunosuppression, beginning three days before Glybera.TM. administration [38]. These immunosuppressive and anti-inflammatory drugs (cyclosporine A, mycophenolate mofetil, and methylprednisolone) compromise the patient's immune system during treatment, and all patients still developed neutralizing antibodies against AAV capsid, precluding future re-administration. Furthermore, many AAV gene therapy clinical trials do not utilize immunosuppression prophylactically and only administer corticosteroids upon signs of inflammation or tissue damage, which has been associated with variable therapeutic efficacy [22, 23]. Interestingly, in agreement with their ability to trigger inflammatory and immune responses, AAV vectors have also been developed as vaccine vehicles against infectious diseases and cancer [39-41].
[0007] Inflammation has been implicated as a critical determinant of successful AAV-mediated transgene expression. A study found that following AAV administration in mice, artificially inducing systemic inflammation such as the upregulation of tumor necrosis factor (TNF), led to a decline in transgene expression in the liver [42]. This indicates that immunological tolerance to AAV-encoded transgene can be broken given sufficient inflammatory responses. Another study characterized inflammation in the murine liver following AAV infection and found transient increases in liver enzymes in the serum, and also liver pathology consistent with portal and lobular inflammation [43]. Strikingly, the authors observed that the use of AAVrh.32.33 viruses, which induced higher liver enzymes than other tested AAV serotypes, also led to a decline in transgene expression to below detection levels, again suggesting that inflammatory and immune responses are associated with poor transgene expression.
[0008] In hemophilia B clinical trials, it has been observed that a subset of patients exhibited elevated liver enzymes, and this transient transaminitis was accompanied by declining levels of transgene-encoded factor IX [22, 23]. A tapering course of corticosteroid therapy was used on a patient with transaminitis, which subsequently normalized aminotransferase levels in the serum and rescued further decline of factor IX expression. Overall, these observations are compatible with immune-mediated destruction of AAV-transduced hepatocytes and demonstrate that inflammatory and immune responses triggered by AAV administration are a safety concern and can hamper therapeutic efficacy in humans. Thus, it would be advantageous to develop viral vectors that intrinsically evade eliciting inflammation. Furthermore, instead of systemic immunosuppression with drugs, it would be beneficial to avoid triggering specific immune responses.
[0009] It has been previously shown that the DNA genome of AAV is sensed by Toll-like receptor 9 (TLR9) during AAV's entry into the cell through the endocytic pathway [36, 44]. TLR9 is a pattern recognition receptor (PRR) found on endosomal membranes of immune cells such as B cells, monocytes, macrophages and plasmacytoid dendritic cells, and binds to unmethylated CpG motifs found in the AAV genome [45, 46]. This leads to TLR9 dimerization, which triggers a cascade of signal transduction that activates NF-kB (also known as p52-RelA complex) and induces type I interferons (IFNs). NF-kB in turn drives the transcriptional upregulation of multiple proinflammatory cytokines such as TNF leading to inflammation and immune cell recruitment, while secreted IFNs induce the expression of numerous interferon-stimulated genes (ISGs) and establish an antiviral state. Importantly, genetic ablation of TLR9 in mice abolishes induction of inflammatory cytokines upon AAV treatment in the liver, and also reduces formation of antibodies and T cells against AAV [36]. Thus, TLR9 plays a critical role in stimulating an early inflammatory and innate immune response during AAV infection, which also contributes to priming adaptive immunity. Finally, two other pattern recognition receptors, TLR2 and TLR4, have been implicated in triggering responses to AAV structural proteins [47, 48].
[0010] In the TLR9 field, a commonly used tool to block TLR9 activation in cell culture is short, single-stranded DNA oligonucleotides that bind TLR9 but do not activate it [49, 50]. Several such sequences are known--some synthetic and others derived from organisms--and they often bear no sequence homology [51-59]. Structural studies have revealed how an inhibitory oligonucleotide binds TLR9 tightly but does not trigger TLR9 dimerization, which is required for TLR9 activation and downstream signaling [60]. In addition to binding TLR9 directly to antagonize its activation, other mechanisms to block TLR9 activation or TLR9-mediated inflammation have been postulated or shown for other TLR9-inhibitory oligonucleotides [reviewed in 49]. These include competing for receptor-mediated endocytosis or phagocytosis, inhibition of TLR9 trafficking or TLR9 processing into a functionally active product, inhibition of endosomal acidification or activity of key proteases in endosomes, or blocking signaling proteins downstream of TLR9. When these inhibitory oligonucleotides are supplied in trans with TLR9 ligands (such as a DNA virus, or a CpG-containing oligonucleotide) in cell culture media, they are endocytosed and can bind to TLR9, preventing its activation by stimulatory ligands. Supplementation of inhibitory oligonucleotides in trans is widely adopted in immunology experiments, but it is unknown in the field if incorporation of these sequences into a viral genome allows it to evade eliciting inflammatory and immune responses.
[0011] While AAV has been shown to elicit much weaker inflammatory responses in comparison to adenoviruses, Glybera.TM. alipogene tiparvovec treatment still includes twelve weeks of immunosuppression, beginning three days before Glybera.TM. alipogene tiparvovec administration. These immunosuppressive drugs strongly hamper T cell activation and therefore compromise the patient's immune system during treatment. It would be advantageous to engineer viral vectors that evade and elicit diminished or no inflammatory response upon administration. Furthermore, it would be beneficial if the immune suppression was not systemic, and if it was transient. Preventing inflammatory and immune responses could also improve transgene expression and may allow the re-administration of the viral vector for future purposes.
[0012] There is a continuing need in the art to improve the efficacy of viral vectors for therapy and for in vivo production of biological products.
SUMMARY OF THE INVENTION
[0013] According to one aspect of the invention a nucleic acid molecule is provided. It comprises a viral genome covalently linked to an inhibitory nucleic acid sequence which binds to TLR9 but does not trigger TLR9 activation.
[0014] According to another aspect a recombinant virus is provided for delivery of a desired function to a mammalian cell. The recombinant virus comprises a viral genome covalently linked to an inhibitory nucleic acid sequence which binds to TLR9 but does not trigger TLR9 activation.
[0015] Another embodiment is an aspect of treating a mammal. The method comprises administering a recombinant virus to a mammal in need thereof. The recombinant virus comprises a viral genome covalently linked to an inhibitory nucleic acid sequence which binds to TLR9 but does not trigger TLR9 activation.
[0016] Still another aspect is a method of making a viral genome of a recombinant virus. An inhibitory nucleic acid sequence is inserted into a viral genome. The inhibitory nucleic acid sequence binds to TLR9 but does not trigger TLR9 activation.
[0017] According to one aspect a nucleic acid molecule is provided. It comprises an inverted terminal repeat (ITR) and a nucleic acid sequence which inhibits TLR9-mediated inflammation.
[0018] Another aspect of the invention is a nucleic acid molecule. The molecule comprises a viral genome covalently linked to an inhibitory nucleic acid sequence which inhibits TLR9-mediated inflammation.
[0019] Yet another aspect of the invention is a recombinant virus for delivery of a desired function to a mammalian cell. The recombinant virus comprises a viral genome comprising an inhibitory nucleic acid sequence which inhibits TLR9-mediated inflammation.
[0020] Still another aspect of the invention is a method of making a viral genome of a recombinant virus. A nucleic acid sequence is inserted into a viral genome. The nucleic acid sequence inhibits TLR9-mediated inflammation.
[0021] According to another aspect of the invention a nucleic acid vector is provided. The vector comprises at least one nucleic acid sequence. The nucleic acid sequence is capable of inhibiting TLR9-mediated inflammation.
[0022] Another aspect of the invention is a method of reducing immunogenicity of a modified virus having a genome. The method comprises inserting a nucleic acid sequence into the genome. The nucleic acid sequence inhibits TLR9-mediated inflammation. The modified virus causes a reduced inflammatory response in a host as compared to a virus that does not contain the inhibitory sequence.
[0023] Still another aspect of the invention is a method of increasing expression in a host cell of a virally introduced transgene. The method comprises introducing into a host a modified virus having a genome. The genome comprises a nucleic acid sequence. The nucleic acid sequence inhibits TLR9-mediated inflammation. The modified virus results in higher transgene expression in a host cell as compared to a virus that does not contain the inhibitory sequence.
[0024] Yet another aspect of the invention is a composition comprising a viral capsid encapsidating a nucleic acid sequence that inhibits TLR9-mediated inflammation.
[0025] These and other aspects and embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with tools for better treating mammals with viral vectors and virions and for better using viral vectors and virions for producing products of transgenes in host cells, host tissues, and host animals.
[0026] Any and all of the above described aspects may be combined with any of the following features.
[0027] The viral genome may be adeno-associated virus (AAV) genome.
[0028] The viral genome may be selected from the group consisting of adenovirus, herpes simplex virus, varicella, variola virus, hepatitis B, cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus, Herpes Zoster, Epstein-Barr virus, human herpes virus 7, Kaposi's sarcoma-associated herpesvirus, and human parvovirus B19.
[0029] The viral genome may be single stranded.
[0030] The viral genome may be packaged in a virion.
[0031] The viral genome may comprise a gene which may be expressible in a human cell.
[0032] The viral genome may be a cytotoxic virus for lysing target tumor cells.
[0033] The inhibitory nucleic acid sequence may comprise c41 oligonucleotide sequence TGGCGCGCACCCACGGCCTG (SEQ ID NO: 1).
[0034] The inhibitory nucleic acid sequence may comprise a plurality of copies of c41 sequence (SEQ ID NO: 1).
[0035] The inhibitory nucleic acid sequence may comprise two copies of c41 sequence (SEQ ID NO: 1) separated by a linker sequence.
[0036] The linker sequence is AAAAA (SEQ ID NO: 8).
[0037] The inhibitory nucleic acid sequence may be selected from the group consisting of:
TABLE-US-00001 ODN 2088: (SEQ ID NO: 2) TCC TGG CGG GGA AGT; ODN 4084-F: (SEQ ID NO: 3) CCTGGATGGGAA; ODN INH-1: (SEQ ID NO: 4) CCTGGATGGGAATTCCCATCCAGG; ODN INH-18: (SEQ ID NO: 5) CCT GGA TGG GAA CTT ACC GCT GCA; ODN TTAGGG: (SEQ ID NO: 6) TT AGG GTT AGG GTT AGG GTT AGG G; and G-ODN: (SEQ ID NO: 7) CTC CTA TTG GGG GTT TCC TAT.
[0038] The inhibitory nucleic acid sequence may be a bacterial sequence.
[0039] The viral genome, recombinant virus, vector, or nucleic acid sequence may comprise a non-human gene.
[0040] The inhibitory nucleic acid sequence may be inserted downstream of or in a 3' untranslated region of the viral genome.
[0041] The viral genome may be covalently linked to the inhibitory nucleic acid sequence by a phosphodiester bond.
[0042] The viral genome, recombinant virus, vector, or nucleic acid sequence may comprise a detectable marker.
[0043] The detectable marker may be inducible.
[0044] The inhibitory nucleic acid sequence may comprise a human telomere sequence shown in SEQ ID NO: 9.
[0045] The viral genome may be self-complementary.
[0046] The viral genome may be covalently linked to a plurality of inhibitory nucleic acid sequences.
[0047] The plurality of inhibitory nucleic acid sequences may comprise an inhibitory sequence and its reverse complement.
[0048] The inhibitory nucleic acid sequence may comprise three copies of c41 sequence (SEQ ID NO: 1), each copy separated by a linker sequence.
[0049] The inhibitory nucleic acid sequence may be selected from the group consisting of:
TABLE-US-00002 ODN 2114: (SEQ ID NO: 16) TCCTGGAGGGGAAGT; ODN 4024: (SEQ ID NO: 17) TCCTGGATGGGAAGT; ODN INH-4: (SEQ ID NO: 18) TTCCCATCCAGGCCTGGATGGGAA; ODN INH-13: (SEQ ID NO: 19) CTTACCGCTGCACCTGGATGGGAA; ODN Poly-G: (SEQ ID NO: 20) GGGGGGGGGGGGGGGGGGGG; ODN GpG: (SEQ ID NO: 21) TGACTGTGAAGGTTAGAGATGA; ODN IRS-869: (SEQ ID NO: 22) TCCTGGAGGGGTTGT; ODN IRS-954: (SEQ ID NO: 23) TGCTCCTGGAGGGGTTGT; and ODN 21158: (SEQ ID NO: 24) CCTGGCGGGG.
[0050] The inhibitory nucleic acid sequence may be ODN TTAGGG (SEQ ID NO: 6).
[0051] The inhibitory sequence may be covalently linked to a linker.
[0052] The inhibitory sequence may be upstream of the linker.
[0053] The inhibitory nucleic acid sequence may comprise a plurality of copies of ODN TTAGGG (SEQ ID NO: 6).
[0054] The plurality of copies of ODN TTAGGG (SEQ ID NO: 6) may each be separated by a linker.
[0055] The inhibitory nucleic acid sequence may comprise at least 2, at least 3, at least 4, or at least 5 copies of ODN TTAGGG (SEQ ID NO: 6), each copy separated by a linker.
[0056] The inhibitory nucleic acid sequence may be a human sequence.
[0057] The viral genome, recombinant virus, vector, or nucleic acid sequence may comprise a non-human nucleic acid sequence.
[0058] The viral genome, recombinant virus, vector, or nucleic acid sequence may comprise a human gene.
[0059] The viral genome, recombinant virus, vector, or nucleic acid sequence may comprise a human nucleic acid sequence.
[0060] The inhibitory nucleic acid sequence may be inserted in the 5' untranslated region of the viral genome.
[0061] The inhibitory nucleic acid sequence may be inserted upstream of a promoter of the viral genome.
[0062] The viral genome, recombinant virus, vector, or nucleic acid sequence may comprise an inducible promoter.
[0063] The inhibitory nucleic acid sequence may comprise two repeated monomers of SEQ ID NO: 1.
[0064] The inhibitory nucleic acid sequence may comprise three repeated monomers of SEQ ID NO: 1.
[0065] The inhibitory nucleic acid sequence may comprise SEQ ID NO: 6 or SEQ ID NO: 9.
[0066] The inhibitory nucleic acid sequence may comprise three repeated monomers of SEQ ID NO: 6 or SEQ ID NO: 9.
[0067] The inhibitory nucleic acid sequence may comprise five repeated monomers of SEQ ID NO: 6 or SEQ ID NO: 9.
[0068] The step of administering may be repeated.
[0069] The viral genome may be packaged in virions.
[0070] The step of inserting may utilize a DNA ligase.
[0071] The viral genome may be single stranded when in virions.
[0072] The viral genome of the recombinant virus may comprise a gene for delivery to and expression in a human cell.
[0073] The viral genome, recombinant virus, vector, or nucleic acid sequence may comprise a linker separating each of the nucleic acid sequences.
[0074] The viral genome, recombinant virus, vector, or nucleic acid sequence may comprise at least 2, at least 3, at least 4, or at least 5 copies of the nucleic acid sequence.
[0075] The inhibitory nucleic acid may be covalently linked to a gene.
[0076] The inhibitory nucleic acid sequence may be 95% identical to c41 oligonucleotide sequence TGGCGCGCACCCACGGCCTG (SEQ ID NO: 1).
[0077] The inhibitory nucleic acid sequence may be 95% identical to SEQ ID NO: 9.
[0078] The inhibitory nucleic acid sequence may be 95% identical to a sequence selected from the group consisting of:
TABLE-US-00003 ODN 2088: (SEQ ID NO: 2) TCC TGG CGG GGA AGT; ODN 4084-F: (SEQ ID NO: 3) CCTGGATGGGAA; ODN INH-1: (SEQ ID NO: 4) CCTGGATGGGAATTCCCATCCAGG; ODN INH-18: (SEQ ID NO: 5) CCT GGA TGG GAA CTT ACC GCT GCA; ODN TTAGGG: (SEQ ID NO: 6) TT AGG GTT AGG GTT AGG GTT AGG G; and G-ODN: (SEQ ID NO: 7) CTC CTA TTG GGG GTT TCC TAT.
[0079] The inhibitory nucleic acid sequence may be 95% identical to a sequence selected from the group consisting of:
TABLE-US-00004 ODN 2114: (SEQ ID NO: 16) TCCTGGAGGGGAAGT; ODN 4024: (SEQ ID NO: 17) TCCTGGATGGGAAGT; ODN INH-4: (SEQ ID NO: 18) TTCCCATCCAGGCCTGGATGGGAA; ODN INH-13: (SEQ ID NO: 19) CTTACCGCTGCACCTGGATGGGAA; ODN Poly-G: (SEQ ID NO: 20) GGGGGGGGGGGGGGGGGGGG; ODN GpG: (SEQ ID NO: 21) TGACTGTGAAGGTTAGAGATGA; ODN IRS-869: (SEQ ID NO: 22) TCCTGGAGGGGTTGT; ODN IRS-954: (SEQ ID NO: 23) TGCTCCTGGAGGGGTTGT; and ODN 21158: (SEQ ID NO: 24) CCTGGCGGGG.
[0080] The inhibitory nucleic acid sequence may comprise a plurality of copies of SEQ ID NO: 6 and/or SEQ ID NO: 9
[0081] The inhibitory nucleic acid sequence may comprise two copies of SEQ ID NO: 6 and/or SEQ ID NO: 9 separated by a linker sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0082] FIG. 1. Schematic drawing of viral gene therapy and inflammatory and immune responses.
[0083] FIG. 2. Schematic drawing showing TLR9 sensing AAV DNA during viral entry and inflammatory and immune responses. From Rogers et al., 2011, Frontiers in Microbiology, "Innate Immune Responses to AAV Vectors," vol. 2, article 194.
[0084] FIGS. 3A-3B. FIG. 3A shows nucleotide sequence of c41 (SEQ ID NO: 1), a single-stranded oligonucleotide. FIG. 3B shows organization of AAV-eGFP and AAV-eGFP-c41 genomes. ITR, inverted terminal repeat; LPA, late polyadenylation signal. Transgene of interest depicted in this case is eGFP. The insertion is of c41 sequence. Spacer depicted in FIG. 3B is shown in SEQ ID NO: 8.
[0085] FIG. 4 shows NF-kB activity in HEK293 TLR9 cells mock-infected or infected with AAV virus (DJ capsid, self-complementary AAV genome, encoding eGFP). **, p<0.005; n.s., not significant. This experiment used a crude viral preparation.
[0086] FIG. 5. Flow cytometry histograms showing GFP expression in HEK293 TLR9 cells mock-infected or infected with AAV virus with or without c41. This experiment used a crude viral preparation.
[0087] FIGS. 6A-6B. FIG. 6A shows nucleotide sequence of "telomere" (SEQ ID NO: 9), a single-stranded oligonucleotide containing the (TTAGGG).sub.4 (SEQ ID NO: 6) motif from mammalian telomeres. FIG. 6B shows organization of AAV-eGFP-telomere genome. The insertion is of "telomere" sequence. Spacers shown in FIG. 6B are SEQ ID NO: 8.
[0088] FIGS. 7A-7B. FIG. 7A shows the percentage of transduced cells (GFP+) 2 days after infection of a B cell line with similar amounts of indicated AAV viruses, analyzed by flow cytometry. FIG. 7B shows TNF production in the supernatant of primary human CD14+ monocytes 18 hours after infection, as assayed by ELISA.
[0089] FIG. 8A-8B. Engineering a self-complementary AAV vector. (FIG. 8A) DNA sequences of "c41" and "telomere". (FIG. 8B) Genome organization of an AAV vector (scAAV-eGFP) and modified vectors. LpA: polyA signal.
[0090] FIG. 9A-9C Inflammatory response to various AAV vectors in human immune cells in vitro. (FIG. 9A) Primary human macrophages were infected with AAV2 viruses (MOI: 10.sup.5 vg/cell) and supernatants were collected 18 h later and analyzed by ELISA for TNF levels. Five uM ODN 2006, a CpG-containing oligonucleotide, served as a positive control. (FIG. 9B, FIG. 9C) Primary human CD14+ monocytes from two different donors were infected similar to (A) and analyzed for TNF levels. Data shown are mean.+-.s.d. of n=3 technical replicates. *P<0.05 (unpaired t-test) compared to scAAV-eGFP.
[0091] FIG. 10A-10E. Further characterization of AAV vectors. (FIG. 10A) DNA sequence of "control". (FIG. 10B) Genome organization of modified vectors. (FIG. 10C) Primary human macrophages were infected with AAV2 viruses similar to (FIG. 9A) and analyzed by ELISA for TNF levels. (FIG. 10D) Primary human monocytes were infected similar to (FIG. 9B, FIG. 9C) and analyzed by ELISA for TNF levels. Data shown (FIG. 10C, FIG. 10D) are mean.+-.s.d. of n=3 technical replicates. *P<0.05 (unpaired t-test) compared to scAAV-eGFP. (FIG. 10E) Adult C57BL/6 mice were infected with indicated AAV2 viruses similar to (FIG. 12A, FIG. 12B and FIG. 12C) and a piece of the liver was analyzed for indicated gene expression by qRT-PCR. Data shown are mean.+-.s.d. of n=5 mice per condition except n=3 mice for scAAV-eGFP-3.times.control. *P<0.05 (unpaired t-test) compared to saline condition. N.s.: not significant (P>0.05).
[0092] FIG. 11A-11B. (FIG. 11A) Primary human macrophages using a different lot of both AAV2 viruses were infected similar to (FIG. 9A) and analyzed for TNF levels. (FIG. 11B) HeLa cells were infected with AAV2 viruses at indicated MOIs and cells were harvested 48 h later and analyzed by flow cytometry for GFP expression. The percentage of GFP positive cells are shown Data shown are mean.+-.s.d. of n=3 technical replicates. *P<0.05 (unpaired t-test) compared to scAAV-eGFP.
[0093] FIG. 12A-12C Inflammatory response to intravenous administration of various AAV vectors in adult mice in vivo. (FIG. 12A, FIG. 12B and FIG. 12C) Adult C57BL/6 mice were infected with indicated AAV2 viruses (10.sup.11 vg per mouse) by tail vein injections. 2 h later, the animals were euthanized and a piece of the liver was analyzed for indicated gene expression by qRT-PCR. Saline injection was set to 1-fold expression for each gene. Data shown are mean.+-.s.d. of n=3 mice per condition (FIG. 12A) or n=4 mice per condition (FIG. 12B and FIG. 12C). *P<0.05 (unpaired t-test) compared to saline condition. N.s.: not significant (P>0.05).
[0094] FIG. 13. Genome organization of a single-stranded AAV vector (ssAAV-eGFP) and ssAAV-eGFP-5.times. telomere.
[0095] FIG. 14A-14C. Inflammatory and immune response following subretinal administration of various AAV vectors in neonatal mice in vivo. (FIG. 14A, FIG. 14B and FIG. 14C) Neonatal CD1 mice (P1) received indicated AAV8 viruses (1.8.times.10.sup.8 vg per mouse eye) by subretinal injections. At P21, the animals were euthanized and the eyecup was dissected out. The retina and the rest of the eyecup were analyzed for indicated gene expression by qRT-PCR. Saline injection was set to 1 fold expression for each gene. Each triangle represents an animal. Data shown are n=3 mice (saline) and n=5 mice (each virus) and mean values are indicated.
[0096] FIG. 15A-15C. Analysis of immune cell markers in the retina following subretinal administration of various AAV vectors in neonatal mice in vivo. (FIG. 15A, FIG. 15B and FIG. 15C) Similar to (FIG. 14A-14C), the retina was analyzed for indicated gene expression by qRT-PCR. Aif1 (Iba1) is known to be expressed in microglia, while Cd4 and Cd8a are markers of helper and cytolytic T cells respectively. Saline injection was set to 1 fold expression for each gene. Each triangle represents an animal. Data shown are n=3 mice (saline) and n=5 mice (each virus) and mean values are indicated.
[0097] FIG. 16. GFP expression in the eye. Neonatal CD1 mice (P1) received indicated AAV8 viruses (1.8.times.10.sup.8 vg per mouse eye) by subretinal injections. At P30, the animals were euthanized and GFP expression was visualized in flat-mounted eye cups. Data shown are n=2 mice per condition.
[0098] FIGS. 17A-17B. FIG. 17A shows nucleotide sequence of c41 (SEQ ID NO: 1), a single-stranded oligonucleotide. FIG. 17B shows organization of AAV-eGFP and AAV-eGFP-c41 genomes. ITR, inverted terminal repeat; LPA, late polyadenylation signal. Transgene of interest depicted in this case is eGFP. The insertion is of c41 sequence. Spacer depicted in FIG. 17B is shown in SEQ ID NO: 8.
[0099] FIGS. 18A-18B. FIG. 18A shows nucleotide sequence of "telomere" (SEQ ID NO: 9), a single-stranded oligonucleotide containing the (TTAGGG).sub.4 (SEQ ID NO: 6) motif from mammalian telomeres. FIG. 18B shows organization of AAV-eGFP-telomere genome. The insertion is of "telomere" sequence. Spacers shown in FIG. 18B are SEQ ID NO: 8.
DETAILED DESCRIPTION OF THE INVENTION
[0100] The inventors have developed viral vectors and virions that harbor their own protection against host immune and inflammatory systems. These vectors and virions carry short nucleic acid sequences which inhibit the activation of toll-like receptor 9 (TLR9), a host protein which activates inflammatory and immune responses in mammalian cells.
[0101] A short nucleotide sequence for inhibition of TLR9 may be of any origin. It can be bacterial, human, synthetic, or from other sources. One particular sequence is the 20 nucleotide long "c41" [TGGCGCGCACCCACGGCCTG (SEQ ID NO: 1)] from Pseudomonas aeruginosa. Another particular sequence is from human telomeres and comprises (TTAGGG).sub.4 (SEQ ID NO:6). Other inhibitory sequences are shown in SEQ ID NO: 2-5, 7, 9, and 16-24. Inhibitory sequences may also be used which share at least 80% homology/identity with these sequences. Inhibitory sequences may also be used which share at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and/or at least 99% homology/identity with these sequences. Multiple copies of the inhibitory sequence can be used, either in tandem arrays or separated in the viral vector by spacer or linker sequences or other portions of the viral genome. In some embodiments one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or twenty copies are used. In some embodiments one or more copies of the inhibitory sequence are on the plus strand and some on the minus strand of the virus genome.
[0102] The inhibitory oligonucleotide sequences are introduced into host cells as part of the viral genome or virion, rather than as a separate agent. This renders the effect of the oligonucleotide sequences local rather than systemic. Moreover, the immune evasion is transient as it occurs during AAV or other virus entry, unlike immune suppression with drugs which can last for weeks. Additionally, it ensures that the beneficial antagonist activity is where it needs to be--with the virus or viral genome. If the virus or viral genome is not replicated in the host cell, then the effect of the oligonucleotide will be transient. If the virus or viral genome is replicated, the effect will be coextensive with the replication.
[0103] An inhibitory nucleic acid sequence may be inserted into a viral genome using any means of recombinant DNA engineering. This may involve in vitro or in vivo recombination. In vitro recombination may be accomplished using a DNA ligase or other nucleic acid joining enzyme, for example. In vivo recombination may be accomplished by co-transforming a host cell with separate donor molecules that share homology by which they will recombine using host cell machinery. Alternatively, a single donor molecule may recombine in vivo with a host cell sequence. Combinations of these approaches may also be used. Typically the insertion will involve a standard linkage of one deoxyribonucleotide to another (a phosphodiester bond). However, there may be circumstances in which non-standard linkages will be used between the inhibitory nucleic acid sequence and the rest of the viral genome. Optionally, the inhibitory nucleic acid sequence is located in an untranslated region of the viral genome.
[0104] The genome may optionally contain a therapeutic gene and/or a marker gene. Typically this gene will be a non-viral gene, or a gene that is not naturally present in the viral genome. The gene may be expressible in a mammalian host cell or animal. Expression may be under the control of a viral promoter or a promoter that is introduced with the gene. Expression may be inducible, repressible, condition-responsive, or constitutive, as examples. A therapeutic gene is one which encodes an RNA or protein product beneficial to the host. The benefit may be, for example, to improve health, protect against infection, or remedy a deficiency. The marker may enable one to track the location, the level of replication, the level of propagation, the level of transcription, or the level of translation of the virus or its products or components. Suitable markers include those which are readily detectable, such as fluorescent proteins, chromogenic proteins, etc. Optionally, a second agent may be used or added for detection of the marker protein or for development of a detectable substance. Introduced genes may be human or non-human, heterologous (from another species) or homologous (from same species) or endogenous (from the same subject).
[0105] Any DNA viral genome can be used, whether single stranded or double stranded. Examples of suitable viruses which may be used, include without limitation, wild-type or variants of adeno-associated virus (AAV), adenovirus, herpes simplex virus, varicella, variola virus, hepatitis B, cytomegalovirus, JC polyomavirus, BK polyomavirus, monkeypox virus, Herpes Zoster, Epstein-Barr virus, human herpes virus 7, Kaposi's sarcoma-associated herpesvirus, human parvovirus B19, and enterovirus. The virus may be, without limitation, cytotoxic, cytolytic, or cause latent infections. Viral vectors in which viral genomes have been modified may also be used. As an example, a genome that is modified to encode fewer viral proteins may be used. As a further example, a viral genome that is modified to encode no viral proteins may be used. Viral genomes may include, by way of non-limiting example, inverted terminal repeats and/or other non-coding genetic elements that facilitate packaging of engineered viral genomes into the capsid.
[0106] Viral genomes may be delivered to a mammalian host cell as naked DNA, in a liposome, complexed to a polymer, in a condensed or compacted state, in a gold particle, in a virion, or any other means that is suitable for the application. Typically a complete viral genome will be administered, but in some situations, it may be desirable to use a partial genome. The partial genome may be complemented by helper functions provided by the host cell or another genomic or viral entity. Partial genomes may be used, for example, if the therapeutic payload is large and some essential viral functions must be omitted to package.
[0107] Recombinant viruses may be administered to a mammal or mammalian cells according to any route which is effective for the purpose. The administration may be systemic, e.g., via the blood. It may be delivered orally, subcutaneously, topically, bucally, anally, intramuscularly, intravenously, intratumorally, intracranially, intrathecally, subretinally, etc. Any suitable carrier or vehicle may also be used for administration. It may be desirable to pre-treat the cells or mammal to render them more permeable to or receptive to the recombinant virus. A mammal "in need of" a recombinant virus may be one for whom the virus will be beneficial. It may be a mammal with a disease or deficiency. It may be one for whom a diagnosis or analysis will be made. It may be one who can benefit from the administered recombinant virus, even though it does not have a disease or deficiency.
[0108] Taken together, our results show that incorporation of c41 or human telomeric sequences into the AAV genome (a) does not lower viral packaging and infectivity, (b) prevents TLR9-mediated inflammation, (c) reduces induction of pro-inflammatory cytokines, and (d) increases transgene expression. The increased transgene expression may be due to a reduced immune response, as TLR9 activation also induces interferon expression, which triggers an antiviral state. The engineered immune-evasion property we show below is specific (against TLR9), transient (e.g., may occur during viral entry), and does not result in systemic immune suppression (only targets AAV-infected immune cells).
[0109] The inhibitory nucleic acid sequences we used, as well as others known in the art, may be incorporated into other viruses that, like AAV, have potential utility for humans and other mammals but elicit inflammatory/immune responses that may be undesirable. For example, oncolytic viruses that preferentially infect and lyse cancer cells are used to kill or shrink tumors. These viruses are replicative (unlike AAV vectors used for gene therapy) so they can release new virions to shrink the remaining tumor. Examples include wild-type or variants of herpes simplex virus, adenovirus, and enterovirus. Some reports have shown that immunosuppression by chemotherapy can enhance oncolytic virus therapy, as the immune system normally attempts to inactivate the oncolytic virus, which would prevent it from infecting cancer cells. Therefore, it is possible that incorporating inhibitory oligonucleotides in the genomes of oncolytic viruses like herpes simplex virus may allow it to evade immune clearance and persist longer for oncolysis.
[0110] The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention. The disclosure of the invention includes all embodiments explicitly recited in the claims. Additionally, all features disclosed in the dependent claims apply equally to the independent claim from which they are based as to the other independent claims. Thus such combinations of dependent claims with other independent claims are expressly contemplated and disclosed.
Example 1--Construction of Modified Viral Genome
[0111] To engineer an AAV vector that has the ability to specifically evade TLR9 activation in immune cells, we inserted two copies of c41 separated by a 5-nucleotide-long spacer (AAAAA; SEQ ID NO: 8) into the 3' untranslated region of AAV vector encoding enhanced green fluorescent protein (eGFP) (FIG. 3B). Subsequently, we produced wild-type AAV-eGFP virus and AAV-eGFP-c41 virus (harboring two c41 insertions). Infectious titers of both viruses were comparable (.about.10.sup.9 infectious units/ml, as determined by titering on HeLa cells), suggesting that addition of c41 into the viral genome did not hamper viral packaging and infectivity, an important consideration for viral vectors that have to be mass-produced for gene therapy.
Example 2--Modified Viral Genome Reduces NF-kB Activation
[0112] To measure the inflammatory response, we used HEK293 cells stably expressing TLR9 (HEK293 TLR9 cells), which senses AAV DNA genomes, and also expressing alkaline phosphatase under the transcriptional control of NF-kB. When NF-kB is activated, which indicates inflammation, alkaline phosphatase is secreted into the media and acts on a provided substrate, leading to a change in color of the media that can be measured on a plate reader. We mock-infected HEK293 TLR9 cells or infected them with either AAV-eGFP or AAV-eGFP-c41. In agreement with the literature, AAV-eGFP infection induced a small but statistically significant increase in NF-kB activity (FIG. 4). In contrast, AAV-eGFP-c41 infection was not significantly different compared to mock-infected cells, indicating that the virus was able to evade eliciting an inflammatory response.
Example 3--Modified Viral Genome Transduced More Cells and Expresses More Transgene
[0113] We analyzed the above three conditions (described in FIG. 4) for eGFP expression using flow cytometry. We found that AAV-eGFP-c41 transduced more cells than AAV-eGFP (52.7% GFP+ compared to 34.6% GFP+) (FIG. 5). In addition, GFP+ cells from AAV-eGFP-c41 infection expressed .about.twice as much eGFP as GFP+ cells from AAV-eGFP infection (mean fluorescence intensity [MFI] of 5335 compared to 2749).
[0114] In summary, we engineered an AAV vector to evade TLR9-mediated inflammation by incorporating an inhibitory oligonucleotide in the viral genome.
Example 4--Incorporation of c41 or Telomeric Sequences in AAV Genome Transduced More Cells and Reduced TNF Induction
[0115] We inserted three copies of "telomere," a sequence derived from mammalian telomeres that contains the suppressive (TTAGGG).sub.4 motif (SEQ ID NO: 6), which has been shown to block TLR9 signaling (FIG. 6A and FIG. 6B). AAV-eGFP-telomere virus yielded similar viral titers as AAV-eGFP and AAV-eGFP-c41 when titered on HeLa cells, demonstrating that incorporation of "telomere" does not hinder viral packaging and infectivity.
[0116] When we infected a B cell line with similar amounts of AAV-eGFP or AAV-eGFP-c41 or AAV-eGFP-telomere virus, both AAV-eGFP-c41 and AAV-eGFP-telomere viruses transduced more cells than AAV-eGFP (FIG. 7A). This finding suggests too that incorporation of inhibitory sequences in the genome of AAV increases transgene expression.
[0117] Subsequently, we harvested primary human CD14+ monocytes from blood and subjected them to similar infection conditions as above. We performed ELISA on the supernatant to analyze TNF production, as TNF is a prototypical pro-inflammatory cytokine induced by NF-kB activation. AAV-eGFP infection increased TNF production compared to mock-infection, while AAV-eGFP-c41 and AAV-eGFP-telomere infections showed no or little increase in TNF production (FIGS. 7A-7B), showing that the two viruses were able to evade eliciting inflammatory responses.
Example 5--Engineering a Self-Complementary AAV Vector
[0118] Investigators often use short inhibitory oligonucleotides (typically 10-30 nucleotides in length) to antagonize TLR9 signaling in cell culture. However, it is unknown if these inhibitory oligonucleotides retain functionality in the context of a much larger viral genome (i.e., the sequence is covalently linked on both ends to much longer sequences). To test this possibility, we utilized a self-complementary (sc) AAV vector encoding enhanced green fluorescent protein (eGFP), and inserted 3 copies of "c41" or "telomere", derived from bacteria and mammalian telomeres respectively [52, 57, 58, 61], into a plasmid harboring the vector genome (FIGS. 8A and 8B). We started with sc AAV vectors as they have been shown to be more efficient at triggering TLR9 activation and inducing more inflammation in the mouse liver than single-stranded (ss) AAV vectors. As "c41" and "telomere" are predicted to have strong secondary structure, we used an AAAAA linker between copies of the inhibitory oligonucleotide. In addition, 3.times.c41 and 3.times. telomere sequences were placed after the polyA sequence and upstream of the right inverted terminal repeat (ITR) so they would be present in the DNA genome during viral entry, but would be absent from subsequent mRNA transcripts upon successful transduction ("scAAV-eGFP-3.times.c41" and "scAAV-eGFP-3.times. telomere"). Finally, to determine if the location of inhibitory oligonucleotide in the viral genome matters, we also created a vector where 3.times. telomere was located between the left ITR and the promoter ("scAAV-3.times. telomere-eGFP").
Example 6--Inflammatory Responses in Primary Human Macrophages and Monocytes In Vitro
[0119] We packaged the various AAV vectors into AAV2 serotype and infected primary human monocyte-derived macrophages at a multiplicity of infection (MOI) of 10.sup.5 viral genomes (vg) per cell. As expected, we found that scAAV-eGFP infection of macrophages elicited robust induction of TNF in the supernatant, a prototypical inflammatory cytokine with well-described roles in stimulating fever, apoptosis and inflammation, and is produced upon TLR9 signaling and NF-kB activation (FIG. 9A). In contrast, both scAAV-eGFP-3.times.41 and scAAV-eGFP-3.times. telomere markedly decreased TNF induction by >95%, indicating that incorporation of "c41" or "telomere" in these viruses could evade eliciting inflammatory responses compared to the wild-type (WT) vector. Furthermore, scAAV-3.times. telomere-eGFP was also able to prevent TNF induction by >95%, demonstrating that the inserted inhibitory oligonucleotides can be placed in other parts of the viral genome and retain the ability to block inflammation. Mock infection with phosphate-buffered saline (PBS) and treatment with ODN 2006, a commercially available CpG-containing oligonucleotide that is known to strongly activate TLR9/NF-kB and inflammation, served as negative and positive controls respectively. We tested primary human CD14+ monocytes and found that again, scAAV-eGFP triggered robust TNF induction while scAAV-eGFP-3.times.c41 and scAAV-eGFP-3.times. telomere negated most of the TNF induction (FIG. 9B). scAAV-3.times. telomere-eGFP likewise reduced TNF induction, although inhibition was .about.85%, which may be due to differences between cell types or donor tissue. The evasion of TNF induction was also reproduced in primary CD14+ monocytes obtained from another donor (FIG. 9C).
[0120] As further characterization, we inserted 3 copies of "control," a random sequence that does not block TLR9, or 1 copy of "telomere", into a plasmid harboring the vector genome (FIG. 10A, FIG. 10B). We picked the sequence "control" as it has been used as a negative control oligonucleotide in TLR9 experiments. We found that scAAV-eGFP-lx telomere was able to reduce TNF induction compared to scAAV-eGFP in human macrophages, but not as efficiently as scAAV-eGFP-3.times. telomere (FIG. 10C). This indicates that 1 copy of telomere can reduce inflammation. We also observed that scAAV-eGFP-3.times.control elicited TNF secretion as efficiently as scAAV-eGFP in human monocytes, suggesting that insertion of sequences that inhibit TLR9 are required to block inflammation (FIG. 10D).
[0121] As AAV vectors are considered biologics and may exhibit lot-to-lot variability, we produced another batch of both scAAV-eGFP and scAAV-eGFP-3.times. telomere AAV2 viruses, and found that scAAV-eGFP-3.times. telomere was able to reduce .about.75% of TNF induction compared to the WT vector (FIG. 11A). Based on the multiple viral preps and donor monocytes and macrophages (FIGS. 9A-9C, FIGS. 10A-10E and FIGS. 11A-11B), we conclude that our engineered vectors containing 3 copies of "c41" or "telomere" reduce TNF induction by approximately 75-98% compared to the WT vector. On average, scAAV-eGFP-3.times. telomere reduced .about.85% of TNF induction compared to scAAV-eGFP. Importantly, we did not observe differences in viral titers (assayed by qPCR for viral genomes) obtained from producing any of the above AAV2 vectors, suggesting that the engineered vectors are not defective in packaging (data not shown). Furthermore, when we infected HeLa cells, a permissive cell line widely used to titer AAV infectivity, with a range of MOIs of scAAV-eGFP and scAAV-eGFP-3.times. telomere, we did not observe differences in transduction (% GFP+ cells) over 4 logs of viral titers, demonstrating that the engineered vector is equally competent at transducing cells (FIG. 11B).
Example 7--Inflammatory Responses in Liver Tissues of Mice In Vivo
[0122] Intravenous delivery of AAV is often used to transduce hepatocytes for gene therapy. Previous work has shown that upon intravenous administration of AAV, Kupffer cells (resident hepatic antigen-presenting cells) in the liver of mice are capable of sensing sc AAV genomes and triggering inflammatory and innate immune responses 1-9 h later [36]. These responses include induction of proinflammatory cytokines such as TNF and IL6 and type I interferons such as IFN-.beta.. TLR9-/- mice do not exhibit these inflammatory and innate immune responses in the liver, demonstrating a central role for TLR9 in vivo as an innate immune sensor. In addition, immune cells such as neutrophils, macrophages and natural killer (NK) cells infiltrate the liver 2 h after AAV administration. To determine if our engineered vectors can reduce inflammation in the liver in vivo, we administered PBS or equal amounts of scAAV-eGFP or scAAV-eGFP-3.times. telomere via tail vein injection. We selected scAAV-eGFP-3.times. telomere for in vivo characterization as "telomere" is derived from human sequences and might be preferable for clinical use. In agreement with previous work, scAAV-eGFP stimulated increased Tnf and 116 expression in the liver (approximately 3 to 10 fold, compared to saline), indicating inflammation (FIG. 12A). In contrast, scAAV-eGFP-3.times. telomere showed little to no increase in inflammatory markers. We tested more mice in subsequent experiments and found that scAAV-eGFP stimulated statistically significant Tnf induction in the liver compared to saline, while scAAV-eGFP-3.times. telomere and scAAV-eGFP-3.times.c41 did not (FIGS. 12B and 12C), demonstrating their ability to evade eliciting inflammation in the liver. Finally, we confirmed that scAAV-eGFP-3.times.control is not able to prevent inflammation in the liver compared to scAAV-eGFP (FIG. 10E).
Example 8--Engineering a Single-Stranded AAV Vector and Determining Inflammatory Responses in Eye Tissues of Mice In Vivo
[0123] Next, we engineered a single-stranded AAV vector, ssAAV-eGFP, by inserting 5 copies of "telomere" with AAAAA linkers, followed by another 5 copies but in anti-sense orientation, into the plasmid, giving ssAAV-eGFP-5.times. telomere (FIG. 13). Since both positive and negative strands of the viral genome are equally likely to be packaged into a viral particle, this ensures that each packaged viral genome would have 5 copies of "telomere" in the correct orientation. Two AAV8 viruses were produced and purified. Again, we did not observe differences in titers between the two vectors, suggesting similar packaging efficiency (data not shown). ssAAV-eGFP was selected as it has been previously used for subretinal injections in mice and efficiently transduces photoreceptors in the eye [62].
[0124] Several studies have suggested that AAV gene therapy in the eye and brain appears to be generally safe [63]. While the eye is often assumed to be an immune-privileged site, it is known to harbor microglia, resident macrophages of the central nervous system which have been reported to express TLR9 and respond to CpG motifs [64-67]. A recent study delivering AAV vectors by subretinal injection in cynomolgus macaques reported dose-related anterior and posterior segment inflammation in the animals, and a macaque was euthanized prematurely due to severe ocular inflammation [68]. Furthermore, vitreous aspirate from the euthanized animal demonstrated the presence of neutrophils and macrophages. Another study utilizing canine models similarly observed anterior and posterior uvetitis upon subretinal injection of AAV vectors, and 3 of 17 eyes developed a multifocal chorioretinitis, which was likewise associated with higher vector doses [69]. These findings strongly suggest that AAV vectors are subject to innate immune surveillance in the eye and can trigger deleterious inflammatory and immune responses.
[0125] Saline or similar amounts of ssAAV-eGFP or ss-AAV-eGFP-5.times. telomere were delivered via subretinal injection into neonates eyes and measured the expression of inflammatory and immune genes. The three mice that received saline injections were uniformly low for Tnf expression in the retina and were set to 1 fold expression (FIG. 14A). In contrast, of the five mice that received ssAAV-eGFP, two exhibited mild upregulation of Tnf (1.9 fold and 8.3 fold), while three animals demonstrated large induction of Tnf (62.2 fold, 534 fold, and 1003 fold), with a mean of 321 fold for the five animals. This finding indicates that while there is variability in inflammation, some animals mount a very strong inflammatory response in the retina upon ssAAV-eGFP subretinal injection. The variability may be due to differences in each injection procedure or the immune status of each animal. Strikingly, the five animals receiving ssAAV-eGFP-5.times. telomere had a mean Tnf induction of 5.6 fold with much less variability, suggesting that ssAAV-eGFP-5.times. telomere was able to avoid eliciting strong inflammation. Similar results were observed in the rest of the eyecup but at a lower magnitude, indicating inflammation was not restricted to the retina (FIG. 14B). We also measured Ifng expression in the retina, a type II interferon critical for antiviral immune responses [70], and observed a similar pattern (FIG. 14C). Prior studies have suggested that subretinal injection of AAV may trigger immune cell infiltration in the eye. Therefore, we also analyzed expression of genes that are known to be expressed specifically in different types of immune cells. We found 18.2 fold higher expression of Aif1 (encoding Iba1, a specific marker for microglia [71, 72]) in ssAAV-eGFP injections compared to saline, suggesting microglia proliferation and/or activation in the retina (FIG. 15A). In contrast, ssAAV-eGFP-5.times. telomere only showed 1.9 fold induction of Aif1 expression. In addition, we found 45.8 fold and 41.8 fold induction of Cd4 and Cd8a, markers of CD4+ helper T cells and CD8+ cytolytic T cells respectively, by ssAAV-eGFP, while ssAAV-eGFP-5.times. telomere only showed 1.5 fold and 3.4 fold induction (FIGS. 15B and 15C). Again, there was considerable variability among mice treated with ssAAV-eGFP with a subset of animals showing robust induction. These results demonstrate that subretinal injection of ssAAV-eGFP administration can stimulate T cells infiltration in the retina, while ssAAV-eGFP-5.times. telomere strongly diminishes it. Taken together, our data indicate that while ssAAV-eGFP induces robust inflammatory and immune responses in the retina and the surrounding tissue, and significant variability is observed, ssAAV-eGFP-5.times. telomere is capable of mitigating a large portion of these responses.
[0126] Given the marked differences in inflammation both in vitro and in vivo, we sought to determine if there are any differences in long-term gene expression. We examined flat-mounted eye cups at P30, 29 d after subretinal injection of the mice, and found that more cells were GFP+ and GFP expression was stronger in ssAAV-eGFP-5.times. telomere treated eyes compared to ssAAV-eGFP, suggesting enhanced gene expression (FIG. 16). Thus, the engineered vector is able to reduce inflammatory and immune responses in the retina and also augment transgene expression.
Example 9--Material and Methods
Animals
[0127] C57BL/6 mice (male, 7-9 weeks old) were purchased from the Jackson Laboratory and CD1 mice were purchased from Charles River Laboratories.
AAV Vectors
[0128] Self-complementary (sc) or single-stranded (ss) AAV vectors were used in this study. Self-complementary vectors lack the terminal resolution sequence in one ITR. All vector genomes were flanked by AAV2 ITRs. scAAV-eGFP was purchased from Cell Biolabs (VPK-430) and has been previously described [73]. scAAV-eGFP expressed enhanced green fluorescent protein (eGFP) from the cytomegalovirus (CMV) promoter, and included an SV40 intron and SV40 polyA sequence. ssAAV-eGFP has been previously described [62] and was originally obtained from the Harvard DF/HCC DNA Resource Core (clone ID: EvN000061595). ssAAV-eGFP contained a CMV enhancer/promoter, human .beta.-globin intron, eGFP, and .beta.-globin polyA sequence. The sequences of "c41" (5'-TGGCGCGCACCCACGGCCTG-3'; SEQ ID NO: 1) derived from Pseudomonas aeruginosa and "telomere" (5'-TTTAGGGTTAGGGTTAGGGTTAGGG-3'; SEQ ID NO: 9; initial T nucleotide is optional for function) derived from mammalian telomeres have been described [52, 57, 58, 61]. A widely used "telomere" oligonucleotide (manufactured by Invivogen, catalog code "tlrl-ttag") harbored an additional T (in bold) compared to published studies and thus was included in the sequence. During the course of this study, Invivogen removed the additional T in their manufactured "telomere" oligonucleotide (catalog code "tlrl-ttag151"). In addition, "control" (5'-GCTAGATGTTAGCGT-3'; SEQ ID NO: 34) was used as a negative control sequence that does not inhibit TLR9 activation (Invivogen, catalog code "tlrl-2088c").
[0129] To engineer scAAV-eGFP, sequences were inserted into the unique SpeI site found immediately 5' of the right ITR. To facilitate sub-cloning, a unique ClaI site was created immediately 5' of the inserted sequences, thus allowing ClaI/SpeI sub-cloning of sequences. 3 copies of "c41," "telomere," or "control" were inserted, separated by AAAAA linkers, giving scAAV-eGFP-3.times.c41, scAAV-eGFP-3.times. telomere and scAAV-eGFP-3.times. control, respectively. Alternatively, one copy of "telomere" was inserted, with an AAAAA linker (SEQ ID NO: 8), giving scAAV-eGFP-lx telomere. We also inserted 3.times. telomere between the left ITR and CMV promoter using the unique AvrII site, giving scAAV-3.times. telomere-eGFP.
[0130] To engineer ssAAV-eGFP, KpnI-5.times. telomere(sense)-5.times. telomere(anti-sense)-NheI was inserted immediately 5' of the XbaI site adjacent to the right ITR. Again, AAAAA was used as a linker between copies of "telomere". Both sense and anti-sense sequences of "telomere" were added as single-stranded AAV vectors have an equal chance of packaging positive or negative strands of the viral genome, thus ensuring that all packaged AAV genomes will carry 5 copies of "telomere" in the right orientation.
[0131] Self-complementary vectors were packaged into AAV2 (Vigene Biosciences) by triple transfection of HEK293 cells and purified using iodixanol gradient ultracentrifugation and then concentrated to 500 ul using Amicon Ultra-15 columns in PBS. The purified viruses were titered by qPCR using primers derived from ITR and an AAV standard. The final yield of the viruses ranged from 0.5-3.times.10.sup.13 vg.
[0132] Single-stranded vectors were packaged into AAV8 based on previously described protocols [74, 75]. Briefly, AAV vector, rep2-cap8 packaging plasmid and adenoviral helper plasmid were transfected into HEK293T cells with polyethylenimine and supernatant was collected 72 h after transfection. AAV8 viruses were precipitated with 8.5% w/v PEG8000 and 0.4M NaCl and centrifuged at 7000 g. The pellet was resuspended in lysis buffer (150 mM NaCl and 20 mM Tris, pH 8.0) and MgCl2 was added to a final concentration of 1 mM. The resuspended viruses were incubated with 25 U/ml Benzonase (Sigma) at 37.degree. C. for 15 min and run on an iodixanol gradient. Recovered AAV vectors were washed 3 times with PBS using Amicon 100K columns (EMD Millipore) and concentrated to 100-500 ul of PBS. Protein gels were run to determine virus titers, using serial dilutions of previous AAV standards for comparison.
Primary Human Monocytes and Monocyte-Derived Macrophages for In Vitro Studies
[0133] Human peripheral blood mononuclear cells (PBMCs) from unidentified healthy donors were purchased (ZenBio). This study was done in accordance with the ethical guidelines of Harvard Medical School. CD14+ monocytes were positively selected from PBMCs using anti-CD14 magnetic microbeads according to the manufacturer's instructions (Miltenyi Biotec) or purchased from Stemcell Technologies. To obtain monocyte-derived macrophages, monocytes were cultured with 50 ng/ml of recombinant human macrophage colony stimulation factor (rhM-CSF, purchased from Peprotech) for 5 to 6 d to allow differentiation into macrophages. Monocytes and macrophages were either used fresh or cryopreserved for subsequent studies.
[0134] 1.times.10.sup.5 monocytes or macrophages were seeded in 190 ul of RPMI growth media per well in 96 well round bottom plates or 96 well flat bottom plates respectively, and infected with 10 ul AAV2 viruses at indicated MOIs in PBS. Mock infection (addition of 10 ul PBS) and ODN 2006 (final concentration of 5 uM, Invivogen), a CpG-containing oligonucleotide known to activate TLR9 and trigger inflammation, served as negative and positive controls. 18 h after infection, supernatants were collected and clarified by low speed centrifugation, followed by ELISA for human TNF (Thermo Scientific).
HeLa Cells Infection
[0135] HeLa cells are highly permissive for AAV2 vectors and are commonly used to determine the transducing titer of AAV2 vector preparations [76]. Briefly, HeLa cells were seeded overnight in 12 wells and were approximately 80% confluent at time of infection (3.times.10.sup.5 cells). Cells were infected with serial ten-fold dilutions of viruses at indicated MOIs and incubated for 48 h before fixing with 1% paraformaldehyde in PBS and followed by flow cytometry analysis for GFP+ cells. PBS mock-infected cells were used to determine GFP+ signal.
Liver Studies In Vivo
[0136] Adult C57BL/6 mice were injected intravenously with 100 ul PBS or AAV2 viruses (10.sup.11 vg per animal) by tail vein injection as previously described [36]. 2 h later, the animals were sacrificed and a portion of the right median lobe of the liver was saved in RNAlater solution (Thermo Scientific). Total RNA was extracted from 10-30 mg of mechanically disrupted liver sample by using an RNA extraction kit (OMEGA Bio-Tek). Similar amounts of RNA were reverse transcribed into cDNA with a high-capacity RNA-to-cDNA kit (Thermo Scientific) and similar amounts of cDNA were assayed with quantitative PCR (qPCR) using TaqMan Fast Advanced Master Mix (Thermo Scientific) and commercially available pre-designed primers/probes with FAM reporter dye for the indicated target genes (IDT). Expression level for each gene was calculated by normalizing against the housekeeping genes Actb or Gapdh using the AACT method and expressed as fold levels compared to saline-injected mice. All qPCR reactions were run on a realplex.sup.4 Mastercycle (Eppendorf).
Eye Studies In Vivo
[0137] Subretinal injection into postnatal day 1 (P1) CD1 neonate eyes were performed as previously described [74, 75]. Approximately 0.2 ul AAV8 virus (1.8.times.10.sup.8 vg per eye) was introduced into the subretinal space using a pulled angled glass pipette controlled by a FemtoJet (Eppendorf). At P21, animals were sacrificed and the eyecup was dissected out. The retina and the rest of the eyecup were subjected to RNA extraction, reverse transcription, and qPCR as described in the liver studies. To visualize GFP expression by histology, eyes were excised at P30, fixed in 4% paraformaldehyde for 2 h, and washed in PBS 3 times. Eye cups were dissected out by removing the cornea, lens, iris, vitreous body and peripheral muscles. Images of flat-mounted eye cups were taken using a .times.10 objective on a Keyence BZ-x700 microscope. Images used for comparison between groups were taken at the same imaging settings in the same imaging session.
Statistics
[0138] Unpaired two-tailed Student's t-tests were used to compare differences between two unpaired experimental groups in all cases. A P value of <0.05 was considered statistically significant. No pre-specified effect size was assumed and in general three to five replicates for each condition was used.
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Sequence CWU
1
1
34120DNAArtificial SequenceSynthetic Polynucleotide 1tggcgcgcac ccacggcctg
20215DNAArtificial
SequenceSynthetic Polynucleotide 2tcctggcggg gaagt
15312DNAArtificial SequenceSynthetic
Polynucleotide 3cctggatggg aa
12424DNAArtificial SequenceSynthetic Polynucleotide
4cctggatggg aattcccatc cagg
24524DNAArtificial SequenceSynthetic Polynucleotide 5cctggatggg
aacttaccgc tgca
24624DNAArtificial SequenceSynthetic Polynucleotide 6ttagggttag
ggttagggtt aggg
24721DNAArtificial SequenceSynthetic Polynucleotide 7ctcctattgg
gggtttccta t
2185DNAArtificial SequenceSynthetic Polynucleotide 8aaaaa
5925DNAArtificial
SequenceSynthetic Polynucleotide 9tttagggtta gggttagggt taggg
25104369DNAArtificial SequenceSynthetic
Polynucleotide 10aaagcttccc ggggggatct gggccactcc ctctctgcgc gctcgctcgc
tcactgaggc 60cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
tgagcgagcg 120agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctggaggg
gtggagtcgt 180gacctaggca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
tggcccgcct 240ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
atctacgtat 300tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
cgtggatagc 360ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
agtttgtttt 420ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
ttgacgcaaa 480tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
gtgaaccgtc 540agatcgcctg gagacgccat ccggactcta aggtaaatat aaaattttta
agtgtataat 600gtgttaaact actgattcta attgtttctc tcttttagat tccaaccttt
ggaactgaat 660tccgcgggcc cgggatccac cggtcgccac catggtgagc aagggcgagg
agctgttcac 720cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
agttcagcgt 780gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
tcatctgcac 840caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
acggcgtgca 900gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
ccgccatgcc 960cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
acaagacccg 1020cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
agggcatcga 1080cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
acagccacaa 1140cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
agatccgcca 1200caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
cccccatcgg 1260cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
ccctgagcaa 1320agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
ccgccgggat 1380cactctcggc atggacgagc tgtacaagta aagcggccgc taggcctcac
ctgcgatctc 1440gatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
ataagctgca 1500ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
ggggaggtgt 1560gggaggtttt ttaaactagt ccactccctc tctgcgcgct cgctcgctca
ctgaggccgg 1620gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
gcgagcgagc 1680gcgcagagag ggacagatcc gggcccgcat gcgtcgacaa ttcactggcc
gtcgttttac 1740aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
gcacatcccc 1800ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
caacagttgc 1860gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat
ctgtgcggta 1920tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca
tagttaagcc 1980agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
ctcccggcat 2040ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
ttttcaccgt 2100catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta
taggttaatg 2160tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
gtgcgcggaa 2220cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
agacaataac 2280cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
catttccgtg 2340tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
ccagaaacgc 2400tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
atcgaactgg 2460atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
ccaatgatga 2520gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
gggcaagagc 2580aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
ccagtcacag 2640aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
ataaccatga 2700gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
gagctaaccg 2760cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
ccggagctga 2820atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
gcaacaacgt 2880tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
ttaatagact 2940ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
gctggctggt 3000ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
gcagcactgg 3060ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
caggcaacta 3120tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
cattggtaac 3180tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
ttttaattta 3240aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
taacgtgagt 3300tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
tgagatcctt 3360tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
gcggtggttt 3420gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
agcagagcgc 3480agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
aagaactctg 3540tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
gccagtggcg 3600ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
gcgcagcggt 3660cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
tacaccgaac 3720tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
agaaaggcgg 3780acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
cttccagggg 3840gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
gagcgtcgat 3900ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
gcggcctttt 3960tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
ttatcccctg 4020attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
cgcagccgaa 4080cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
cgcaaaccgc 4140ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
cccgactgga 4200aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
gcaccccagg 4260ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
taacaatttc 4320acacaggaaa cagctatgac catgattacg ccaagctctc gagatctag
4369114450DNAArtificial SequenceSynthetic Polynucleotide
11aaagcttccc ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc
60cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
120agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt
180gacctaggca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
240ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
300tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
360ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
420ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
480tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
540agatcgcctg gagacgccat ccggactcta aggtaaatat aaaattttta agtgtataat
600gtgttaaact actgattcta attgtttctc tcttttagat tccaaccttt ggaactgaat
660tccgcgggcc cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac
720cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
780gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
840caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
900gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
960cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
1020cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
1080cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
1140cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
1200caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
1260cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
1320agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
1380cactctcggc atggacgagc tgtacaagta aagcggccgc taggcctcac ctgcgatctc
1440gatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
1500ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
1560gggaggtttt ttaaatcgat tggcgcgcac ccacggcctg aaaaatggcg cgcacccacg
1620gcctgaaaaa tggcgcgcac ccacggcctg aaaaaactag tccactccct ctctgcgcgc
1680tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
1740ggcctcagtg agcgagcgag cgcgcagaga gggacagatc cgggcccgca tgcgtcgaca
1800attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
1860atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
1920atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc
1980tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct
2040ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac
2100gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca
2160tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac
2220gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
2280ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
2340atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
2400tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
2460tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
2520gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
2580aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
2640gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
2700ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
2760gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
2820gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
2880atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
2940ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
3000cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
3060cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
3120gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
3180cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
3240cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
3300taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
3360ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
3420aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
3480caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
3540taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
3600gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
3660cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
3720taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
3780agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
3840ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
3900gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
3960acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
4020acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
4080tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
4140ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
4200agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
4260acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
4320tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
4380ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct
4440cgagatctag
4450124465DNAArtificial SequenceSynthetic Polynucleotide 12aaagcttccc
ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60cgggcgacca
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120agcgcgcaga
gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt 180gacctaggca
tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 240ggcattatgc
ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 300tagtcatcgc
tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 360ggtttgactc
acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 420ggcaccaaaa
tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 480tgggcggtag
gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 540agatcgcctg
gagacgccat ccggactcta aggtaaatat aaaattttta agtgtataat 600gtgttaaact
actgattcta attgtttctc tcttttagat tccaaccttt ggaactgaat 660tccgcgggcc
cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac 720cggggtggtg
cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 780gtccggcgag
ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 840caccggcaag
ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca 900gtgcttcagc
cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 960cgaaggctac
gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 1020cgccgaggtg
aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 1080cttcaaggag
gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 1140cgtctatatc
atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 1200caacatcgag
gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 1260cgacggcccc
gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa 1320agaccccaac
gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 1380cactctcggc
atggacgagc tgtacaagta aagcggccgc taggcctcac ctgcgatctc 1440gatgctttat
ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 1500ataaacaagt
taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 1560gggaggtttt
ttaaatcgat tttagggtta gggttagggt tagggaaaaa tttagggtta 1620gggttagggt
tagggaaaaa tttagggtta gggttagggt tagggaaaaa actagtccac 1680tccctctctg
cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 1740gggctttgcc
cgggcggcct cagtgagcga gcgagcgcgc agagagggac agatccgggc 1800ccgcatgcgt
cgacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 1860gcgttaccca
acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 1920aagaggcccg
caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 1980tgatgcggta
ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 2040tcagtacaat
ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 2100ctgacgcgcc
ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 2160tctccgggag
ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 2220agggcctcgt
gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 2280cgtcaggtgg
cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 2340tacattcaaa
tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 2400gaaaaaggaa
gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 2460cattttgcct
tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 2520atcagttggg
tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 2580agagttttcg
ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 2640gcgcggtatt
atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 2700ctcagaatga
cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 2760cagtaagaga
attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 2820ttctgacaac
gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 2880atgtaactcg
ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 2940gtgacaccac
gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 3000tacttactct
agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 3060gaccacttct
gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 3120gtgagcgtgg
gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 3180tcgtagttat
ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 3240ctgagatagg
tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 3300tactttagat
tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 3360ttgataatct
catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 3420ccgtagaaaa
gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 3480tgcaaacaaa
aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 3540ctctttttcc
gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 3600tgtagccgta
gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 3660tgctaatcct
gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 3720actcaagacg
atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 3780cacagcccag
cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 3840gagaaagcgc
cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 3900tcggaacagg
agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 3960ctgtcgggtt
tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 4020ggagcctatg
gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 4080cttttgctca
catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 4140cctttgagtg
agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 4200gcgaggaagc
ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 4260attaatgcag
ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 4320ttaatgtgag
ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 4380gtatgttgtg
tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 4440attacgccaa
gctctcgaga tctag
4465134465DNAArtificial SequenceSynthetic Polynucleotide 13aaagcttccc
ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60cgggcgacca
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120agcgcgcaga
gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt 180gacctaggtt
tagggttagg gttagggtta gggaaaaatt tagggttagg gttagggtta 240gggaaaaatt
tagggttagg gttagggtta gggaaaaacc taggcatatg ccaagtacgc 300cccctattga
cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 360tatgggactt
tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 420tgcggttttg
gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 480gtctccaccc
cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 540caaaatgtcg
taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 600aggtctatat
aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccgg 660actctaaggt
aaatataaaa tttttaagtg tataatgtgt taaactactg attctaattg 720tttctctctt
ttagattcca acctttggaa ctgaattccg cgggcccggg atccaccggt 780cgccaccatg
gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 840gctggacggc
gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc 900cacctacggc
aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg 960gcccaccctc
gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca 1020catgaagcag
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 1080catcttcttc
aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga 1140caccctggtg
aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct 1200ggggcacaag
ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca 1260gaagaacggc
atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca 1320gctcgccgac
cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 1380caaccactac
ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca 1440catggtcctg
ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta 1500caagtaaagc
ggccgctagg cctcacctgc gatctcgatg ctttatttgt gaaatttgtg 1560atgctattgc
tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt 1620gcattcattt
tatgtttcag gttcaggggg aggtgtggga ggttttttaa actagtccac 1680tccctctctg
cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 1740gggctttgcc
cgggcggcct cagtgagcga gcgagcgcgc agagagggac agatccgggc 1800ccgcatgcgt
cgacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 1860gcgttaccca
acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 1920aagaggcccg
caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 1980tgatgcggta
ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 2040tcagtacaat
ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 2100ctgacgcgcc
ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 2160tctccgggag
ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 2220agggcctcgt
gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 2280cgtcaggtgg
cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 2340tacattcaaa
tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 2400gaaaaaggaa
gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 2460cattttgcct
tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 2520atcagttggg
tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 2580agagttttcg
ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 2640gcgcggtatt
atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 2700ctcagaatga
cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 2760cagtaagaga
attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 2820ttctgacaac
gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 2880atgtaactcg
ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 2940gtgacaccac
gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 3000tacttactct
agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 3060gaccacttct
gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 3120gtgagcgtgg
gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 3180tcgtagttat
ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 3240ctgagatagg
tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 3300tactttagat
tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 3360ttgataatct
catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 3420ccgtagaaaa
gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 3480tgcaaacaaa
aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 3540ctctttttcc
gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 3600tgtagccgta
gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 3660tgctaatcct
gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 3720actcaagacg
atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 3780cacagcccag
cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 3840gagaaagcgc
cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 3900tcggaacagg
agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 3960ctgtcgggtt
tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 4020ggagcctatg
gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 4080cttttgctca
catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 4140cctttgagtg
agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 4200gcgaggaagc
ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 4260attaatgcag
ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 4320ttaatgtgag
ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 4380gtatgttgtg
tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 4440attacgccaa
gctctcgaga tctag
4465146940DNAArtificial SequenceSynthetic Polynucleotide 14cagcagctgc
gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60acctttggtc
gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc 120atcactaggg
gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca 180tgctctagcg
gcctcggcct ctgcataaat aaaaaaaatt agtcagccat gagcttggcc 240cattgcatac
gttgtatcca tatcataata tgtacattta tattggctca tgtccaacat 300taccgccatg
ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 360tagttcatag
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 420gctgaccgcc
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 480cgccaatagg
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 540tggcagtaca
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 600aatggcccgc
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 660acatctacgt
attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg 720ggcgtggata
gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 780ggagtttgtt
ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc 840cattgacgca
aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 900tagtgaaccg
tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac 960accgggaccg
atccagcctc ccctcgaagc tgatcctgag aacttcaggg tgagtctatg 1020ggacccttga
tgttttcttt ccccttcttt tctatggtta agttcatgtc ataggaaggg 1080gagaagtaac
agggtacaca tattgaccaa atcagggtaa ttttgcattt gtaattttaa 1140aaaatgcttt
cttcttttaa tatacttttt tgtttatctt atttctaata ctttccctaa 1200tctctttctt
tcagggcaat aatgatacaa tgtatcatgc ctctttgcac cattctaaag 1260aataacagtg
ataatttctg ggttaaggca atagcaatat ttctgcatat aaatatttct 1320gcatataaat
tgtaactgat gtaagaggtt tcatattgct aatagcagct acaatccagc 1380taccattctg
cttttatttt atggttggga taaggctgga ttattctgag tccaagctag 1440gcccttttgc
taatcatgtt catacctctt atcttcctcc cacagctcct gggcaacgtg 1500ctggtctgtg
tgctggccca tcactttggc aaagaattcc gcgggcccgg gatccaccgg 1560tcgccaccat
ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg 1620agctggacgg
cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg 1680ccacctacgg
caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct 1740ggcccaccct
cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc 1800acatgaagca
gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca 1860ccatcttctt
caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg 1920acaccctggt
gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc 1980tggggcacaa
gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc 2040agaagaacgg
catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc 2100agctcgccga
ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg 2160acaaccacta
cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc 2220acatggtcct
gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt 2280acaagtaaag
cggccgctct agaggatcca agcttatcga taccgtcgac ctcgagggcc 2340cagatctaat
tcaccccacc agtgcaggct gcctatcaga aagtggtggc tggtgtggct 2400aatgccctgg
cccacaagta tcactaagct cgctttcttg ctgtccaatt tctattaaag 2460gttcctttgt
tccctaagtc caactactaa actgggggat attatgaagg gccttgagca 2520tctggattct
gcctaataaa aaacatttat tttcattgca atgatgtatt taaattattt 2580ctgaatattt
tactaaaaag ggaatgtggg aggtcagtgc atttaaaaca taaagaaatg 2640aagagctagt
tcaaaccttg ggaaaataca ctatatctta aactccatga aagaaggtga 2700ggctgcaaac
agctaatgca cattggcaac agcccctgat gcctatgcct tattcatccc 2760tcagaaaagg
attcaagtag aggcttgatt tggaggttaa agttttgcta tgctgtattt 2820tacattactt
attgttttag ctgtcctcat gaatgtcttt tcactaccca tttgcttatc 2880ctgcatctct
cagccttgac tccactcagt tctcttgctt agagatacca cctttcccct 2940gaagtgttcc
ttccatgttt tacggcgaga tggtttctcc tcgcctggcc actcagcctt 3000agttgtctct
gttgtcttat agaggtctac ttgaagaagg aaaaacaggg ggcatggttt 3060gactgtcctg
tgagcccttc ttccctgcct cccccactca cagtgacccg gaatccctcg 3120acatctagag
catggctacg tagataagta gcatggcggg ttaatcatta actacaagga 3180acccctagtg
atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 3240gcgaccaaag
gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 3300gcgccagctg
gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 3360gcctgaatgg
cgaatggaat tccagacgat tgagcgtcaa aatgtaggta tttccatgag 3420cgtttttcct
gttgcaatgg ctggcggtaa tattgttctg gatattacca gcaaggccga 3480tagtttgagt
tcttctactc aggcaagtga tgttattact aatcaaagaa gtattgcgac 3540aacggttaat
ttgcgtgatg gacagactct tttactcggt ggcctcactg attataaaaa 3600cacttctcag
gattctggcg taccgttcct gtctaaaatc cctttaatcg gcctcctgtt 3660tagctcccgc
tctgattcta acgaggaaag cacgttatac gtgctcgtca aagcaaccat 3720agtacgcgcc
ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 3780ccgctacact
tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 3840ccacgttcgc
cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 3900ttagtgcttt
acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 3960ggccatcgcc
ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4020gtggactctt
gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4080tataagggat
tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 4140ttaacgcgaa
ttttaacaaa atattaacgt ttacaattta aatatttgct tatacaatct 4200tcctgttttt
ggggcttttc tgattatcaa ccggggtaca tatgattgac atgctagttt 4260tacgattacc
gttcatcgat tctcttgttt gctccagact ctcaggcaat gacctgatag 4320cctttgtaga
gacctctcaa aaatagctac cctctccggc atgaatttat cagctagaac 4380ggttgaatat
catattgatg gtgatttgac tgtctccggc ctttctcacc cgtttgaatc 4440tttacctaca
cattactcag gcattgcatt taaaatatat gagggttcta aaaattttta 4500tccttgcgtt
gaaataaagg cttctcccgc aaaagtatta cagggtcata atgtttttgg 4560tacaaccgat
ttagctttat gctctgaggc tttattgctt aattttgcta attctttgcc 4620ttgcctgtat
gatttattgg atgttggaat tcctgatgcg gtattttctc cttacgcatc 4680tgtgcggtat
ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 4740agttaagcca
gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 4800tcccggcatc
cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 4860tttcaccgtc
atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 4920aggttaatgt
catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 4980tgcgcggaac
ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 5040gacaataacc
ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 5100atttccgtgt
cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 5160cagaaacgct
ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 5220tcgaactgga
tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 5280caatgatgag
cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 5340ggcaagagca
actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 5400cagtcacaga
aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 5460taaccatgag
tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 5520agctaaccgc
ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 5580cggagctgaa
tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 5640caacaacgtt
gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 5700taatagactg
gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 5760ctggctggtt
tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 5820cagcactggg
gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 5880aggcaactat
ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 5940attggtaact
gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 6000tttaatttaa
aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 6060aacgtgagtt
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 6120gagatccttt
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 6180cggtggtttg
tttgccggat caagagctac caactctttt tccgaaggta actggcttca 6240gcagagcgca
gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 6300agaactctgt
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 6360ccagtggcga
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 6420cgcagcggtc
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 6480acaccgaact
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 6540gaaaggcgga
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 6600ttccaggggg
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 6660agcgtcgatt
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 6720cggccttttt
acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 6780tatcccctga
ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 6840gcagccgaac
gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 6900gcaaaccgcc
tctccccgcg cgttggccga ttcattaatg
6940157257DNAArtificial SequenceSynthetic Polynucleotide 15cagcagctgc
gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60acctttggtc
gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc 120atcactaggg
gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca 180tgctctagcg
gcctcggcct ctgcataaat aaaaaaaatt agtcagccat gagcttggcc 240cattgcatac
gttgtatcca tatcataata tgtacattta tattggctca tgtccaacat 300taccgccatg
ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 360tagttcatag
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 420gctgaccgcc
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 480cgccaatagg
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 540tggcagtaca
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 600aatggcccgc
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 660acatctacgt
attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg 720ggcgtggata
gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 780ggagtttgtt
ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc 840cattgacgca
aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 900tagtgaaccg
tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac 960accgggaccg
atccagcctc ccctcgaagc tgatcctgag aacttcaggg tgagtctatg 1020ggacccttga
tgttttcttt ccccttcttt tctatggtta agttcatgtc ataggaaggg 1080gagaagtaac
agggtacaca tattgaccaa atcagggtaa ttttgcattt gtaattttaa 1140aaaatgcttt
cttcttttaa tatacttttt tgtttatctt atttctaata ctttccctaa 1200tctctttctt
tcagggcaat aatgatacaa tgtatcatgc ctctttgcac cattctaaag 1260aataacagtg
ataatttctg ggttaaggca atagcaatat ttctgcatat aaatatttct 1320gcatataaat
tgtaactgat gtaagaggtt tcatattgct aatagcagct acaatccagc 1380taccattctg
cttttatttt atggttggga taaggctgga ttattctgag tccaagctag 1440gcccttttgc
taatcatgtt catacctctt atcttcctcc cacagctcct gggcaacgtg 1500ctggtctgtg
tgctggccca tcactttggc aaagaattcc gcgggcccgg gatccaccgg 1560tcgccaccat
ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg 1620agctggacgg
cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg 1680ccacctacgg
caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct 1740ggcccaccct
cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc 1800acatgaagca
gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca 1860ccatcttctt
caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg 1920acaccctggt
gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc 1980tggggcacaa
gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc 2040agaagaacgg
catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc 2100agctcgccga
ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg 2160acaaccacta
cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc 2220acatggtcct
gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt 2280acaagtaaag
cggccgctct agaggatcca agcttatcga taccgtcgac ctcgagggcc 2340cagatctaat
tcaccccacc agtgcaggct gcctatcaga aagtggtggc tggtgtggct 2400aatgccctgg
cccacaagta tcactaagct cgctttcttg ctgtccaatt tctattaaag 2460gttcctttgt
tccctaagtc caactactaa actgggggat attatgaagg gccttgagca 2520tctggattct
gcctaataaa aaacatttat tttcattgca atgatgtatt taaattattt 2580ctgaatattt
tactaaaaag ggaatgtggg aggtcagtgc atttaaaaca taaagaaatg 2640aagagctagt
tcaaaccttg ggaaaataca ctatatctta aactccatga aagaaggtga 2700ggctgcaaac
agctaatgca cattggcaac agcccctgat gcctatgcct tattcatccc 2760tcagaaaagg
attcaagtag aggcttgatt tggaggttaa agttttgcta tgctgtattt 2820tacattactt
attgttttag ctgtcctcat gaatgtcttt tcactaccca tttgcttatc 2880ctgcatctct
cagccttgac tccactcagt tctcttgctt agagatacca cctttcccct 2940gaagtgttcc
ttccatgttt tacggcgaga tggtttctcc tcgcctggcc actcagcctt 3000agttgtctct
gttgtcttat agaggtctac ttgaagaagg aaaaacaggg ggcatggttt 3060gactgtcctg
tgagcccttc ttccctgcct cccccactca cagtgacccg gaatccctcg 3120acaggtacct
ttagggttag ggttagggtt agggaaaaat ttagggttag ggttagggtt 3180agggaaaaat
ttagggttag ggttagggtt agggaaaaat ttagggttag ggttagggtt 3240agggaaaaat
ttagggttag ggttagggtt agggaaaaac cctaacccta accctaaccc 3300taaatttttt
ccctaaccct aaccctaacc ctaaattttt tccctaaccc taaccctaac 3360cctaaatttt
ttccctaacc ctaaccctaa ccctaaattt tttccctaac cctaacccta 3420accctaaatt
ttttgctagc tctagagcat ggctacgtag ataagtagca tggcgggtta 3480atcattaact
acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 3540tcgctcactg
aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 3600tcagtgagcg
agcgagcgcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 3660ttcccaacag
ttgcgcagcc tgaatggcga atggaattcc agacgattga gcgtcaaaat 3720gtaggtattt
ccatgagcgt ttttcctgtt gcaatggctg gcggtaatat tgttctggat 3780attaccagca
aggccgatag tttgagttct tctactcagg caagtgatgt tattactaat 3840caaagaagta
ttgcgacaac ggttaatttg cgtgatggac agactctttt actcggtggc 3900ctcactgatt
ataaaaacac ttctcaggat tctggcgtac cgttcctgtc taaaatccct 3960ttaatcggcc
tcctgtttag ctcccgctct gattctaacg aggaaagcac gttatacgtg 4020ctcgtcaaag
caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 4080ggttacgcgc
agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 4140cttcccttcc
tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 4200ccctttaggg
ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 4260tgatggttca
cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 4320gtccacgttc
tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 4380ggtctattct
tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 4440gctgatttaa
caaaaattta acgcgaattt taacaaaata ttaacgttta caatttaaat 4500atttgcttat
acaatcttcc tgtttttggg gcttttctga ttatcaaccg gggtacatat 4560gattgacatg
ctagttttac gattaccgtt catcgattct cttgtttgct ccagactctc 4620aggcaatgac
ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg 4680aatttatcag
ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt 4740tctcacccgt
ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag 4800ggttctaaaa
atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag 4860ggtcataatg
tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat 4920tttgctaatt
ctttgccttg cctgtatgat ttattggatg ttggaattcc tgatgcggta 4980ttttctcctt
acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat 5040ctgctctgat
gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc 5100ctgacgggct
tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 5160ctgcatgtgt
cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt 5220gatacgccta
tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 5280cacttttcgg
ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 5340tatgtatccg
ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 5400gagtatgagt
attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 5460tcctgttttt
gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 5520tgcacgagtg
ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 5580ccccgaagaa
cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 5640atcccgtatt
gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 5700cttggttgag
tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 5760attatgcagt
gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 5820gatcggagga
ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 5880ccttgatcgt
tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 5940gatgcctgta
gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 6000agcttcccgg
caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 6060gcgctcggcc
cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 6120gtctcgcggt
atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 6180ctacacgacg
gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 6240tgcctcactg
attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 6300tgatttaaaa
cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 6360catgaccaaa
atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 6420gatcaaagga
tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 6480aaaaccaccg
ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 6540gaaggtaact
ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 6600gttaggccac
cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 6660gttaccagtg
gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 6720atagttaccg
gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 6780cttggagcga
acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 6840cacgcttccc
gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 6900agagcgcacg
agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 6960tcgccacctc
tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 7020gaaaaacgcc
agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 7080catgttcttt
cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 7140agctgatacc
gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 7200ggaagagcgc
ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatg
72571615DNAArtificial SequenceSynthetic Polynucleotide 16tcctggaggg gaagt
151715DNAArtificial
SequenceSynthetic Polynucleotide 17tcctggatgg gaagt
151824DNAArtificial SequenceSynthetic
Polynucleotide 18ttcccatcca ggcctggatg ggaa
241924DNAArtificial SequenceSynthetic Polynucleotide
19cttaccgctg cacctggatg ggaa
242020DNAArtificial SequenceSynthetic Polynucleotide 20gggggggggg
gggggggggg
202122DNAArtificial SequenceSynthetic Polynucleotide 21tgactgtgaa
ggttagagat ga
222215DNAArtificial SequenceSynthetic Polynucleotide 22tcctggaggg gttgt
152318DNAArtificial
SequenceSynthetic Polynucleotide 23tgctcctgga ggggttgt
182410DNAArtificial SequenceSynthetic
Polynucleotide 24cctggcgggg
10254369DNAArtificial SequenceSynthetic Polynucleotide
25aaagcttccc ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc
60cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
120agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt
180gacctaggca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
240ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
300tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
360ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
420ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
480tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
540agatcgcctg gagacgccat ccggactcta aggtaaatat aaaattttta agtgtataat
600gtgttaaact actgattcta attgtttctc tcttttagat tccaaccttt ggaactgaat
660tccgcgggcc cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac
720cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
780gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
840caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
900gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
960cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
1020cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
1080cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
1140cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
1200caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
1260cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
1320agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
1380cactctcggc atggacgagc tgtacaagta aagcggccgc taggcctcac ctgcgatctc
1440gatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
1500ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
1560gggaggtttt ttaaactagt ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
1620gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
1680gcgcagagag ggacagatcc gggcccgcat gcgtcgacaa ttcactggcc gtcgttttac
1740aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
1800ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
1860gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
1920tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
1980agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
2040ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
2100catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
2160tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
2220cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
2280cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
2340tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
2400tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
2460atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
2520gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
2580aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
2640aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
2700gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
2760cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
2820atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
2880tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
2940ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
3000ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
3060ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
3120tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
3180tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
3240aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
3300tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
3360tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
3420gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
3480agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
3540tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
3600ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
3660cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
3720tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
3780acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
3840gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
3900ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
3960tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
4020attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
4080cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
4140ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
4200aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
4260ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
4320acacaggaaa cagctatgac catgattacg ccaagctctc gagatctag
4369264450DNAArtificial SequenceSynthetic Polynucleotide 26aaagcttccc
ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60cgggcgacca
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120agcgcgcaga
gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt 180gacctaggca
tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 240ggcattatgc
ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 300tagtcatcgc
tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 360ggtttgactc
acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 420ggcaccaaaa
tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 480tgggcggtag
gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 540agatcgcctg
gagacgccat ccggactcta aggtaaatat aaaattttta agtgtataat 600gtgttaaact
actgattcta attgtttctc tcttttagat tccaaccttt ggaactgaat 660tccgcgggcc
cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac 720cggggtggtg
cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 780gtccggcgag
ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 840caccggcaag
ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca 900gtgcttcagc
cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 960cgaaggctac
gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 1020cgccgaggtg
aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 1080cttcaaggag
gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 1140cgtctatatc
atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 1200caacatcgag
gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 1260cgacggcccc
gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa 1320agaccccaac
gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 1380cactctcggc
atggacgagc tgtacaagta aagcggccgc taggcctcac ctgcgatctc 1440gatgctttat
ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 1500ataaacaagt
taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 1560gggaggtttt
ttaaatcgat tggcgcgcac ccacggcctg aaaaatggcg cgcacccacg 1620gcctgaaaaa
tggcgcgcac ccacggcctg aaaaaactag tccactccct ctctgcgcgc 1680tcgctcgctc
actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 1740ggcctcagtg
agcgagcgag cgcgcagaga gggacagatc cgggcccgca tgcgtcgaca 1800attcactggc
cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 1860atcgccttgc
agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 1920atcgcccttc
ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc 1980tccttacgca
tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 2040ctgatgccgc
atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 2100gggcttgtct
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 2160tgtgtcagag
gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 2220gcctattttt
ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 2280ttcggggaaa
tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 2340atccgctcat
gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 2400tgagtattca
acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 2460tttttgctca
cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 2520gagtgggtta
catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 2580aagaacgttt
tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 2640gtattgacgc
cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 2700ttgagtactc
accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 2760gcagtgctgc
cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 2820gaggaccgaa
ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 2880atcgttggga
accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 2940ctgtagcaat
ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 3000cccggcaaca
attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 3060cggcccttcc
ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 3120gcggtatcat
tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 3180cgacggggag
tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 3240cactgattaa
gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 3300taaaacttca
tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 3360ccaaaatccc
ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 3420aaggatcttc
ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 3480caccgctacc
agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 3540taactggctt
cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag 3600gccaccactt
caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 3660cagtggctgc
tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 3720taccggataa
ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 3780agcgaacgac
ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 3840ttcccgaagg
gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 3900gcacgaggga
gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 3960acctctgact
tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 4020acgccagcaa
cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 4080tctttcctgc
gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 4140ataccgctcg
ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 4200agcgcccaat
acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 4260acgacaggtt
tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 4320tcactcatta
ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 4380ttgtgagcgg
ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct 4440cgagatctag
4450274425DNAArtificial SequenceSynthetic Polynucleotide 27aaagcttccc
ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60cgggcgacca
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120agcgcgcaga
gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt 180gacctaggca
tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 240ggcattatgc
ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 300tagtcatcgc
tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 360ggtttgactc
acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 420ggcaccaaaa
tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 480tgggcggtag
gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 540agatcgcctg
gagacgccat ccggactcta aggtaaatat aaaattttta agtgtataat 600gtgttaaact
actgattcta attgtttctc tcttttagat tccaaccttt ggaactgaat 660tccgcgggcc
cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac 720cggggtggtg
cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 780gtccggcgag
ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 840caccggcaag
ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca 900gtgcttcagc
cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 960cgaaggctac
gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 1020cgccgaggtg
aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 1080cttcaaggag
gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 1140cgtctatatc
atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 1200caacatcgag
gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 1260cgacggcccc
gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa 1320agaccccaac
gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 1380cactctcggc
atggacgagc tgtacaagta aagcggccgc taggcctcac ctgcgatctc 1440gatgctttat
ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 1500ataaacaagt
taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 1560gggaggtttt
ttaaatcgat tggcgcgcac ccacggcctg aaaaatggcg cgcacccacg 1620gcctgaaaaa
actagtccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 1680ccaaaggtcg
cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 1740agagagggac
agatccgggc ccgcatgcgt cgacaattca ctggccgtcg ttttacaacg 1800tcgtgactgg
gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt 1860cgccagctgg
cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 1920cctgaatggc
gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 1980acaccgcata
tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc 2040ccgacacccg
ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 2100ttacagacaa
gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 2160accgaaacgc
gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 2220gataataatg
gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 2280tatttgttta
tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 2340ataaatgctt
caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 2400ccttattccc
ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 2460gaaagtaaaa
gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 2520caacagcggt
aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 2580ttttaaagtt
ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 2640cggtcgccgc
atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 2700gcatcttacg
gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 2760taacactgcg
gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 2820tttgcacaac
atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 2880agccatacca
aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 2940caaactatta
actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 3000ggaggcggat
aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 3060tgctgataaa
tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 3120agatggtaag
ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 3180tgaacgaaat
agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 3240agaccaagtt
tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 3300gatctaggtg
aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 3360gttccactga
gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 3420tctgcgcgta
atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 3480gccggatcaa
gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 3540accaaatact
gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 3600accgcctaca
tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 3660gtcgtgtctt
accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 3720ctgaacgggg
ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 3780atacctacag
cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 3840gtatccggta
agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 3900cgcctggtat
ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 3960gtgatgctcg
tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 4020gttcctggcc
ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 4080tgtggataac
cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 4140cgagcgcagc
gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 4200ccccgcgcgt
tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc 4260gggcagtgag
cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt 4320acactttatg
cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac 4380aggaaacagc
tatgaccatg attacgccaa gctctcgaga tctag
4425284465DNAArtificial SequenceSynthetic Polynucleotide 28aaagcttccc
ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60cgggcgacca
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120agcgcgcaga
gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt 180gacctaggca
tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 240ggcattatgc
ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 300tagtcatcgc
tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 360ggtttgactc
acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 420ggcaccaaaa
tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 480tgggcggtag
gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 540agatcgcctg
gagacgccat ccggactcta aggtaaatat aaaattttta agtgtataat 600gtgttaaact
actgattcta attgtttctc tcttttagat tccaaccttt ggaactgaat 660tccgcgggcc
cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac 720cggggtggtg
cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 780gtccggcgag
ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 840caccggcaag
ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca 900gtgcttcagc
cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 960cgaaggctac
gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 1020cgccgaggtg
aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 1080cttcaaggag
gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 1140cgtctatatc
atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 1200caacatcgag
gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 1260cgacggcccc
gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa 1320agaccccaac
gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 1380cactctcggc
atggacgagc tgtacaagta aagcggccgc taggcctcac ctgcgatctc 1440gatgctttat
ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 1500ataaacaagt
taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 1560gggaggtttt
ttaaatcgat tttagggtta gggttagggt tagggaaaaa tttagggtta 1620gggttagggt
tagggaaaaa tttagggtta gggttagggt tagggaaaaa actagtccac 1680tccctctctg
cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 1740gggctttgcc
cgggcggcct cagtgagcga gcgagcgcgc agagagggac agatccgggc 1800ccgcatgcgt
cgacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 1860gcgttaccca
acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 1920aagaggcccg
caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 1980tgatgcggta
ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 2040tcagtacaat
ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 2100ctgacgcgcc
ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 2160tctccgggag
ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 2220agggcctcgt
gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 2280cgtcaggtgg
cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 2340tacattcaaa
tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 2400gaaaaaggaa
gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 2460cattttgcct
tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 2520atcagttggg
tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 2580agagttttcg
ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 2640gcgcggtatt
atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 2700ctcagaatga
cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 2760cagtaagaga
attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 2820ttctgacaac
gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 2880atgtaactcg
ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 2940gtgacaccac
gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 3000tacttactct
agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 3060gaccacttct
gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 3120gtgagcgtgg
gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 3180tcgtagttat
ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 3240ctgagatagg
tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 3300tactttagat
tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 3360ttgataatct
catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 3420ccgtagaaaa
gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 3480tgcaaacaaa
aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 3540ctctttttcc
gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 3600tgtagccgta
gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 3660tgctaatcct
gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 3720actcaagacg
atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 3780cacagcccag
cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 3840gagaaagcgc
cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 3900tcggaacagg
agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 3960ctgtcgggtt
tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 4020ggagcctatg
gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 4080cttttgctca
catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 4140cctttgagtg
agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 4200gcgaggaagc
ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 4260attaatgcag
ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 4320ttaatgtgag
ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 4380gtatgttgtg
tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 4440attacgccaa
gctctcgaga tctag
4465294405DNAArtificial SequenceSynthetic Polynucleotide 29aaagcttccc
ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60cgggcgacca
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120agcgcgcaga
gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt 180gacctaggca
tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 240ggcattatgc
ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 300tagtcatcgc
tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 360ggtttgactc
acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 420ggcaccaaaa
tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 480tgggcggtag
gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 540agatcgcctg
gagacgccat ccggactcta aggtaaatat aaaattttta agtgtataat 600gtgttaaact
actgattcta attgtttctc tcttttagat tccaaccttt ggaactgaat 660tccgcgggcc
cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac 720cggggtggtg
cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 780gtccggcgag
ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 840caccggcaag
ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca 900gtgcttcagc
cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 960cgaaggctac
gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 1020cgccgaggtg
aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 1080cttcaaggag
gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 1140cgtctatatc
atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 1200caacatcgag
gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 1260cgacggcccc
gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa 1320agaccccaac
gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 1380cactctcggc
atggacgagc tgtacaagta aagcggccgc taggcctcac ctgcgatctc 1440gatgctttat
ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 1500ataaacaagt
taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 1560gggaggtttt
ttaaatcgat tttagggtta gggttagggt tagggaaaaa actagtccac 1620tccctctctg
cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 1680gggctttgcc
cgggcggcct cagtgagcga gcgagcgcgc agagagggac agatccgggc 1740ccgcatgcgt
cgacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 1800gcgttaccca
acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 1860aagaggcccg
caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 1920tgatgcggta
ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 1980tcagtacaat
ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 2040ctgacgcgcc
ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 2100tctccgggag
ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 2160agggcctcgt
gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 2220cgtcaggtgg
cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 2280tacattcaaa
tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 2340gaaaaaggaa
gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 2400cattttgcct
tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 2460atcagttggg
tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 2520agagttttcg
ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 2580gcgcggtatt
atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 2640ctcagaatga
cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 2700cagtaagaga
attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 2760ttctgacaac
gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 2820atgtaactcg
ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 2880gtgacaccac
gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 2940tacttactct
agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 3000gaccacttct
gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 3060gtgagcgtgg
gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 3120tcgtagttat
ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 3180ctgagatagg
tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 3240tactttagat
tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 3300ttgataatct
catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 3360ccgtagaaaa
gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 3420tgcaaacaaa
aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 3480ctctttttcc
gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 3540tgtagccgta
gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 3600tgctaatcct
gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 3660actcaagacg
atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 3720cacagcccag
cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 3780gagaaagcgc
cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 3840tcggaacagg
agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 3900ctgtcgggtt
tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 3960ggagcctatg
gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 4020cttttgctca
catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 4080cctttgagtg
agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 4140gcgaggaagc
ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 4200attaatgcag
ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 4260ttaatgtgag
ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 4320gtatgttgtg
tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 4380attacgccaa
gctctcgaga tctag
4405304465DNAArtificial SequenceSynthetic Polynucleotide 30aaagcttccc
ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60cgggcgacca
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120agcgcgcaga
gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt 180gacctaggtt
tagggttagg gttagggtta gggaaaaatt tagggttagg gttagggtta 240gggaaaaatt
tagggttagg gttagggtta gggaaaaacc taggcatatg ccaagtacgc 300cccctattga
cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 360tatgggactt
tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 420tgcggttttg
gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 480gtctccaccc
cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 540caaaatgtcg
taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 600aggtctatat
aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccgg 660actctaaggt
aaatataaaa tttttaagtg tataatgtgt taaactactg attctaattg 720tttctctctt
ttagattcca acctttggaa ctgaattccg cgggcccggg atccaccggt 780cgccaccatg
gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 840gctggacggc
gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc 900cacctacggc
aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg 960gcccaccctc
gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca 1020catgaagcag
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 1080catcttcttc
aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga 1140caccctggtg
aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct 1200ggggcacaag
ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca 1260gaagaacggc
atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca 1320gctcgccgac
cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 1380caaccactac
ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca 1440catggtcctg
ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta 1500caagtaaagc
ggccgctagg cctcacctgc gatctcgatg ctttatttgt gaaatttgtg 1560atgctattgc
tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt 1620gcattcattt
tatgtttcag gttcaggggg aggtgtggga ggttttttaa actagtccac 1680tccctctctg
cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 1740gggctttgcc
cgggcggcct cagtgagcga gcgagcgcgc agagagggac agatccgggc 1800ccgcatgcgt
cgacaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg 1860gcgttaccca
acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg 1920aagaggcccg
caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc 1980tgatgcggta
ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc 2040tcagtacaat
ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg 2100ctgacgcgcc
ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg 2160tctccgggag
ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa 2220agggcctcgt
gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga 2280cgtcaggtgg
cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 2340tacattcaaa
tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 2400gaaaaaggaa
gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 2460cattttgcct
tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 2520atcagttggg
tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 2580agagttttcg
ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 2640gcgcggtatt
atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 2700ctcagaatga
cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 2760cagtaagaga
attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 2820ttctgacaac
gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 2880atgtaactcg
ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 2940gtgacaccac
gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac 3000tacttactct
agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 3060gaccacttct
gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 3120gtgagcgtgg
gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 3180tcgtagttat
ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 3240ctgagatagg
tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 3300tactttagat
tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 3360ttgataatct
catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 3420ccgtagaaaa
gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 3480tgcaaacaaa
aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 3540ctctttttcc
gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag 3600tgtagccgta
gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 3660tgctaatcct
gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 3720actcaagacg
atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 3780cacagcccag
cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 3840gagaaagcgc
cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 3900tcggaacagg
agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 3960ctgtcgggtt
tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 4020ggagcctatg
gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 4080cttttgctca
catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 4140cctttgagtg
agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 4200gcgaggaagc
ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc 4260attaatgcag
ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa 4320ttaatgtgag
ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc 4380gtatgttgtg
tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg 4440attacgccaa
gctctcgaga tctag
4465314435DNAArtificial SequenceSynthetic Polynucleotide 31aaagcttccc
ggggggatct gggccactcc ctctctgcgc gctcgctcgc tcactgaggc 60cgggcgacca
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 120agcgcgcaga
gagggagtgg ccaactccat cactaggggt tcctggaggg gtggagtcgt 180gacctaggca
tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 240ggcattatgc
ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 300tagtcatcgc
tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 360ggtttgactc
acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 420ggcaccaaaa
tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 480tgggcggtag
gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 540agatcgcctg
gagacgccat ccggactcta aggtaaatat aaaattttta agtgtataat 600gtgttaaact
actgattcta attgtttctc tcttttagat tccaaccttt ggaactgaat 660tccgcgggcc
cgggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac 720cggggtggtg
cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt 780gtccggcgag
ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac 840caccggcaag
ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca 900gtgcttcagc
cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc 960cgaaggctac
gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg 1020cgccgaggtg
aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga 1080cttcaaggag
gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa 1140cgtctatatc
atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca 1200caacatcgag
gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg 1260cgacggcccc
gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa 1320agaccccaac
gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat 1380cactctcggc
atggacgagc tgtacaagta aagcggccgc taggcctcac ctgcgatctc 1440gatgctttat
ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca 1500ataaacaagt
taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 1560gggaggtttt
ttaaatcgat gctagatgtt agcgtaaaaa gctagatgtt agcgtaaaaa 1620gctagatgtt
agcgtaaaaa actagtccac tccctctctg cgcgctcgct cgctcactga 1680ggccgggcga
ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga 1740gcgagcgcgc
agagagggac agatccgggc ccgcatgcgt cgacaattca ctggccgtcg 1800ttttacaacg
tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 1860atcccccttt
cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 1920agttgcgcag
cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 1980gcggtatttc
acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 2040taagccagcc
ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 2100cggcatccgc
ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 2160caccgtcatc
accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 2220ttaatgtcat
gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 2280gcggaacccc
tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 2340aataaccctg
ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 2400tccgtgtcgc
ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 2460aaacgctggt
gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 2520aactggatct
caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 2580tgatgagcac
ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 2640aagagcaact
cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 2700tcacagaaaa
gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 2760ccatgagtga
taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 2820taaccgcttt
tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 2880agctgaatga
agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 2940caacgttgcg
caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 3000tagactggat
ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 3060gctggtttat
tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 3120cactggggcc
agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 3180caactatgga
tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 3240ggtaactgtc
agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 3300aatttaaaag
gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 3360gtgagttttc
gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 3420atcctttttt
tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 3480tggtttgttt
gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 3540gagcgcagat
accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 3600actctgtagc
accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 3660gtggcgataa
gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 3720agcggtcggg
ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 3780ccgaactgag
atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 3840aggcggacag
gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 3900cagggggaaa
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 3960gtcgattttt
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 4020cctttttacg
gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 4080cccctgattc
tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 4140gccgaacgac
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 4200aaccgcctct
ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg 4260actggaaagc
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac 4320cccaggcttt
acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac 4380aatttcacac
aggaaacagc tatgaccatg attacgccaa gctctcgaga tctag
4435326940DNAArtificial SequenceSynthetic Polynucleotide 32cagcagctgc
gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60acctttggtc
gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc 120atcactaggg
gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca 180tgctctagcg
gcctcggcct ctgcataaat aaaaaaaatt agtcagccat gagcttggcc 240cattgcatac
gttgtatcca tatcataata tgtacattta tattggctca tgtccaacat 300taccgccatg
ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 360tagttcatag
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 420gctgaccgcc
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 480cgccaatagg
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 540tggcagtaca
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 600aatggcccgc
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 660acatctacgt
attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg 720ggcgtggata
gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 780ggagtttgtt
ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc 840cattgacgca
aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 900tagtgaaccg
tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac 960accgggaccg
atccagcctc ccctcgaagc tgatcctgag aacttcaggg tgagtctatg 1020ggacccttga
tgttttcttt ccccttcttt tctatggtta agttcatgtc ataggaaggg 1080gagaagtaac
agggtacaca tattgaccaa atcagggtaa ttttgcattt gtaattttaa 1140aaaatgcttt
cttcttttaa tatacttttt tgtttatctt atttctaata ctttccctaa 1200tctctttctt
tcagggcaat aatgatacaa tgtatcatgc ctctttgcac cattctaaag 1260aataacagtg
ataatttctg ggttaaggca atagcaatat ttctgcatat aaatatttct 1320gcatataaat
tgtaactgat gtaagaggtt tcatattgct aatagcagct acaatccagc 1380taccattctg
cttttatttt atggttggga taaggctgga ttattctgag tccaagctag 1440gcccttttgc
taatcatgtt catacctctt atcttcctcc cacagctcct gggcaacgtg 1500ctggtctgtg
tgctggccca tcactttggc aaagaattcc gcgggcccgg gatccaccgg 1560tcgccaccat
ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg 1620agctggacgg
cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg 1680ccacctacgg
caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct 1740ggcccaccct
cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc 1800acatgaagca
gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca 1860ccatcttctt
caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg 1920acaccctggt
gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc 1980tggggcacaa
gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc 2040agaagaacgg
catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc 2100agctcgccga
ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg 2160acaaccacta
cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc 2220acatggtcct
gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt 2280acaagtaaag
cggccgctct agaggatcca agcttatcga taccgtcgac ctcgagggcc 2340cagatctaat
tcaccccacc agtgcaggct gcctatcaga aagtggtggc tggtgtggct 2400aatgccctgg
cccacaagta tcactaagct cgctttcttg ctgtccaatt tctattaaag 2460gttcctttgt
tccctaagtc caactactaa actgggggat attatgaagg gccttgagca 2520tctggattct
gcctaataaa aaacatttat tttcattgca atgatgtatt taaattattt 2580ctgaatattt
tactaaaaag ggaatgtggg aggtcagtgc atttaaaaca taaagaaatg 2640aagagctagt
tcaaaccttg ggaaaataca ctatatctta aactccatga aagaaggtga 2700ggctgcaaac
agctaatgca cattggcaac agcccctgat gcctatgcct tattcatccc 2760tcagaaaagg
attcaagtag aggcttgatt tggaggttaa agttttgcta tgctgtattt 2820tacattactt
attgttttag ctgtcctcat gaatgtcttt tcactaccca tttgcttatc 2880ctgcatctct
cagccttgac tccactcagt tctcttgctt agagatacca cctttcccct 2940gaagtgttcc
ttccatgttt tacggcgaga tggtttctcc tcgcctggcc actcagcctt 3000agttgtctct
gttgtcttat agaggtctac ttgaagaagg aaaaacaggg ggcatggttt 3060gactgtcctg
tgagcccttc ttccctgcct cccccactca cagtgacccg gaatccctcg 3120acatctagag
catggctacg tagataagta gcatggcggg ttaatcatta actacaagga 3180acccctagtg
atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 3240gcgaccaaag
gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 3300gcgccagctg
gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca 3360gcctgaatgg
cgaatggaat tccagacgat tgagcgtcaa aatgtaggta tttccatgag 3420cgtttttcct
gttgcaatgg ctggcggtaa tattgttctg gatattacca gcaaggccga 3480tagtttgagt
tcttctactc aggcaagtga tgttattact aatcaaagaa gtattgcgac 3540aacggttaat
ttgcgtgatg gacagactct tttactcggt ggcctcactg attataaaaa 3600cacttctcag
gattctggcg taccgttcct gtctaaaatc cctttaatcg gcctcctgtt 3660tagctcccgc
tctgattcta acgaggaaag cacgttatac gtgctcgtca aagcaaccat 3720agtacgcgcc
ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 3780ccgctacact
tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg 3840ccacgttcgc
cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 3900ttagtgcttt
acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 3960ggccatcgcc
ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4020gtggactctt
gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4080tataagggat
tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 4140ttaacgcgaa
ttttaacaaa atattaacgt ttacaattta aatatttgct tatacaatct 4200tcctgttttt
ggggcttttc tgattatcaa ccggggtaca tatgattgac atgctagttt 4260tacgattacc
gttcatcgat tctcttgttt gctccagact ctcaggcaat gacctgatag 4320cctttgtaga
gacctctcaa aaatagctac cctctccggc atgaatttat cagctagaac 4380ggttgaatat
catattgatg gtgatttgac tgtctccggc ctttctcacc cgtttgaatc 4440tttacctaca
cattactcag gcattgcatt taaaatatat gagggttcta aaaattttta 4500tccttgcgtt
gaaataaagg cttctcccgc aaaagtatta cagggtcata atgtttttgg 4560tacaaccgat
ttagctttat gctctgaggc tttattgctt aattttgcta attctttgcc 4620ttgcctgtat
gatttattgg atgttggaat tcctgatgcg gtattttctc cttacgcatc 4680tgtgcggtat
ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat 4740agttaagcca
gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 4800tcccggcatc
cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 4860tttcaccgtc
atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 4920aggttaatgt
catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 4980tgcgcggaac
ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 5040gacaataacc
ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 5100atttccgtgt
cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 5160cagaaacgct
ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 5220tcgaactgga
tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 5280caatgatgag
cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 5340ggcaagagca
actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 5400cagtcacaga
aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 5460taaccatgag
tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 5520agctaaccgc
ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 5580cggagctgaa
tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 5640caacaacgtt
gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 5700taatagactg
gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 5760ctggctggtt
tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 5820cagcactggg
gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 5880aggcaactat
ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 5940attggtaact
gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 6000tttaatttaa
aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 6060aacgtgagtt
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 6120gagatccttt
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 6180cggtggtttg
tttgccggat caagagctac caactctttt tccgaaggta actggcttca 6240gcagagcgca
gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca 6300agaactctgt
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 6360ccagtggcga
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 6420cgcagcggtc
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 6480acaccgaact
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 6540gaaaggcgga
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 6600ttccaggggg
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 6660agcgtcgatt
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 6720cggccttttt
acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 6780tatcccctga
ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 6840gcagccgaac
gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 6900gcaaaccgcc
tctccccgcg cgttggccga ttcattaatg
6940337257DNAArtificial SequenceSynthetic Polynucleotide 33cagcagctgc
gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60acctttggtc
gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaactcc 120atcactaggg
gttccttgta gttaatgatt aacccgccat gctacttatc tacgtagcca 180tgctctagcg
gcctcggcct ctgcataaat aaaaaaaatt agtcagccat gagcttggcc 240cattgcatac
gttgtatcca tatcataata tgtacattta tattggctca tgtccaacat 300taccgccatg
ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat 360tagttcatag
cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg 420gctgaccgcc
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa 480cgccaatagg
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact 540tggcagtaca
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta 600aatggcccgc
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt 660acatctacgt
attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg 720ggcgtggata
gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg 780ggagtttgtt
ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc 840cattgacgca
aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt 900tagtgaaccg
tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac 960accgggaccg
atccagcctc ccctcgaagc tgatcctgag aacttcaggg tgagtctatg 1020ggacccttga
tgttttcttt ccccttcttt tctatggtta agttcatgtc ataggaaggg 1080gagaagtaac
agggtacaca tattgaccaa atcagggtaa ttttgcattt gtaattttaa 1140aaaatgcttt
cttcttttaa tatacttttt tgtttatctt atttctaata ctttccctaa 1200tctctttctt
tcagggcaat aatgatacaa tgtatcatgc ctctttgcac cattctaaag 1260aataacagtg
ataatttctg ggttaaggca atagcaatat ttctgcatat aaatatttct 1320gcatataaat
tgtaactgat gtaagaggtt tcatattgct aatagcagct acaatccagc 1380taccattctg
cttttatttt atggttggga taaggctgga ttattctgag tccaagctag 1440gcccttttgc
taatcatgtt catacctctt atcttcctcc cacagctcct gggcaacgtg 1500ctggtctgtg
tgctggccca tcactttggc aaagaattcc gcgggcccgg gatccaccgg 1560tcgccaccat
ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg 1620agctggacgg
cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg 1680ccacctacgg
caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct 1740ggcccaccct
cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc 1800acatgaagca
gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca 1860ccatcttctt
caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg 1920acaccctggt
gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc 1980tggggcacaa
gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc 2040agaagaacgg
catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc 2100agctcgccga
ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg 2160acaaccacta
cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc 2220acatggtcct
gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt 2280acaagtaaag
cggccgctct agaggatcca agcttatcga taccgtcgac ctcgagggcc 2340cagatctaat
tcaccccacc agtgcaggct gcctatcaga aagtggtggc tggtgtggct 2400aatgccctgg
cccacaagta tcactaagct cgctttcttg ctgtccaatt tctattaaag 2460gttcctttgt
tccctaagtc caactactaa actgggggat attatgaagg gccttgagca 2520tctggattct
gcctaataaa aaacatttat tttcattgca atgatgtatt taaattattt 2580ctgaatattt
tactaaaaag ggaatgtggg aggtcagtgc atttaaaaca taaagaaatg 2640aagagctagt
tcaaaccttg ggaaaataca ctatatctta aactccatga aagaaggtga 2700ggctgcaaac
agctaatgca cattggcaac agcccctgat gcctatgcct tattcatccc 2760tcagaaaagg
attcaagtag aggcttgatt tggaggttaa agttttgcta tgctgtattt 2820tacattactt
attgttttag ctgtcctcat gaatgtcttt tcactaccca tttgcttatc 2880ctgcatctct
cagccttgac tccactcagt tctcttgctt agagatacca cctttcccct 2940gaagtgttcc
ttccatgttt tacggcgaga tggtttctcc tcgcctggcc actcagcctt 3000agttgtctct
gttgtcttat agaggtctac ttgaagaagg aaaaacaggg ggcatggttt 3060gactgtcctg
tgagcccttc ttccctgcct cccccactca cagtgacccg gaatccctcg 3120acaggtacct
ttagggttag ggttagggtt agggaaaaat ttagggttag ggttagggtt 3180agggaaaaat
ttagggttag ggttagggtt agggaaaaat ttagggttag ggttagggtt 3240agggaaaaat
ttagggttag ggttagggtt agggaaaaac cctaacccta accctaaccc 3300taaatttttt
ccctaaccct aaccctaacc ctaaattttt tccctaaccc taaccctaac 3360cctaaatttt
ttccctaacc ctaaccctaa ccctaaattt tttccctaac cctaacccta 3420accctaaatt
ttttgctagc tctagagcat ggctacgtag ataagtagca tggcgggtta 3480atcattaact
acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 3540tcgctcactg
aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 3600tcagtgagcg
agcgagcgcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc 3660ttcccaacag
ttgcgcagcc tgaatggcga atggaattcc agacgattga gcgtcaaaat 3720gtaggtattt
ccatgagcgt ttttcctgtt gcaatggctg gcggtaatat tgttctggat 3780attaccagca
aggccgatag tttgagttct tctactcagg caagtgatgt tattactaat 3840caaagaagta
ttgcgacaac ggttaatttg cgtgatggac agactctttt actcggtggc 3900ctcactgatt
ataaaaacac ttctcaggat tctggcgtac cgttcctgtc taaaatccct 3960ttaatcggcc
tcctgtttag ctcccgctct gattctaacg aggaaagcac gttatacgtg 4020ctcgtcaaag
caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 4080ggttacgcgc
agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 4140cttcccttcc
tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 4200ccctttaggg
ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 4260tgatggttca
cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 4320gtccacgttc
tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 4380ggtctattct
tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 4440gctgatttaa
caaaaattta acgcgaattt taacaaaata ttaacgttta caatttaaat 4500atttgcttat
acaatcttcc tgtttttggg gcttttctga ttatcaaccg gggtacatat 4560gattgacatg
ctagttttac gattaccgtt catcgattct cttgtttgct ccagactctc 4620aggcaatgac
ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg 4680aatttatcag
ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt 4740tctcacccgt
ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag 4800ggttctaaaa
atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag 4860ggtcataatg
tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat 4920tttgctaatt
ctttgccttg cctgtatgat ttattggatg ttggaattcc tgatgcggta 4980ttttctcctt
acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat 5040ctgctctgat
gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc 5100ctgacgggct
tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 5160ctgcatgtgt
cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt 5220gatacgccta
tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 5280cacttttcgg
ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 5340tatgtatccg
ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 5400gagtatgagt
attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 5460tcctgttttt
gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 5520tgcacgagtg
ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 5580ccccgaagaa
cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 5640atcccgtatt
gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 5700cttggttgag
tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 5760attatgcagt
gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 5820gatcggagga
ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 5880ccttgatcgt
tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 5940gatgcctgta
gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 6000agcttcccgg
caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 6060gcgctcggcc
cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 6120gtctcgcggt
atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 6180ctacacgacg
gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 6240tgcctcactg
attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 6300tgatttaaaa
cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 6360catgaccaaa
atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 6420gatcaaagga
tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 6480aaaaccaccg
ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 6540gaaggtaact
ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 6600gttaggccac
cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 6660gttaccagtg
gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 6720atagttaccg
gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 6780cttggagcga
acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 6840cacgcttccc
gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 6900agagcgcacg
agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 6960tcgccacctc
tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 7020gaaaaacgcc
agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 7080catgttcttt
cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 7140agctgatacc
gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 7200ggaagagcgc
ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatg
72573415DNAArtificial SequenceSynthetic Polynucleotide 34gctagatgtt agcgt
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