NUCLEIC ACID MOLECULES ENCODING AN ENGINEERED ANTIGEN RECEPTOR AND AN INHIBITORY NUCLEIC ACID MOLECULE AND METHODS OF USE THEREOF

Abstract

The present disclosure provides nucleic acid molecules encoding an engineered antigen receptor, such as a chimeric antigen receptor or exogenous T cell receptor, and an inhibitory nucleic acid molecule, such as an RNA interference molecule. The present disclosure further relates to nucleic acids, DNA constructs, vectors, pharmaceutical compositions, genetically-modified cells, and methods of treatment that utilize the nucleic acid molecules of the invention.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a Continuation of U.S. application Ser. No. 16/678,600, filed Nov. 8, 2019, which is a Continuation of PCT/US2018/031674 filed May 8, 2018, which International Application was published by the International Bureau in English on Nov. 15, 2018, and application claims priority from U.S. Provisional Patent Application No. 62/503,060, filed May 8, 2017, and U.S. Provisional Patent Application No. 62/579,460, filed Oct. 31, 2017, which applications are hereby incorporated in their entirety by reference in this application.

FIELD OF THE INVENTION

[0002] The present disclosure relates to the field of molecular biology and recombinant nucleic acid technology. In particular, the present disclosure relates to nucleic acid molecules encoding an engineered antigen receptor, such as a chimeric antigen receptor or exogenous T cell receptor, and an inhibitory nucleic acid molecule, such as an RNA interference molecule. The present disclosure further relates to nucleic acids, DNA constructs, viral vectors, pharmaceutical compositions, genetically-modified cells, and methods of treatment that utilize the nucleic acid molecule of the invention.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

[0003] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 19, 2020, is named P89339_1050US_C1_Seq_List, and is 188580 bytes in size.

BACKGROUND OF THE INVENTION

[0004] T cell adoptive immunotherapy is a promising approach for cancer treatment. This strategy utilizes isolated human T cells that have been genetically-modified to enhance their specificity for a specific tumor associated antigen. Genetic modification may involve the expression of a chimeric antigen receptor (CAR) or an exogenous T cell receptor to graft antigen specificity onto the T cell. By contrast to exogenous T cell receptors, CARs derive their specificity from the variable domains of a monoclonal antibody. Thus, T cells expressing CARs induce tumor immunoreactivity in a major histocompatibility complex (MEW) non-restricted manner. To date, T cell adoptive immunotherapy has been utilized as a clinical therapy for a number of cancers, including B cell malignancies (e.g., acute lymphoblastic leukemia (ALL), B cell non-Hodgkin lymphoma (NEIL), and chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma, advanced gliomas, ovarian cancer, mesothelioma, melanoma, and pancreatic cancer.

[0005] Despite its potential usefulness as a cancer treatment, adoptive immunotherapy has been limited, in part, by alloreactivity between host tissues and allogeneic CAR T cells. One cause of alloreactivity arises from the presence of non-host MHC class I molecules on the cell surface of CART cells. MEW class I molecules consist of two polypeptide chains, .alpha. and .beta.. In humans, the .alpha. chain consists of three subunits, .alpha.1, .alpha.2, and .alpha.3, which are encoded by polymorphic human leukocyte antigen (HLA) genes on chromosome 6. The variability of HLA loci, and the encoded .alpha. chain subunits, can cause allogeneic CAR T cells to be seen by the host immune system as foreign cells because they bear foreign MHC class I molecules. As a result, CAR T cells administered to a patient can be subject to host versus graft (HvG) rejection, where they are recognized and killed by the host's cytotoxic T cells.

[0006] The .beta. chain of MHC class I molecules consists of beta-2 microglobulin, which is encoded by the non-polymorphic beta-2 microglobulin (B2M) gene on chromosome 15 (SEQ ID NO: 1). Beta-2 microglobulin is non-covalently linked to the .alpha.3 subunit and is common to all MHC class I molecules. Furthermore, expression of MHC class I molecules at the cell surface requires its association with beta-2 microglobulin. As such, beta-2 microglobulin represents a logical target for suppressing the expression of MHC class I molecules on CAR T cells, which could render the cells invisible to host cytotoxic T cells and reduce alloreactivity. However, complete knockout of beta-2 microglobulin expression may result in NK cell killing of CAR T cells due to the lack of cell surface MHC class I molecules, which could prompt NK cells to recognize them as non-self and initiate cytotoxic action.

[0007] Another cause of alloreactivity to CAR T cells is the expression of the endogenous T cell receptor on the cell surface. T cell receptors typically consist of variable a and .beta. chains or, in smaller numbers, variable .gamma. and .delta. chains. The T cell receptor complexes with accessory proteins, including CD3, and functions with cell surface co-receptors (e.g., CD4 and CD8) to recognize antigens bound to MEW molecules on antigen presenting cells. In the case of allogeneic CAR T cells, expression of endogenous T cell receptors may cause the cell to recognize host MHC antigens following administration to a patient, which can lead to the development of graft-versus-host-disease (GVHD).

[0008] To forestall alloreactivity, clinical trials have largely focused on the use of autologous CAR T cells, wherein a donor's T cells are isolated, genetically-modified to incorporate a chimeric antigen receptor, and then re-infused into the same subject. An autologous approach provides immune tolerance to the administered CAR T cells; however, this approach is constrained by both the time and expense necessary to produce patient-specific CAR T cells after a patient's cancer has been diagnosed.

[0009] Thus, a need exists in the art for the development of allogeneic CAR T cells which exhibit reduced allogenicity but, at the same time, avoid NK cell killing in vivo.

SUMMARY OF THE INVENTION

[0010] In one aspect, the invention provides a nucleic acid molecule comprising: (a) a first expression cassette comprising a nucleic acid sequence encoding an engineered antigen receptor; (b) a second expression cassette comprising a nucleic acid sequence encoding an inhibitory nucleic acid molecule; (c) a 5' homology arm; and (d) a 3' homology arm; wherein the 5' homology arm and the 3' homology arm have homology to chromosomal regions flanking a nuclease recognition sequence in a gene of interest.

[0011] In some embodiments, the inhibitory nucleic acid molecule is an RNA interference molecule. In certain embodiments, the RNA interference molecule is a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a hairpin siRNA, a microRNA (miRNA), a precursor miRNA, or an miRNA-adapted shRNA. In particular embodiments, the RNA interference molecule is an shRNA.

[0012] In some embodiments the engineered antigen receptor is a chimeric antigen receptor. In other embodiments, the engineered antigen receptor is an exogenous T cell receptor.

[0013] In some embodiments, the nuclease recognition sequence is an engineered meganuclease recognition sequence, a TALEN recognition sequence, a zinc finger nuclease (ZFN) recognition sequence, a CRISPR/Cas recognition sequence, a compact TALEN recognition sequence, or a megaTAL recognition sequence. In certain embodiments, the nuclease recognition sequence is an engineered meganuclease recognition sequence.

[0014] In some embodiments, the gene of interest is any gene of interest. In certain embodiments, the gene of interest is a human T cell receptor alpha constant region gene. In particular embodiments the nuclease recognition sequence is an engineered meganuclease recognition sequence. In certain embodiments, the engineered meganuclease recognition sequence comprises SEQ ID NO: 1 in a human T cell receptor alpha constant region gene.

[0015] In some embodiments, the first expression cassette further comprises a promoter which drives expression of the engineered antigen receptor. In certain embodiments, the promoter is a JeT promoter.

[0016] In some embodiments, the second expression cassette further comprises a promoter which drives expression of the inhibitory nucleic acid molecule. In certain embodiments, the promoter is a U6 promoter.

[0017] In some embodiments, the first expression cassette comprises a polyadenylation signal to terminate translation of the engineered antigen receptor. In some embodiments, the second expression cassette comprises a central polypurine tract and central terminator sequence (cPPT/CTS) sequence to terminate translation of the inhibitory nucleic acid.

[0018] In some embodiments, the first expression cassette and the second expression cassette are in the same orientation in the nucleic acid molecule. In certain embodiments, the first expression cassette and the second expression cassette are in a 5' to 3' orientation relative to the 5' and 3' homology arms. In some such embodiments, the first expression cassette is 5' upstream of the second expression cassette. In other such embodiments, the second expression cassette is 5' upstream of the first expression cassette.

[0019] In some embodiments, wherein the first expression cassette and the second expression cassette are in the same orientation in the nucleic acid molecule, the first expression cassette and the second expression cassette are in a 3' to 5' orientation relative to the 5' and 3' homology arms. In some such embodiments, the first expression cassette is 5' upstream of the second expression cassette. In other such embodiments, the second expression cassette is 5' upstream of the first expression cassette.

[0020] In some embodiments, the first expression cassette and the second expression cassette are in opposite orientations in the nucleic acid molecule. In some such embodiments, the first expression cassette is in a 3' to 5' orientation and the second expression cassette is in a 5' to 3' orientation relative to the 5' and 3' homology arms. In certain embodiments, the first expression cassette is 5' upstream of the second expression cassette. In other embodiments, the second expression cassette is 5' upstream of the first expression cassette.

[0021] In particular embodiments, wherein the first expression cassette and the second expression cassette are in opposite orientations in the nucleic acid molecule, the first expression cassette is in a 5' to 3' orientation and the second expression cassette is in a 3' to 5' orientation relative to the 5' and 3' homology arms. In some such embodiments, the first expression cassette is 5' upstream of the second expression cassette. In other such embodiments, the second expression cassette is 5' upstream of the first expression cassette.

[0022] In some embodiments, the nucleic acid molecule comprises multiple copies of the second expression cassette. In some such embodiments, the copies are identical. In further embodiments, the copies include a promoter, a coding sequence for the inhibitory nucleic acid molecule, and a sequence, such as a (cPPT/CTS) sequence, to terminate translation of the inhibitory nucleic acid molecule. In some such embodiments, the copies of the second expression cassette are in tandem in the nucleic acid molecule, and can be in the same orientation, or in opposite orientations. In other such embodiments, the copies may not be in tandem, and can be in the same orientation, or in opposite orientations.

[0023] In some embodiments, the nucleic acid molecule further comprises a 5' inverted terminal repeat and a 3' inverted terminal repeat flanking the first expression cassette and the second expression cassette.

[0024] In some embodiments, the inhibitory nucleic acid molecule is inhibitory against human beta-2 microglobulin.

[0025] In some embodiments, the inhibitory nucleic acid molecule is inhibitory against a component of the MHC class I molecule. In certain embodiments, the inhibitory molecule is inhibitory against an MHC class I alpha-1 (.alpha.1) domain, alpha-2 (.alpha.2) domain, alpha-3 (.alpha.3) domain, or against beta-2 microglobulin.

[0026] In some embodiments, the inhibitory nucleic acid molecule is inhibitory against human CD52.

[0027] In certain embodiments, the inhibitory nucleic acid molecule is an shRNA inhibitory against beta-2 microglobulin, wherein the shRNA has a sequence comprising any one of SEQ ID NOs: 2-4. In particular embodiments, the shRNA has a sequence comprising SEQ ID NO: 2. In some such embodiments, the first expression cassette and the second expression cassette are in a 3' to 5' orientation relative to the 5' and 3' homology arms, and wherein the first expression cassette is 5' upstream of the second expression cassette. In some such embodiments, the first expression cassette comprises: (i) a nucleic acid sequence encoding a chimeric antigen receptor or an exogenous T cell receptor; (ii) a JeT promoter which drives expression of the chimeric antigen receptor or the exogenous T cell receptor; and (iii) a polyA sequence; and the second expression cassette comprises: (iv) a nucleic acid sequence encoding the shRNA; (v) a U6 promoter which drives expression of the shRNA; and (vi) a central polypurine tract and central terminator sequence (cPPT/CTS) sequence.

[0028] In certain embodiments, the inhibitory nucleic acid molecule is an shRNA inhibitory against beta-2 microglobulin, wherein the shRNA has a sequence comprising any one of SEQ ID NOs: 2-4. In particular embodiments, the shRNA has a sequence comprising SEQ ID NO: 2. In some such embodiments, the first expression cassette is in a 3' to 5' orientation and the second expression cassette is in a 5' to 3' orientation relative to the 5' and 3' homology arms, and the first expression cassette is 5' upstream of the second expression cassette. In some such embodiments, the first expression cassette comprises: (i) a nucleic acid sequence encoding a chimeric antigen receptor or an exogenous T cell receptor; (ii) a JeT promoter which drives expression of the chimeric antigen receptor or the exogenous T cell receptor; and (iii) a polyA sequence; and the second expression cassette comprises: (iv) a nucleic acid sequence encoding the shRNA; (v) a U6 promoter which drives expression of the shRNA; and (vi) a central polypurine tract and central terminator sequence (cPPT/CTS) sequence. In some such embodiments, the nucleic acid molecule comprises a first copy and a second copy of the second expression cassette, wherein the first copy and the second copy are identical, and wherein the first copy and the second copy are in tandem, and further wherein the first copy and the second copy are in the same orientation.

[0029] In another aspect, the invention provides a recombinant DNA construct comprising any nucleic acid molecule of the invention described herein.

[0030] In some embodiments, the recombinant DNA construct encodes a viral vector. In certain embodiments, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an adeno-associated viral (AAV) vector. In particular embodiments, the viral vector is a recombinant AAV vector.

[0031] In another aspect, the invention provides a viral vector comprising any nucleic acid molecule of the invention described herein. In certain embodiments, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an adeno-associated viral (AAV) vector. In particular embodiments, the viral vector is a recombinant AAV vector.

[0032] In another aspect, the invention provides a method for producing a genetically-modified eukaryotic cell, the method comprising introducing into a cell any nucleic acid molecule of the invention described herein and: (a) a nucleic acid encoding an engineered nuclease having specificity for the nuclease recognition sequence, wherein the engineered nuclease is expressed in the cell; or (b) an engineered nuclease protein having specificity for the nuclease recognition sequence; wherein the engineered nuclease recognizes and cleaves the nuclease recognition sequence in the genome of the cell to generate a cleavage site, and wherein the nucleic acid molecule of the invention is inserted into the genome of the cell at the cleavage site.

[0033] In some embodiments of the method, the genetically-modified eukaryotic cell is a human T cell.

[0034] In some embodiments of the method, the engineered nuclease is an engineered meganuclease, a TALEN, a zinc finger nuclease (ZFN), a CRISPR/Cas, a compact TALEN, or a megaTAL. In certain embodiments of the method, the engineered nuclease is an engineered meganuclease.

[0035] In some embodiments of the method, the nuclease recognition sequence is in a human T cell receptor alpha constant region gene.

[0036] In certain embodiments of the method, the nuclease recognition sequence is an engineered meganuclease recognition sequence. In particular embodiments, wherein the engineered meganuclease recognition sequence is within a human T cell receptor alpha constant region, the nuclease recognition sequence comprises SEQ ID NO: 1.

[0037] In some embodiments of the method, wherein the nuclease recognition sequence is within a human T cell receptor alpha constant region, cell surface expression of an endogenous T cell receptor is reduced compared to a control cell.

[0038] In some embodiments of the method, the nucleic acid encoding the engineered nuclease is an mRNA. In certain embodiments, the mRNA is a polycistronic mRNA encoding the engineered nuclease and at least one additional polypeptide or nucleic acid molecule.

[0039] In some embodiments of the method, the nucleic acid molecule of the invention described herein is introduced into the cell using a viral vector. In certain embodiments of the method, the viral vector is an adenoviral vector, a lentiviral vector, a retroviral vector, or an AAV vector. In particular embodiments of the method, the viral vector is a recombinant AAV vector, such as a recombinant AAV vector previously described herein.

[0040] In some embodiments of the method, the nucleic acid molecule of the invention described herein is introduced into the cell using a recombinant DNA construct. In certain embodiments of the method, the recombinant DNA construct is a recombinant DNA construct previously described herein.

[0041] In some embodiments of the method, the nucleic acid molecule of the invention described herein is inserted into the genome of the cell at the cleavage site by homologous recombination.

[0042] In some embodiments of the method, the engineered antigen receptor is a chimeric antigen receptor. In other embodiments of the method, the engineered antigen receptor is an exogenous T cell receptor.

[0043] In some embodiments of the method, the inhibitory nucleic acid molecule is inhibitory against human beta-2 microglobulin. In certain embodiments of the method, cell surface expression of beta-2 microglobulin is between about 1% and about 50% of cell surface beta-2 microglobulin expression on a control cell. In other embodiments of the method, cell surface expression of beta-2 microglobulin is between about 1% and about 25% of cell surface beta-2 microglobulin expression on a control cell. In other embodiments of the method, cell surface expression of beta-2 microglobulin is between about 1% and about 10% of cell surface beta-2 microglobulin expression on a control cell. In other embodiments of the method, cell surface expression of beta-2 microglobulin is between about 1% and about 5% of cell surface beta-2 microglobulin expression on a control cell. In particular embodiments of the method, a control cell is not genetically-modified to reduce cell surface beta-2 microglobulin expression.

[0044] In some embodiments of the method, the inhibitory nucleic acid molecule is inhibitory against human beta-2 microglobulin. In certain embodiments of the method, cell surface expression of beta-2 microglobulin is reduced by 10% to 95% compared to cell surface beta-2 microglobulin expression on a control cell. In other embodiments of the method, cell surface expression of beta-2 microglobulin is reduced by 50% to 95% compared to cell surface beta-2 microglobulin expression on a control cell. In other embodiments of the method, cell surface expression of beta-2 microglobulin is reduced by 75% to 95% compared to cell surface beta-2 microglobulin expression on a control cell. In other embodiments of the method, cell surface expression of beta-2 microglobulin is reduced by 90% to 95% compared to cell surface beta-2 microglobulin expression on a control cell. In particular embodiments of the method, a control cell is not genetically-modified to reduce cell surface beta-2 microglobulin expression.

[0045] In some embodiments of the method, the inhibitory nucleic acid molecule is inhibitory against a component of the MHC class I molecule. In certain embodiments of the method, cell surface expression of MHC class I molecules is between about 1% and about 50% of expression of MHC class I molecules on a control cell. In certain embodiments of the method, cell surface expression of MHC class I molecules is between about 1% and about 25% of expression of MHC class I molecules on a control cell. In certain embodiments of the method, cell surface expression of MHC class I molecules is between about 1% and about 10% of expression of MHC class I molecules on a control cell. In certain embodiments of the method, cell surface expression of MHC class I molecules is between about 1% and about 5% of expression of MHC class I molecules on a control cell. In particular embodiments of the method, a control cell is not genetically-modified to reduce cell surface expression of MHC class I molecules.

[0046] In some embodiments of the method, the inhibitory nucleic acid molecule is inhibitory against a component of the MHC class I molecule. In certain embodiments of the method, cell surface expression of MHC class I molecules is reduced by 10% to 95% compared to expression of MHC class I molecules on a control cell. In certain embodiments of the method, cell surface expression of MHC class I molecules is reduced by 50% to 95% compared to expression of MHC class I molecules on a control cell. In certain embodiments of the method, cell surface expression of MHC class I molecules is reduced by 75% to 95% compared to expression of MHC class I molecules on a control cell. In certain embodiments of the method, cell surface expression of MHC class I molecules is reduced by 90% to 95% compared to expression of MHC class I molecules on a control cell. In particular embodiments of the method, a control cell is not genetically-modified to reduce cell surface expression of MHC class I molecules.

[0047] In some embodiments of the method, the inhibitory nucleic acid molecule is inhibitory against human CD52. In certain embodiments of the method, cell surface expression of CD52 is between about 1% and about 50% of cell surface CD52 expression on a control cell. In other embodiments of the method, cell surface expression of CD52 is between about 1% and about 25% of cell surface CD52 expression on a control cell. In other embodiments of the method, cell surface expression of CD52 is between about 1% and about 10% of cell surface CD52 expression on a control cell. In other embodiments of the method, cell surface expression of CD52 is between about 1% and about 5% of cell surface CD52 expression on a control cell. In particular embodiments of the method, a control cell is not genetically-modified to reduce cell surface expression of CD52.

[0048] In some embodiments of the method, the inhibitory nucleic acid molecule is inhibitory against human CD52. In certain embodiments of the method, cell surface expression of CD52 is reduced by 10% to 95% compared to cell surface CD52 expression on a control cell. In other embodiments of the method, cell surface expression of CD52 is reduced by 50% to 95% compared to cell surface CD52 expression on a control cell. In other embodiments of the method, cell surface expression of CD52 is reduced by 75% to 95% compared to cell surface CD52 expression on a control cell. In other embodiments of the method, cell surface expression of CD52 is reduced by 90% to 95% compared to cell surface CD52 expression on a control cell. In particular embodiments of the method, a control cell is not genetically-modified to reduce cell surface expression of CD52.

[0049] In another aspect the invention provides a genetically-modified eukaryotic cell made by any of the methods described herein above.

[0050] In another aspect, the invention provides a genetically-modified eukaryotic cell comprising any nucleic acid molecule of the invention described herein, wherein the engineered antigen receptor and the inhibitory nucleic acid molecule are expressed in the genetically-modified eukaryotic cell.

[0051] In some embodiments, the genetically-modified eukaryotic cell is a genetically-modified human T cell.

[0052] In some embodiments, the nucleic acid molecule of the invention is inserted into the genome of the genetically-modified eukaryotic cell at the nuclease recognition sequence.

[0053] In some embodiments, the gene of interest is a human T cell receptor alpha constant region gene.

[0054] In some embodiments, the nuclease recognition sequence is an engineered meganuclease recognition sequence, a TALEN recognition sequence, a zinc finger nuclease (ZFN) recognition sequence, a CRISPR/Cas recognition sequence, a compact TALEN recognition sequence, or a megaTAL recognition sequence. In certain embodiments, the nuclease recognition sequence is an engineered meganuclease recognition sequence.

[0055] In particular embodiments, wherein the nuclease recognition sequence is within a human T cell receptor alpha constant region gene, the nuclease recognition sequence is an engineered meganuclease recognition sequence comprising SEQ ID NO: 1.

[0056] In some embodiments, cell surface expression of an endogenous T cell receptor is reduced compared to a control cell.

[0057] In some embodiments, the inhibitory nucleic acid molecule is inhibitory against human beta-2 microglobulin. In certain embodiments, cell surface expression of beta-2 microglobulin is between about 1% and about 50% of cell surface beta-2 microglobulin expression on a control cell. In other embodiments, cell surface expression of beta-2 microglobulin is between about 1% and about 25% of cell surface beta-2 microglobulin expression on a control cell. In other embodiments, cell surface expression of beta-2 microglobulin is between about 1% and about 10% of cell surface beta-2 microglobulin expression on a control cell. In other embodiments, cell surface expression of beta-2 microglobulin is between about 1% and about 5% of cell surface beta-2 microglobulin expression on a control cell. In particular embodiments, a control cell is not genetically-modified to reduce cell surface beta-2 microglobulin expression.

[0058] In some embodiments, the inhibitory nucleic acid molecule is inhibitory against human beta-2 microglobulin. In certain embodiments, cell surface expression of beta-2 microglobulin is reduced by 10% to 95% compared to cell surface beta-2 microglobulin expression on a control cell. In other embodiments, cell surface expression of beta-2 microglobulin is reduced by 50% to 95% compared to cell surface beta-2 microglobulin expression on a control cell. In other embodiments, cell surface expression of beta-2 microglobulin is reduced by 75% to 95% compared to cell surface beta-2 microglobulin expression on a control cell. In other embodiments, cell surface expression of beta-2 microglobulin is reduced by 90% to 95% compared to cell surface beta-2 microglobulin expression on a control cell. In particular embodiments, a control cell is not genetically-modified to reduce cell surface beta-2 microglobulin expression.

[0059] In some embodiments, the inhibitory nucleic acid molecule is inhibitory against a component of the MHC class I molecule. In certain embodiments, cell surface expression of MHC class I molecules is between about 1% and about 50% of expression of MHC class I molecules on a control cell. In certain embodiments, cell surface expression of MHC class I molecules is between about 1% and about 25% of expression of MHC class I molecules on a control cell. In certain embodiments, cell surface expression of MHC class I molecules is between about 1% and about 10% of expression of MHC class I molecules on a control cell. In certain embodiments, cell surface expression of MHC class I molecules is between about 1% and about 5% of expression of MHC class I molecules on a control cell. In particular embodiments, a control cell is not genetically-modified to reduce cell surface expression of MHC class I molecules.

[0060] In some embodiments, the inhibitory nucleic acid molecule is inhibitory against a component of the MHC class I molecule. In certain embodiments, cell surface expression of MHC class I molecules is reduced by 10% to 95% compared to expression of MHC class I molecules on a control cell. In certain embodiments, cell surface expression of MHC class I molecules is reduced by 50% to 95% compared to expression of MHC class I molecules on a control cell. In certain embodiments, cell surface expression of MHC class I molecules is reduced by 75% to 95% compared to expression of MHC class I molecules on a control cell.

[0061] In certain embodiments, cell surface expression of MHC class I molecules is reduced by 90% to 95% compared to expression of MHC class I molecules on a control cell. In particular embodiments, a control cell is not genetically-modified to reduce cell surface expression of MHC class I molecules.

[0062] In some embodiments, the inhibitory nucleic acid molecule is inhibitory against human CD52. In certain embodiments, cell surface expression of CD52 is between about 1% and about 50% of cell surface CD52 expression on a control cell. In other embodiments, cell surface expression of CD52 is between about 1% and about 25% of cell surface CD52 expression on a control cell. In other embodiments, cell surface expression of CD52 is between about 1% and about 10% of cell surface CD52 expression on a control cell. In other embodiments, cell surface expression of CD52 is between about 1% and about 5% of cell surface CD52 expression on a control cell. In particular embodiments, a control cell is not genetically-modified to reduce cell surface expression of CD52.

[0063] In some embodiments, the inhibitory nucleic acid molecule is inhibitory against human CD52. In certain embodiments, cell surface expression of CD52 is reduced by 10% to 95% compared to cell surface CD52 expression on a control cell. In other embodiments, cell surface expression of CD52 is reduced by 50% to 95% compared to cell surface CD52 expression on a control cell. In other embodiments, cell surface expression of CD52 is reduced by 75% to 95% compared to cell surface CD52 expression on a control cell. In other embodiments, cell surface expression of CD52 is reduced by 90% to 95% compared to cell surface CD52 expression on a control cell. In particular embodiments, a control cell is not genetically-modified to reduce cell surface expression of CD52.

[0064] In another aspect, the invention provides a genetically-modified eukaryotic cell comprising in its genome a nucleic acid sequence encoding an engineered antigen receptor which is expressed by the genetically-modified eukaryotic cell, wherein cell surface expression of beta-2 microglobulin on the genetically-modified eukaryotic cell is reduced by 10% to 95% compared to cell surface beta-2 microglobulin expression on a control cell. In certain embodiments, cell surface expression of beta-2 microglobulin on the genetically-modified eukaryotic cell is reduced between 50% and 95% compared to cell surface beta-2 microglobulin expression on a control cell. In certain embodiments, cell surface expression of beta-2 microglobulin on the genetically-modified eukaryotic cell is reduced between 75% and 95% compared to cell surface beta-2 microglobulin expression on a control cell. In certain embodiments, cell surface expression of beta-2 microglobulin on the genetically-modified eukaryotic cell is reduced between 90% and 95% compared to cell surface beta-2 microglobulin expression on a control cell. In particular embodiments, the control cell is not genetically-modified to reduce cell surface expression of beta-2 microglobulin.

[0065] In some embodiments, cell surface expression of beta-2 microglobulin is between about 1% and about 50% of cell surface beta-2 microglobulin expression on a control cell. In other embodiments, cell surface expression of beta-2 microglobulin is between about 1% and about 25% of cell surface beta-2 microglobulin expression on a control cell. In other embodiments, cell surface expression of beta-2 microglobulin is between about 1% and about 10% of cell surface beta-2 microglobulin expression on a control cell. In other embodiments, cell surface expression of beta-2 microglobulin is between about 1% and about 5% of cell surface beta-2 microglobulin expression on a control cell. In particular embodiments, a control cell is not genetically-modified to reduce cell surface beta-2 microglobulin expression.

[0066] In certain embodiments, the genetically-modified eukaryotic cell comprises in its genome a nucleic acid sequence encoding an inhibitory nucleic acid molecule which is inhibitory against beta-2 microglobulin. In particular embodiments, the inhibitory nucleic acid molecule is an RNA interference molecule. In some embodiments, the RNA interference molecule is a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a hairpin siRNA, a microRNA (miRNA), a precursor miRNA, or an miRNA-adapted shRNA. In certain embodiments, the RNA interference molecule is an shRNA. In particular embodiments, the shRNA comprises a sequence of any one of SEQ ID NOs: 2-4. In specific embodiments, the shRNA comprises a sequence of SEQ ID NO: 2.

[0067] In some embodiments, the nucleic acid sequence encoding the engineered antigen receptor is integrated at the same location within the genome as the nucleic acid sequence encoding the inhibitory nucleic acid molecule. In particular embodiments, the genetically-modified eukaryotic cell comprises in its genome the nucleic acid molecule of the invention.

[0068] In other embodiments, the nucleic acid sequence encoding the engineered antigen receptor is integrated at a different location within the genome than the nucleic acid sequence encoding the inhibitory nucleic acid molecule.

[0069] In some embodiments, the genetically-modified eukaryotic cell is less susceptible to endogenous NK cell killing when compared to a control cell, has extended persistence in a subject when compared to a control cell, exhibits enhanced expansion in a subject when compared to a control cell, and/or exhibits reduced allogenicity when compared to a control cell.

[0070] In some embodiments, the engineered antigen receptor is a chimeric antigen receptor or an exogenous T cell receptor.

[0071] In some embodiments, the genetically-modified eukaryotic cell is a genetically-modified human T cell.

[0072] In particular embodiments, the genetically-modified eukaryotic cell is a genetically-modified human T cell, and the engineered antigen receptor is a chimeric antigen receptor or an exogenous T cell receptor.

[0073] In another aspect, the invention provides a genetically-modified eukaryotic cell comprising in its genome a nucleic acid sequence encoding an engineered antigen receptor which is expressed by the genetically-modified eukaryotic cell, wherein cell surface expression of MHC class I molecules on the genetically-modified eukaryotic cell is reduced by 10% to 95% compared to cell surface expression of MHC class I molecules on a control cell. In certain embodiments, cell surface expression of MHC class I molecules on the genetically-modified eukaryotic cell is reduced by 50% to 95% compared to cell surface expression of MHC class I molecules on a control cell. In certain embodiments, cell surface expression of MHC class I molecules on the genetically-modified eukaryotic cell is reduced by 75% to 95% compared to cell surface expression of MHC class I molecules on a control cell. In certain embodiments, cell surface expression of MHC class I molecules on the genetically-modified eukaryotic cell is reduced by 90% to 95% compared to cell surface expression of MHC class I molecules on a control cell. In particular embodiments, the control cell is not genetically-modified to reduce cell surface expression of a component of the MHC class I molecule.

[0074] In some embodiments, cell surface expression of MHC class I molecules is between about 1% and about 50% of cell surface MHC class I molecule expression on a control cell. In other embodiments, cell surface expression of MHC class I molecules is between about 1% and about 25% of cell surface MHC class I molecule expression on a control cell. In other embodiments, cell surface expression of MHC class I molecules is between about 1% and about 10% of cell surface MHC class I molecule expression on a control cell. In other embodiments, cell surface expression of MHC class I molecules is between about 1% and about 5% of cell surface MHC class I molecule expression on a control cell. In particular embodiments, a control cell is not genetically-modified to reduce cell surface expression of MHC class I molecules.

[0075] In certain embodiments, the genetically-modified eukaryotic cell comprises in its genome a nucleic acid sequence encoding an inhibitory nucleic acid molecule which is inhibitory against a component of the MHC class I molecule. In certain embodiments, the inhibitory molecule is inhibitory against an MHC class I alpha-1 (.quadrature.1) domain, alpha-2 (.quadrature.2) domain, alpha-3 (.quadrature.3) domain, or against beta-2 microglobulin. In a particular embodiment, the inhibitory molecule is inhibitory against beta-2 microglobulin.

[0076] In particular embodiments, the inhibitory nucleic acid molecule is an RNA interference molecule. In some embodiments, the RNA interference molecule is a short hairpin RNA (shRNA), a small interfering RNA (siRNA), a hairpin siRNA, a microRNA (miRNA), a precursor miRNA, or an miRNA-adapted shRNA. In certain embodiments, the RNA interference molecule is an shRNA.

[0077] In certain embodiments, the inhibitory nucleic acid molecule is an shRNA inhibitory against beta-2 microglobulin, wherein the shRNA has a sequence comprising any one of SEQ ID NOs: 2-4. In particular embodiments, the shRNA has a sequence comprising SEQ ID NO: 2. In some such embodiments, the first expression cassette and the second expression cassette are in a 3' to 5' orientation relative to the 5' and 3' homology arms, and wherein the first expression cassette is 5' upstream of the second expression cassette. In some such embodiments, the first expression cassette comprises: (i) a nucleic acid sequence encoding a chimeric antigen receptor or an exogenous T cell receptor; (ii) a JeT promoter which drives expression of the chimeric antigen receptor or the exogenous T cell receptor; and (iii) a polyA sequence; and the second expression cassette comprises: (iv) a nucleic acid sequence encoding the shRNA; (v) a U6 promoter which drives expression of the shRNA; and (vi) a central polypurine tract and central terminator sequence (cPPT/CTS) sequence.

[0078] In certain embodiments, the inhibitory nucleic acid molecule is an shRNA inhibitory against beta-2 microglobulin, wherein the shRNA has a sequence comprising any one of SEQ ID NOs: 2-4. In particular embodiments, the shRNA has a sequence comprising SEQ ID NO: 2. In some such embodiments, the first expression cassette is in a 3' to 5' orientation and the second expression cassette is in a 5' to 3' orientation relative to the 5' and 3' homology arms, and the first expression cassette is 5' upstream of the second expression cassette. In some such embodiments, the first expression cassette comprises: (i) a nucleic acid sequence encoding a chimeric antigen receptor or an exogenous T cell receptor; (ii) a JeT promoter which drives expression of the chimeric antigen receptor or the exogenous T cell receptor; and (iii) a polyA sequence; and the second expression cassette comprises: (iv) a nucleic acid sequence encoding the shRNA; (v) a U6 promoter which drives expression of the shRNA; and (vi) a central polypurine tract and central terminator sequence (cPPT/CTS) sequence. In some such embodiments, the nucleic acid molecule comprises a first copy and a second copy of the second expression cassette, wherein the first copy and the second copy are identical, and wherein the first copy and the second copy are in tandem, and further wherein the first copy and the second copy are in the same orientation.

[0079] In some embodiments, the nucleic acid sequence encoding the engineered antigen receptor is integrated at the same location within the genome as the nucleic acid sequence encoding the inhibitory nucleic acid molecule. In particular embodiments, the genetically-modified eukaryotic cell comprises in its genome the nucleic acid molecule of the invention.

[0080] In other embodiments, the nucleic acid sequence encoding the engineered antigen receptor is integrated at a different location within the genome than the nucleic acid sequence encoding the inhibitory nucleic acid molecule.

[0081] In some embodiments, the genetically-modified eukaryotic cell is less susceptible to endogenous NK cell killing when compared to a control cell, has extended persistence in a subject when compared to a control cell, exhibits enhanced expansion in a subject when compared to a control cell, and/or exhibits reduced allogenicity when compared to a control cell.

[0082] In some embodiments, the engineered antigen receptor is a chimeric antigen receptor or an exogenous T cell receptor.

[0083] In some embodiments, the genetically-modified eukaryotic cell is a genetically-modified human T cell.

[0084] In particular embodiments, the genetically-modified eukaryotic cell is a genetically-modified human T cell, and the engineered antigen receptor is a chimeric antigen receptor or an exogenous T cell receptor.

[0085] In another aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a therapeutically effective amount of any genetically-modified eukaryotic cell described herein above.

[0086] In some particular embodiments, the genetically-modified eukaryotic cell of the pharmaceutical composition is a genetically-modified human T cell, the engineered antigen receptor is a chimeric antigen receptor or exogenous T cell receptor, and cell surface expression of beta-2 microglobulin is between about 1% and about 50%, about 1% and about 25%, about 1% and about 10%, or about 1% and about 5% of cell surface beta-2 microglobulin expression on a control cell.

[0087] In other particular embodiments, the genetically-modified eukaryotic cell of the pharmaceutical composition is a genetically-modified human T cell, the engineered antigen receptor is a chimeric antigen receptor or exogenous T cell receptor, and cell surface expression of beta-2 microglobulin on the genetically-modified human T cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cell surface expression of beta-2 microglobulin on a control cell.

[0088] In some particular embodiments, the genetically-modified eukaryotic cell of the pharmaceutical composition is a genetically-modified human T cell, the engineered antigen receptor is a chimeric antigen receptor or exogenous T cell receptor, and cell surface expression of MHC class I molecules is between about 1% and about 50%, about 1% and about 25%, about 1% and about 10%, or about 1% and about 5% of cell surface expression of MHC class I molecules on a control cell.

[0089] In other particular embodiments, the genetically-modified eukaryotic cell of the pharmaceutical composition is a genetically-modified human T cell, the engineered antigen receptor is a chimeric antigen receptor or exogenous T cell receptor, and cell surface expression of MHC class I molecules on the genetically-modified human T cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cell surface expression of MHC class I molecules on a control cell.

[0090] In other particular embodiments, the genetically-modified eukaryotic cell of the pharmaceutical composition is a genetically-modified human T cell, and the engineered antigen receptor is a chimeric antigen receptor, and cell surface expression of CD52 is between about 1% and about 50%, about 1% and about 25%, about 1% and about 10%, or about 1% and about 5% of cell surface CD52 expression on a control cell.

[0091] In some embodiments, the genetically-modified eukaryotic cell of the pharmaceutical composition is a genetically-modified human T cell, the engineered antigen receptor is a chimeric antigen receptor or exogenous T cell receptor, and cell surface expression of CD52 on the genetically-modified human T cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cell surface expression of CD52 on a control cell.

[0092] In certain embodiments, the pharmaceutical composition of the invention is for immunotherapy in the treatment of cancer in a subject in need thereof.

[0093] In another aspect, the invention provides a population of genetically-modified eukaryotic cells comprising a plurality of any genetically-modified eukaryotic cell described herein.

[0094] In some embodiments, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100%, of cells in the population are a genetically-modified eukaryotic cell as described herein.

[0095] In particular embodiments, the genetically-modified eukaryotic cells of the population are genetically-modified human T cells, or cells derived therefrom, or genetically-modified NK cells, or cells derived therefrom.

[0096] In certain embodiments, the genetically-modified eukaryotic cells of the population comprise a cell surface chimeric antigen receptor or exogenous T cell receptor. In some of these embodiments, the chimeric antigen receptor or exogenous T cell receptor comprises an extracellular ligand-binding domain having specificity for a tumor-specific antigen.

[0097] In some embodiments, the genetically-modified eukaryotic cells of the population have no cell surface expression of an endogenous T cell receptor when compared to an unmodified control cell. In some embodiments, the genetically-modified eukaryotic cells of the population have reduced cell surface expression of beta-2 microglobulin, MEW class I molecules, or CD52.

[0098] In another aspect, the invention provides a method of using immunotherapy to treat a disease in a subject in need thereof, the method comprising administering to the subject a genetically-modified eukaryotic cell described herein; wherein the genetically-modified eukaryotic cell is a genetically-modified human T cell expressing a chimeric antigen receptor or an exogenous T cell receptor; and wherein cell surface expression of beta-2 microglobulin on the genetically-modified human T cell is between about 1% and about 50%, about 1% and about 25%, about 1% and about 10%, or about 1% and about 5% of cell surface beta-2 microglobulin expression on a control cell.

[0099] In some embodiments of the method, cell surface expression of beta-2 microglobulin on the genetically-modified human T cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cell surface beta-2 microglobulin expression on a control cell.

[0100] In some embodiments of the method, endogenous NK cell killing of the genetically-modified human T cell is reduced in the subject when compared to a genetically-modified human T cell having no cell surface beta-2 microglobulin expression.

[0101] In some embodiments of the method, the subject is administered any pharmaceutical composition described herein in which cell surface beta-2 microglobulin expression is reduced on the genetically-modified human T cell when compared to a control cell.

[0102] In some embodiments of the method, the genetically-modified human T cell is allogeneic to the subject.

[0103] In some embodiments of the method, persistence of the genetically-modified human T cell is extended in the subject when compared to a genetically-modified human T cell having no cell surface beta-2 microglobulin expression, or when compared to a genetically-modified human T cell having a wild-type level of cell surface expression of beta-2 microglobulin.

[0104] In some embodiments of the method, expansion of the genetically-modified human T cell is enhanced in the subject when compared to a genetically-modified human T cell having no cell surface beta-2 microglobulin expression, or when compared to a genetically-modified human T cell having a wild-type level of cell surface expression of beta-2 microglobulin.

[0105] In some embodiments of the method, allogenicity of the genetically-modified human T cell is reduced when compared to a genetically-modified human T cell having a wild-type level of cell surface expression of beta-2 microglobulin.

[0106] In some embodiments of the method, the disease is cancer.

[0107] In some embodiments of the method, the cancer is selected from the group consisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, and leukemia. In certain embodiments of the method, the cancer is selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma, leukemia, and Hodgkin's lymphoma. In particular embodiments of the method, the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

[0108] In another aspect, the invention provides a method of using immunotherapy to treat a disease in a subject in need thereof, the method comprising administering to the subject a genetically-modified eukaryotic cell described herein; wherein the genetically-modified eukaryotic cell is a genetically-modified human T cell expressing a chimeric antigen receptor or an exogenous T cell receptor; and wherein cell surface expression of MHC class I molecules on the genetically-modified human T cell is between about 1% and about 50%, about 1% and about 25%, about 1% and about 10%, or about 1% and about 5% of cell surface expression of MHC class I molecules on a control cell.

[0109] In some embodiments of the method, cell surface expression of MHC class I molecules on the genetically-modified human T cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cell surface expression of MHC class I molecules on a control cell.

[0110] In some embodiments of the method, endogenous NK cell killing of the genetically-modified human T cell is reduced in the subject when compared to a genetically-modified human T cell having no cell surface expression of MHC class I molecules.

[0111] In some embodiments of the method, the subject is administered any pharmaceutical composition described herein in which cell surface expression of MHC class I molecules is reduced on the genetically-modified human T cell when compared to a control cell.

[0112] In some embodiments of the method, the genetically-modified human T cell is allogeneic to the subject.

[0113] In some embodiments of the method, persistence of the genetically-modified human T cell is extended in the subject when compared to a genetically-modified human T cell having no cell surface MHC class I molecule expression, or when compared to a genetically-modified human T cell having a wild-type level of cell surface expression of MHC class I molecules.

[0114] In some embodiments of the method, expansion of the genetically-modified human T cell is enhanced in the subject when compared to a genetically-modified human T cell having no cell surface MHC class I molecule expression, or when compared to a genetically-modified human T cell having a wild-type level of cell surface expression of MHC class I molecules.

[0115] In some embodiments of the method, allogenicity of the genetically-modified human T cell is reduced when compared to a genetically-modified human T cell having a wild-type level of cell surface expression of MHC class I molecules.

[0116] In some embodiments of the method, the disease is cancer.

[0117] In some embodiments of the method, the cancer is selected from the group consisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, and leukemia. In certain embodiments of the method, the cancer is selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma, leukemia, and Hodgkin's lymphoma. In particular embodiments of the method, the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

[0118] In another aspect, the invention provides a method of using immunotherapy to treat cancer in a subject in need thereof, the method comprising administering to the subject a genetically-modified eukaryotic cell described herein; wherein the genetically-modified eukaryotic cell is a genetically-modified human T cell expressing a chimeric antigen receptor or an exogenous T cell receptor; and wherein cell surface expression of CD52 on the genetically-modified human T cell is between 1% and 50%, 1% and 25%, 1% and 10%, or 1% and 5% of cell surface CD52 expression on a control cell.

[0119] In some embodiments of the method, cell surface expression of CD52 on the genetically-modified human T cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% compared to cell surface expression of CD52 on a control cell.

[0120] In some embodiments of the method, the subject is administered a pharmaceutical composition described herein in which cell surface expression of CD52 is reduced on the genetically-modified human T cell when compared to a control cell.

[0121] In some embodiments of the method, the genetically-modified human T cell is allogeneic to the subject.

[0122] In some embodiments of the method, the cancer is selected from the group consisting of a cancer of carcinoma, lymphoma, sarcoma, blastomas, and leukemia. In certain embodiments of the method, the cancer is selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma, leukemia, and Hodgkin's lymphoma. In particular embodiments of the method, the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma.

[0123] In another aspect, the invention provides a method for preparing an enriched population of genetically-modified eukaryotic cells comprising an engineered antigen receptor, the method comprising preparing a population of cells comprising a genetically-modified eukaryotic cell described herein and cells expressing a wild-type level of cell surface CD52, wherein cell surface expression of CD52 on the genetically-modified eukaryotic cell is between about 1% and about 50%, about 1% and about 25%, about 1% and about 10%, or about 1% and about 5% when compared to a control cell, the method comprising: (a) contacting the population of cells with beads conjugated to an anti-CD52 binding molecule, wherein cells expressing a wild-type level of cell surface CD52 are bound to the beads and the genetically-modified eukaryotic cell is not bound to the beads; and (b) removing the beads from the population of cells to produce the enriched population of cells; wherein the enriched population of cells is enriched for the genetically-modified eukaryotic cell.

[0124] In some embodiments of the method, cell surface expression of CD52 on the genetically-modified eukaryotic cell is reduced by 10% to 95%, by 50% to 95%, by 75% to 95%, or by 90% to 95% when compared to a control cell.

[0125] In some embodiments of the method, the beads are magnetic beads. In certain embodiments of the method, the magnetic beads are removed from the population of cells by magnetic separation.

[0126] In some embodiments of the method, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or up to 100% of cells in the enriched population are the genetically-modified eukaryotic cell.

[0127] In some embodiments of the method, the genetically-modified eukaryotic cell expresses a chimeric antigen receptor. In other embodiments of the method, the genetically-modified eukaryotic cell expresses an exogenous T cell receptor.

[0128] In some embodiments of the method, the genetically-modified eukaryotic cell is a genetically-modified human T cell, such as any genetically-modified T cell described herein.

[0129] In another aspect, the present disclosure provides a genetically-modified eukaryotic cell described herein for use as a medicament. The present disclosure further provides the use of a genetically-modified eukaryotic cell described herein in the manufacture of a medicament for treating a disease in a subject in need thereof. In one such embodiment, the medicament is useful in the treatment of cancer.

[0130] The foregoing and other aspects and embodiments of the present invention can be more fully understood by reference to the following detailed description and claims. Certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All sub-combinations of features listed in the embodiments are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. Embodiments of each aspect of the present invention disclosed herein apply to each other aspect of the invention mutatis mutandis.

BRIEF DESCRIPTION OF THE FIGURES

[0131] FIG. 1 shows diagrams of various embodiments of the nucleic acid molecule of the invention. The JeT promoter is shown as an example of a promoter driving expression of the engineered antigen receptor. A U6 promoter is shown as an example of a promoter driving expression of the inhibitory nucleic acid molecule. A chimeric antigen receptor (CAR) is shown as an example of an engineered antigen receptor. An shRNA is shown as an example of an inhibitory nucleic acid molecule. A poly-A sequence is shown as an example of a sequence which terminates translation of the engineered antigen receptor. A cPPT/CTS sequence is shown as an example of a sequence which terminates translation of the inhibitory nucleic acid molecule. 5' and 3' homology arms are shown flanking the first expression cassette and second expression cassette of each construct. Optional 5' and 3' inverted terminal repeats are further shown in each construct. Constructs above the dashed line have first and second expression cassettes in the same orientation, whereas constructs below the dashed line have first and second expression cassettes in opposite orientations.

[0132] FIG. 2A-2L shows flow plots which represent NK cell killing of primary human T cells. The indicated ratios represent the ratio of NK cells to T cells (E:T) in each experiment. FIG. 2A shows NK cell killing of B2M+ T cells using a 2:1 ratio. FIG. 2B shows NK cell killing of B2M+ T cells using a 1:1 ratio. FIG. 2C shows NK cell killing of B2M+ T cells using a 0.5:1 ratio. FIG. 2D shows NK cell killing of B2M+ T cells using a 0:1 ratio. FIG. 2E shows NK cell killing of B2M- T cells using a 2:1 ratio. FIG. 2F shows NK cell killing of B2M- T cells using a 1:1 ratio. FIG. 2G shows NK cell killing of B2M- T cells using a 0.5:1 ratio. FIG. 2H shows NK cell killing of B2M- T cells using a 0:1 ratio. FIG. 2I shows NK cell killing of Daudi Class I-negative cells using a 2:1 ratio.

[0133] FIG. 2J shows NK cell killing of Daudi Class I-negative cells using a 1:1 ratio. FIG. 2K shows NK cell killing of Daudi Class I-negative cells using a 0.5:1 ratio. FIG. 2L shows NK cell killing of Daudi Class I-negative cells using a 0:1 ratio.

[0134] FIG. 3 shows a chart summarizing NK cell killing of B2M+ and B2M- cells at different ratios.

[0135] FIG. 4 shows percentage knockdown of human B2M in primary human T cells by three candidate B2M shRNAs.

[0136] FIGS. 5A-5D show flow diagrams representing NK cell lysis or allogeneic cell lysis of K562 cells or mock-treated primary human T cells. FIG. 5A shows NK cell lysis of K562 cells. FIG. 5B shows allogeneic cell lysis of K562 cells. FIG. 5C shows NK cell lysis of mock-treated primary human T cells. FIG. 5D shows allogeneic cell lysis of mock-treated primary human T cells.

[0137] FIGS. 6A-6D show flow diagrams representing NK cell lysis or allogeneic cell lysis of primary human T cells treated with B2M shRNAs. FIG. 6A shows NK cell lysis of primary human T cells treated with shRNA254. FIG. 6B shows allogeneic cell lysis of primary human T cells treated with shRNA254. FIG. 6C shows NK cell lysis of primary human T cells treated with shRNA472. FIG. 6D shows allogeneic cell lysis of primary human T cells treated with shRNA472.

[0138] FIGS. 7A-7F show diagrams of various nucleic acid molecule constructs encoding a chimeric antigen receptor and an shRNA against beta-2 microglobulin. FIG. 7A shows construct 7007 (SEQ ID NO: 18). FIG. 7B shows construct 7217 (SEQ ID NO: 19).

[0139] FIG. 7C shows construct 7008 (SEQ ID NO: 20). FIG. 7D shows construct 7218 (SEQ ID NO: 21). FIG. 7E shows construct 7009 (SEQ ID NO: 22). FIG. 7F shows construct 7219 (SEQ ID NO: 23).

[0140] FIG. 8 shows percentage knockdown of human CD52 in primary human T cells by three different candidate CD52 shRNAs.

[0141] FIG. 9 A-C show knockdown of CD52 using shRNA and magnetic enrichment of the knockdown population of primary human T cells by CD52 magnetic depletion. FIG. 9A shows T cells that were mock transduced. FIG. 9B shows T cells transduced with an shRNA-568 lentivirus. FIG. 9C shows lentivirus-shRNA568 transduced cells that have undergone a CD52 magnetic depletion.

[0142] FIGS. 10A-10H shows diagrams of nucleic acid molecule constructs encoding a chimeric antigen receptor and an shRNA against CD52. FIG. 10A shows construct 7005 (SEQ ID NO: 10) which encodes a CAR only. FIG. 10B shows construct 7002 (SEQ ID NO: 11) which encodes a CAR only. FIG. 10C shows construct 7004 (SEQ ID NO: 12). FIG. 10D shows construct 7204 (SEQ ID NO: 13). FIG. 10E shows construct 7013 (SEQ ID NO: 14). FIG. 10F shows construct 7213 (SEQ ID NO: 15).

[0143] FIG. 10G shows construct 7014 (SEQ ID NO: 16). FIG. 10H shows construct 7214 (SEQ ID NO: 17).

[0144] FIG. 11A-11D shows CD52 knockdown profiles using CAR/CD52 shRNA constructs with different orientations. FIG. 11A shows CD52 expression when a CAR is expressed in the absence of a CD52 shRNA. FIG. 11B shows CD52 expression when using the 7013 construct. FIG. 11C shows CD52 expression when using the 7004 construct. FIG. 11D shows CD52 expression when using the 7014 construct.

[0145] FIG. 12A-12C shows B2M knockdown on CAR T cells using CAR/B2M shRNA constructs having one or multiple shRNA cassettes. FIG. 12A shows B2M expression in CAR T cells expressing no B2M shRNA (7002--shaded curve) or a single B2M shRNA cassette (7008--open curve). FIG. 12B shows B2M expression in CAR T cells expressing no B2M shRNA (7002--shaded) or two B2M shRNA cassettes (7029--open). FIG. 12C shows B2M expression in CAR-/CD3+ (i.e. non-edited) populations from cultures electroporated with 7002, 7008, or 7029.

[0146] FIG. 13A-13C shows cell surface expression of beta-2 microglobulin on T cells transfected with linearized DNA to express a control CAR-negative construct (7002), CAR constructs expressing a single shRNA472 copy in a 3' to 5' head-to-tail configuration with the CAR (7056), or in a 3' to 5'/5' to 3' tail-to-tail configuration with the CAR (7059), or a CAR construct expressing two shRNA cassette copies in a 3' to 5'/5' to 3' tail-to-tail configuration with the CAR (7060). FIG. 13A shows CAR T cells expressing the 7002 and 7056 constructs. FIG. 13B shows CAR T cells expressing the 7002 and 7059 constructs. FIG. 13C shows CAR T cells expressing the 7002 and 7060 constructs.

[0147] FIG. 14 A-D shows beta-2 microglobulin expression or HLA-A, B, and C expression (i.e., MHC class I molecule expression) on T cells transduced with an AAV comprising construct 7056 which expresses a single copy of the shRNA472 in a 3' to 5' head-to-tail configuration with the CAR. FIG. 14A shows the B2M surface levels in CD3-/CAR+ cells compared to meganuclease-edited cells expressing no shRNA from a control culture. FIG. 14B shows B2M levels on CD3-/CAR+ versus CD3+/CAR-populations in the same culture. FIG. 14C shows HLA-ABC (i.e., MHC class I molecule) surface levels in CD3-/CAR+ cells compared to meganuclease-edited cells expressing no shRNA from a control culture. FIG. 14D shows HLA-ABC levels on CD3-/CAR+ versus CD3+/CAR- populations in the same culture.

BRIEF DESCRIPTION OF THE SEQUENCES

[0148] SEQ ID NO: 1 sets forth the nucleic acid sequence of the TRC 1-2 recognition sequence.

[0149] SEQ ID NO: 2 sets forth the nucleic acid sequence of the anti-beta-2 microglobulin shRNA472.

[0150] SEQ ID NO: 3 sets forth the nucleic acid sequence of the anti-beta-2 microglobulin shRNA256.

[0151] SEQ ID NO: 4 sets forth the nucleic acid sequence of the anti-beta-2 microglobulin shRNA254.

[0152] SEQ ID NO: 5 sets forth the nucleic acid sequence of the anti-CD52 shRNA572.

[0153] SEQ ID NO: 6 sets forth the nucleic acid sequence of the anti-CD52 shRNA876.

[0154] SEQ ID NO: 7 sets forth the nucleic acid sequence of the anti-CD52 shRNA568.

[0155] SEQ ID NO: 8 sets forth the nucleic acid sequence of the anti-CD52 shRNA569.

[0156] SEQ ID NO: 9 sets forth the nucleic acid sequence of the anti-CD52 shRNA571.

[0157] SEQ ID NO: 10 sets forth the nucleic acid sequence of the CAR 7005 construct.

[0158] SEQ ID NO: 11 sets forth the nucleic acid sequence of the CAR 7002 construct.

[0159] SEQ ID NO: 12 sets forth the nucleic acid sequence of the CAR 7004 construct.

[0160] SEQ ID NO: 13 sets forth the nucleic acid sequence of the CAR 7204 construct.

[0161] SEQ ID NO: 14 sets forth the nucleic acid sequence of the CAR 7013 construct.

[0162] SEQ ID NO: 15 sets forth the nucleic acid sequence of the CAR 7213 construct.

[0163] SEQ ID NO: 16 sets forth the nucleic acid sequence of the CAR 7014 construct.

[0164] SEQ ID NO: 17 sets forth the nucleic acid sequence of the CAR 7214 construct.

[0165] SEQ ID NO: 18 sets forth the nucleic acid sequence of the CAR 7007 construct.

[0166] SEQ ID NO: 19 sets forth the nucleic acid sequence of the CAR 7217 construct.

[0167] SEQ ID NO: 20 sets forth the nucleic acid sequence of the CAR 7008 construct.

[0168] SEQ ID NO: 21 sets forth the nucleic acid sequence of the CAR 7218 construct.

[0169] SEQ ID NO: 22 sets forth the nucleic acid sequence of the CAR 7009 construct.

[0170] SEQ ID NO: 23 sets forth the nucleic acid sequence of the CAR 7219 construct.

[0171] SEQ ID NO: 24 sets forth the nucleic acid sequence of the CAR 7029 construct.

[0172] SEQ ID NO: 25 sets forth the nucleic acid sequence of the CAR 7056 construct.

[0173] SEQ ID NO: 26 sets forth the nucleic acid sequence of the CAR 7059 construct.

[0174] SEQ ID NO: 27 sets forth the nucleic acid sequence of the CAR 7060 construct.

[0175] SEQ ID NO: 28 sets forth the nucleic acid sequence of the cPPT/CTS sequence.

DETAILED DESCRIPTION OF THE INVENTION

1.1 References and Definitions

[0176] The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

[0177] The present disclosure can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present disclosure to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the present disclosure.

[0178] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the present disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present disclosure.

[0179] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.

[0180] As used herein, "a," "an," or "the" can mean one or more than one. For example, "a" cell can mean a single cell or a multiplicity of cells.

[0181] As used herein, unless specifically indicated otherwise, the word "or" is used in the inclusive sense of "and/or" and not the exclusive sense of "either/or."

[0182] The terms "expression cassette," "recombinant DNA construct," "recombinant construct," "expression construct," "chimeric construct," "construct," and "recombinant DNA fragment" are used interchangeably herein and are nucleic acid fragments. A recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector.

[0183] As used herein, a "vector" or "recombinant DNA vector" may be a construct that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. If a vector is used then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art. Vectors can include, without limitation, plasmid vectors and recombinant lentiviral or recombinant AAV vectors, or any other vector known in that art suitable for delivering a gene encoding a co-stimulatory domain of the present disclosure to a target cell. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleotides or nucleic acid sequences of the present disclosure.

[0184] As used herein, a "vector" can also refer to a viral vector. Viral vectors can include, without limitation, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV).

[0185] As used herein, the term "operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a nucleic acid sequence encoding a nuclease as disclosed herein and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the nucleic acid sequence encoding the nuclease. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame.

[0186] As used herein, the term "RNA interference" or "RNAi" refers to a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Pat. No. 6,506,559.

[0187] The term "siRNA" as used herein refers to small interfering RNA, also known as short interfering RNA or silencing RNA. siRNAs can be, for example, 18 to 30, 20 to 25, 21 to 23 or 21 nucleotide-long double-stranded RNA molecules. An "shRNA" as used herein is a short hairpin RNA, which is a sequence of RNA that makes a tight hairpin turn that can also be used to silence gene expression via RNA interference. shRNA can by operably linked to the U6 promoter expression. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA. shRNA disclosed herein can comprise a sequence complementary to at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, or 23 nucleotides of the mRNA a target protein.

[0188] As used herein, an "engineered antigen receptor" refers to an exogenous receptor introduced into a cell, such as a chimeric antigen receptor or exogenous T cell receptor, which induces an activating signal in the cell upon stimulation/binding to a ligand or antigen (e.g., a tumor-specific antigen).

[0189] As used herein, a "chimeric antigen receptor" or "CAR" refers to an engineered receptor that grafts specificity for an antigen or other ligand or molecule onto an immune effector cell (e.g., a T cell or NK cell). A chimeric antigen receptor typically comprises at least an extracellular ligand-binding domain or moiety and an intracellular domain that comprises one or more signaling domains and/or co-stimulatory domains.

[0190] In some embodiments, the extracellular ligand-binding domain or moiety is in the form of a single-chain variable fragment (scFv) derived from a monoclonal antibody, which provides specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cell, such as a cancer cell or other disease-causing cell or particle). In some embodiments, the scFv is attached via a linker sequence. In some embodiments, the extracellular ligand-binding domain is specific for any antigen or epitope of interest. In some embodiments, the scFv is humanized. In some embodiments, the extracellular domain of a chimeric antigen receptor comprises an autoantigen (see, Payne et al. (2016) Science, Vol. 353 (6295): 179-184), which is recognized by autoantigen-specific B cell receptors on B lymphocytes, thus directing T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs can be referred to as chimeric autoantibody receptors (CAARs), and the incorporation of one or more co-stimulatory domains described herein into such CAARs is encompassed by the present disclosure.

[0191] The extracellular domain of a chimeric antigen receptor can also comprise a naturally-occurring ligand for an antigen of interest, or a fragment of a naturally-occurring ligand which retains the ability to bind the antigen of interest.

[0192] Intracellular signaling domains are cytoplasmic domains which transmit an activation signal to the cell following binding of the extracellular domain. An intracellular signaling domain can be any intracellular signaling domain of interest that is known in the art. Such cytoplasmic signaling domains can include, without limitation, CD3 .zeta..

[0193] In some embodiments, the intracellular domain also includes one or more intracellular co-stimulatory domains, such as those described herein, which transmit a co-stimulatory signal which promotes cell proliferation, cell survival, and/or cytokine secretion after binding of the extracellular domain. As used herein, a "co-stimulatory domain" refers to a polypeptide domain which transmits an intracellular proliferative and/or cell-survival signal upon activation. Activation of a co-stimulatory domain may occur following homodimerization of two co-stimulatory domain polypeptides. Activation may also occur, for example, following activation of a construct comprising the co-stimulatory domain (e.g., a chimeric antigen receptor or an inducible regulatory construct). Generally, a co-stimulatory domain can be derived from a transmembrane co-stimulatory receptor, particularly from an intracellular portion of a co-stimulatory receptor. Such intracellular co-stimulatory domains can be any of those known in the art and can include, without limitation, CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83, N1, N6, or any combination thereof.

[0194] As used herein, a "co-stimulatory signal" refers to an intracellular signal induced by a co-stimulatory domain that promotes cell proliferation, expansion of a cell population in vitro and/or in vivo, promotes cell survival, modulates (e.g., upregulates or downregulates) the secretion of cytokines, and/or modulates the production and/or secretion of other immunomodulatory molecules. In some embodiments, a co-stimulatory signal is induced following homodimerization of two co-stimulatory domain polypeptides. In some embodiments, a co-stimulatory signal is induced following activation of a construct comprising the co-stimulatory domain (e.g., a chimeric antigen receptor or an inducible regulatory construct).

[0195] A chimeric antigen receptor can further include additional structural elements, including a transmembrane domain that is attached to the extracellular ligand-binding domain via a hinge or spacer sequence. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide can be a subunit of the T-cell receptor (i.e., an .alpha., .beta., .gamma. or .zeta., polypeptide constituting CD3 complex), IL2 receptor p55 (a chain), p75 (.beta. chain) or .gamma. chain, subunit chain of Fc receptors (e.g., Fcy receptor III) or CD proteins such as the CD8 alpha chain. Alternatively the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine.

[0196] The hinge region refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, a hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Hinge regions may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence. In particular examples, a hinge domain can comprise a part of a human CD8 alpha chain, Fc.gamma.Rllla receptor or IgGl.

[0197] As used herein, the term "activation" refers to the state of a cell (e.g., a T cell) that has been sufficiently stimulated to induce detectable effector function. In some embodiments, activation is associated with induced cytokine production and/or induced cell proliferation and expansion.

[0198] As used herein, an "exogenous T cell receptor" or "exogenous TCR" refers to a TCR whose sequence is introduced into the genome of an immune effector cell (e.g., a human T cell) that may or may not endogenously express the TCR. Expression of an exogenous TCR on an immune effector cell can confer specificity for a specific epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cancer cell or other disease-causing cell or particle). Such exogenous T cell receptors can comprise alpha and beta chains or, alternatively, may comprise gamma and delta chains. Exogenous TCRs useful in the invention may have specificity to any antigen or epitope of interest.

[0199] As used herein, with respect to a protein, the term "engineered" or "recombinant" means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids which encode the protein, and cells or organisms which express the protein. With respect to a nucleic acid, the term "recombinant" means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant.

[0200] As used herein, the term "wild-type" refers to the most common naturally occurring polynucleotide or polypeptide sequence responsible for a given phenotype. Whereas a wild-type allele or polypeptide can confer a normal phenotype in an organism, a mutant or variant allele or polypeptide can, in some instances, confer an altered phenotype.

[0201] As used herein with respect to recombinant proteins, the term "modification" means any insertion, deletion or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., a wild-type or a native sequence).

[0202] As used herein, the term "meganuclease" refers to an endonuclease that binds double-stranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs. A meganuclease can be an endonuclease that is derived from I-CreI, and can refer to an engineered variant of I-CreI that has been modified relative to natural I-CreI with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties. Methods for producing such modified variants of I-CreI are known in the art (e.g. WO 2007/047859). A meganuclease as used herein binds to double-stranded DNA as a heterodimer. A meganuclease may also be a "single-chain meganuclease" in which a pair of DNA-binding domains are joined into a single polypeptide using a peptide linker. The term "homing endonuclease" is synonymous with the term "meganuclease." Meganucleases of the present disclosure are substantially non-toxic when expressed in cells, particularly in human T cells, such that cells can be transfected and maintained at 37.degree. C. without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.

[0203] As used herein, the term "single-chain meganuclease" refers to a polypeptide comprising a pair of nuclease subunits joined by a linker. A single-chain meganuclease has the organization: N-terminal subunit--Linker--C-terminal subunit. The two meganuclease subunits will generally be non-identical in amino acid sequence and will recognize non-identical DNA sequences. Thus, single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences. A single-chain meganuclease may be referred to as a "single-chain heterodimer" or "single-chain heterodimeric meganuclease" although it is not, in fact, dimeric. For clarity, unless otherwise specified, the term "meganuclease" can refer to a dimeric or single-chain meganuclease.

[0204] As used herein, the term "linker" refers to an exogenous peptide sequence used to join two meganuclease subunits into a single polypeptide. A linker may have a sequence that is found in natural proteins, or may be an artificial sequence that is not found in any natural protein. A linker may be flexible and lacking in secondary structure or may have a propensity to form a specific three-dimensional structure under physiological conditions. A linker can include, without limitation, any of those encompassed by U.S. Pat. Nos. 8,445,251 and 9,434,931.

[0205] As used herein, the term "zinc finger nuclease" or "ZFN" refers to a chimeric protein comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, S1 nuclease, mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeast HO endonuclease. Nuclease domains useful for the design of zinc finger nucleases include those from a Type IIs restriction endonuclease, including but not limited to FokI, FoM, and StsI restriction enzyme. Additional Type IIs restriction endonucleases are described in International Publication No. WO 2007/014275, which is incorporated by reference in its entirety. The structure of a zinc finger domain is stabilized through coordination of a zinc ion. DNA binding proteins comprising one or more zinc finger domains bind DNA in a sequence-specific manner. The zinc finger domain can be a native sequence or can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence .about.18 basepairs in length, comprising a pair of nine basepair half-sites separated by 2-10 basepairs. See, for example, U.S. Pat. Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,200,759, and International Publication Nos. WO 95/19431, WO 96/06166, WO 98/53057, WO 98/54311, WO 00/27878, WO 01/60970, WO 01/88197, and WO 02/099084, each of which is incorporated by reference in its entirety. By fusing this engineered protein domain to a nuclease domain, such as FokI nuclease, it is possible to target DNA breaks with genome-level specificity. The selection of target sites, zinc finger proteins and methods for design and construction of zinc finger nucleases are known to those of skill in the art and are described in detail in U.S. Publications Nos. 20030232410, 20050208489, 2005064474, 20050026157, 20060188987 and International Publication No. WO 07/014275, each of which is incorporated by reference in its entirety. Cleavage by a zinc finger nuclease can create a blunt end or a 5' overhand of variable length (frequently four basepairs).

[0206] As used herein, the term "TALEN" refers to an endonuclease comprising a DNA-binding domain comprising a plurality of TAL domain repeats fused to a nuclease domain or an active portion thereof from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, S1 nuclease, mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeast HO endonuclease. See, for example, Christian et al. (2010) Genetics 186:757-761, which is incorporated by reference in its entirety. Nuclease domains useful for the design of TALENs include those from a Type IIs restriction endonuclease, including but not limited to FokI, FoM, StsI, HhaI, HindIII, Nod, BbvCI, EcoRI, BglI, and AlwI. Additional Type IIs restriction endonucleases are described in International Publication No. WO 2007/014275. In some embodiments, the nuclease domain of the TALEN is a FokI nuclease domain or an active portion thereof. TAL domain repeats can be derived from the TALE (transcription activator-like effector) family of proteins used in the infection process by plant pathogens of the Xanthomonas genus. TAL domain repeats are 33-34 amino acid sequences with divergent 12th and 13th amino acids. These two positions, referred to as the repeat variable dipeptide (RVD), are highly variable and show a strong correlation with specific nucleotide recognition. Each base pair in the DNA target sequence is contacted by a single TAL repeat, with the specificity resulting from the RVD. In some embodiments, the TALEN comprises 16-22 TAL domain repeats. DNA cleavage by a TALEN requires two DNA recognition regions flanking a nonspecific central region (i.e., the "spacer"). The term "spacer" in reference to a TALEN refers to the nucleic acid sequence that separates the two nucleic acid sequences recognized and bound by each monomer constituting a TALEN. The TAL domain repeats can be native sequences from a naturally-occurring TALE protein or can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence (see, for example, Boch et al. (2009) Science 326(5959):1509-1512 and Moscou and Bogdanove (2009) Science 326(5959):1501, each of which is incorporated by reference in its entirety). See also, U.S. Publication No. 20110145940 and International Publication No. WO 2010/079430 for methods for engineering a TALEN to recognize a specific sequence and examples of RVDs and their corresponding target nucleotides. In some embodiments, each nuclease (e.g., FokI) monomer can be fused to a TAL effector sequence that recognizes a different DNA sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme.

[0207] As used herein, the term "compact TALEN" refers to an endonuclease comprising a DNA-binding domain with one or more TAL domain repeats fused in any orientation to any portion of the I-TevI homing endonuclease or any of the endonucleases listed in Table 2 in U.S. Application No. 20130117869 (which is incorporated by reference in its entirety), including but not limited to MmeI, EndA, End1, I-BasI, I-TevII, I-TevIII, I-TwoI, MspI, MvaI, NucA, and NucM. Compact TALENs do not require dimerization for DNA processing activity, alleviating the need for dual target sites with intervening DNA spacers. In some embodiments, the compact TALEN comprises 16-22 TAL domain repeats.

[0208] As used herein, the term "CRISPR" refers to a caspase-based endonuclease comprising a caspase, such as Cas9, and a guide RNA that directs DNA cleavage of the caspase by hybridizing to a recognition site in the genomic DNA. The caspase component of a CRISPR is an RNA-guided DNA endonuclease. In certain embodiments, the caspase is a class II Cas enzyme. In some of these embodiments, the caspase is a class II, type II enzyme, such as Cas9. In other embodiments, the caspase is a class II, type V enzyme, such as Cpf1. The guide RNA comprises a direct repeat and a guide sequence (often referred to as a spacer in the context of an endogenous CRISPR system), which is complementary to the target recognition site. In certain embodiments, the CRISPR further comprises a tracrRNA (trans-activating CRISPR RNA) that is complementary (fully or partially) to a direct repeat sequence (sometimes referred to as a tracr-mate sequence) present on the guide RNA. In particular embodiments, the caspase can be mutated with respect to a corresponding wild-type enzyme such that the enzyme lacks the ability to cleave one strand of a target polynucleotide, functioning as a nickase, cleaving only a single strand of the target DNA. Non-limiting examples of caspase enzymes that function as a nickase include Cas9 enzymes with a D10A mutation within the RuvC I catalytic domain, or with a H840A, N854A, or N863A mutation.

[0209] As used herein, the term "megaTAL" refers to a single-chain nuclease comprising a transcription activator-like effector (TALE) DNA binding domain with an engineered, sequence-specific homing endonuclease.

[0210] As used herein, the term "recognition sequence" refers to a DNA sequence that is bound and cleaved by an endonuclease. In the case of a meganuclease, a recognition sequence comprises a pair of inverted, 9 basepair "half sites" which are separated by four basepairs. In the case of a single-chain meganuclease, the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. Cleavage by a meganuclease produces four basepair 3' "overhangs". "Overhangs", or "sticky ends" are short, single-stranded DNA segments that can be produced by endonuclease cleavage of a double-stranded DNA sequence. In the case of meganucleases and single-chain meganucleases derived from I-CreI, the overhang comprises bases 10-13 of the 22 basepair recognition sequence. In the case of a compact TALEN, the recognition sequence comprises a first CNNNGN sequence that is recognized by the I-TevI domain, followed by a non-specific spacer 4-16 basepairs in length, followed by a second sequence 16-22 bp in length that is recognized by the TAL-effector domain (this sequence typically has a 5' T base). Cleavage by a Compact TALEN produces two basepair 3' overhangs. In the case of a CRISPR, the recognition sequence is the sequence, typically 16-24 basepairs, to which the guide RNA binds to direct cleavage. Full complementarity between the guide sequence and the recognition sequence is not necessarily required to effect cleavage. Cleavage by a CRISPR can produce blunt ends (such as by a class II, type II caspase) or overhanging ends (such as by a class II, type V caspase), depending on the caspase. In those embodiments wherein a CpfI caspase is utilized, cleavage by the CRISPR complex comprising the same will result in 5' overhangs and in certain embodiments, 5 nucleotide 5' overhangs. Each caspase enzyme also requires the recognition of a PAM (protospacer adjacent motif) sequence that is near the recognition sequence complementary to the guide RNA. The precise sequence, length requirements for the PAM, and distance from the target sequence differ depending on the caspase enzyme, but PAMs are typically 2-5 base pair sequences adjacent to the target/recognition sequence. PAM sequences for particular caspase enzymes are known in the art (see, for example, U.S. Pat. No. 8,697,359 and U.S. Publication No. 20160208243, each of which is incorporated by reference in its entirety) and PAM sequences for novel or engineered caspase enzymes can be identified using methods known in the art, such as a PAM depletion assay (see, for example, Karvelis et al. (2017) Methods 121-122:3-8, which is incorporated herein in its entirety). In the case of a zinc finger, the DNA binding domains typically recognize an 18-bp recognition sequence comprising a pair of nine basepair "half-sites" separated by 2-10 basepairs and cleavage by the nuclease creates a blunt end or a 5' overhang of variable length (frequently four basepairs).

[0211] As used herein, the term "target site" or "target sequence" refers to a region of the chromosomal DNA of a cell comprising a recognition sequence for a nuclease.

[0212] As used herein, the term "homologous recombination" or "HR" refers to the natural, cellular process in which a double-stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). The homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell.

[0213] As used herein, the term "non-homologous end-joining" or "NHEJ" refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non-homologous DNA segments (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976).

[0214] As used herein, the term "reduced" refers to any reduction in the symptoms or severity of a disease or any reduction in the proliferation or number of cancerous cells. In either case, such a reduction may be up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100%. Accordingly, the term "reduced" encompasses both a partial reduction and a complete reduction of a disease state.

[0215] As used herein, the term "reduced" can also refer to a decrease in the cell surface expression of a polypeptide when compared to an appropriate control cell. In the present context, a reduction is distinct from knockout of polypeptide expression, wherein expression is reduced by 100%. Rather, in the present invention, a reduction indicates that expression is decreased but not completely eliminated. Such as a reduction can be, for example, a reduction in cell surface beta-2 microglobulin, MHC class I molecule, or CD52 expression when compared to a control cell which has not been genetically-modified to reduce beta-2 microglobulin, MHC class I molecules, or CD52, respectively. A reduction in expression can be between about 10% and about 99% or any number or range therein. For example, a reduction can be between about 10% and 95%, about 50% and about 95%, about 75% and about 95%, or about 90% and about 95%, when compared to a control cell. A reduction can also be by about 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% when compared to a control cell.

[0216] As used herein, the term "MHC class I molecule" refers to a major histocompatibility complex (MHC) found on the cell surface which displays peptide fragments of non-self proteins. MHC class I molecules consist of two polypeptide chains. The alpha chain consists of 3 polypeptides referred to as the alpha-1 (.quadrature.1), alpha-2 (.quadrature.2), and alpha-3 (.quadrature.3) domains. The alpha chain is linked non-covalently via the .quadrature.3 domain to a beta chain which consists of beta-2 microglobulin (B2M). The alpha chain is polymorphic and is encoded by the HLA gene (i.e., HLA-A, HLA-B, and HLA-C), whereas beta-2 microglobulin is not polymorphic and it encoded by the B2M gene.

[0217] As used herein, the term "beta-2 microglobulin" refers to the beta chain component of MHC class I molecules. Human beta-2 microglobulin is encoded by the B2M gene (e.g., NCBI Gene ID 567). Expression of beta-2 microglobulin is necessary for assembly and function of MHC class I molecules on the cell surface.

[0218] As used herein, the term "CD52" refers to the polypeptide encoded by the human CD52 gene (e.g., NCBI gene ID 1043) which is also referred to as cluster of differentiation 52.

[0219] As used herein, the term "anti-tumor activity" or "anti-tumor effect" refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the genetically-modified cells of the present disclosure in prevention of the occurrence of tumor in the first place.

[0220] The term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results. The therapeutically effective amount will vary depending on the therapeutic (e.g., genetically-modified cell, CAR T cell, etc.) formulation or composition, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject to be treated. In specific embodiments, an effective amount of a cell comprising a co-stimulatory domain disclosed herein or pharmaceutical compositions disclosed herein reduces at least one symptom or the progression of a disease.

[0221] As used herein, the term "treat" or "treatment" means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0222] As used herein, the term "cancer" should be understood to encompass any neoplastic disease (whether invasive or metastatic) which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor.

[0223] As used herein, the term "carcinoma" refers to a malignant growth made up of epithelial cells.

[0224] As used herein, the term "leukemia" refers to malignancies of the hematopoietic organs/systems and is generally characterized by an abnormal proliferation and development of leukocytes and their precursors in the blood and bone marrow.

[0225] As used herein, the term "sarcoma" refers to a tumor which is made up of a substance like the embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillary, heterogeneous, or homogeneous substance.

[0226] As used herein, the term "melanoma" refers to a tumor arising from the melanocytic system of the skin and other organs.

[0227] As used herein, the term "lymphoma" refers to a group of blood cell tumors that develop from lymphocytes.

[0228] As used herein, the term "blastoma" refers to a type of cancer that is caused by malignancies in precursor cells or blasts (immature or embryonic tissue).

[0229] As used herein, "transfected" or "transformed" or "transduced" or "nucleofected" refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0230] As used herein, a "human T cell" or "T cell" refers to a T cell isolated from a human donor. Human T cells, and cells derived therefrom, include isolated T cells that have not been passaged in culture, T cells that have been passaged and maintained under cell culture conditions without immortalization, and T cells that have been immortalized and can be maintained under cell culture conditions indefinitely.

[0231] As used herein, a "human natural killer cell" or "human NK cell" or "natural killer cell" or "NK cell" refers to a type of cytotoxic lymphocyte critical to the innate immune system. The role NK cells play is analogous to that of cytotoxic T-cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virally infected cells and respond to tumor formation, acting at around 3 days after infection.

[0232] As used herein, a "control" or "control cell" refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically-modified cell. A control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the genetically-modified cell; (b) a cell of the same genotype as the genetically-modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically-modified cell but which is not exposed to conditions, stimuli, or further genetic modifications that would induce expression of altered genotype or phenotype. In particular embodiments, a control cell is otherwise identical to a genetically-modified cell but has not been genetically-modified to reduce cell surface expression of a particular polypeptide (e.g., beta-2 microglobulin, MHC class I molecules, CD52).

[0233] As used herein with respect to both amino acid sequences and nucleic acid sequences, the terms "percent identity," "sequence identity," "percentage similarity," "sequence similarity," and the like, refer to a measure of the degree of similarity of two sequences based upon an alignment of the sequences which maximizes similarity between aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov), and are described in, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth. Enzymol. 266:131-141; Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402); Zhang et al. (2000), J. Comput. Biol. 7(1-2):203-14. As used herein, percent similarity of two amino acid sequences is the score based upon the following parameters for the BLASTp algorithm: word size=3; gap opening penalty=-11; gap extension penalty=-1; and scoring matrix=BLOSUM62. As used herein, percent similarity of two nucleic acid sequences is the score based upon the following parameters for the BLASTn algorithm: word size=11; gap opening penalty=-5; gap extension penalty=-2; match reward=1; and mismatch penalty=-3.

[0234] As used herein with respect to modifications of two proteins or amino acid sequences, the term "corresponding to" is used to indicate that a specified modification in the first protein is a substitution of the same amino acid residue as in the modification in the second protein, and that the amino acid position of the modification in the first proteins corresponds to or aligns with the amino acid position of the modification in the second protein when the two proteins are subjected to standard sequence alignments (e.g., using the BLASTp program). Thus, the modification of residue "X" to amino acid "A" in the first protein will correspond to the modification of residue "Y" to amino acid "A" in the second protein if residues X and Y correspond to each other in a sequence alignment, and despite the fact that X and Y may be different numbers.

[0235] As used herein, the recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values .gtoreq.0 and .ltoreq.2 if the variable is inherently continuous.

2.1 Principle of the Invention

[0236] The present disclosure is based, in part, on the observation that knockdown of cell surface beta-2 microglobulin, and consequently MHC class I molecules, can reduce allogenicity of genetically-modified cells, such as CAR T cells. Importantly, the inventors have discovered that an incomplete knockdown of beta-2 microglobulin and MEW class I molecules (i.e., to a low percentage of wild-type expression, but not complete knockout) not only reduces allogenicity of genetically-modified cells, but also serves to dramatically reduce killing by NK cells, which can recognize cells that are B2M-negative as non-self and induce a cytotoxic action.

[0237] The present invention is also based, in part, on the inventors' discovery that a population of CAR-positive T cells can be enriched by an advantageous negative-selection method when the CAR-encoding construct includes a coding sequence for an RNA interfering molecule against CD52. In this manner, a population of CAR T cells can be contacted with beads conjugated to an anti-CD52 antibody in order to capture CD52-positive cells. Separation of the beads, and thus the CD52-positive cells, results in an enriched population of CAR-positive cells having reduced cell surface expression of CD52.

[0238] Accordingly, a nucleic acid molecule is provided comprising a first expression cassette which encodes an engineered antigen receptor, such as a chimeric antigen receptor, and a second expression cassette which encodes an inhibitory nucleic acid molecule, such as an RNA interfering molecule. Further, the nucleic acid molecule is flanked by 5' and 3' homology arms to promote targeted insertion of the nucleic acid into the genome of a cell at a double-strand break, such as a cleavage site produced by an engineered nuclease. In certain embodiments of the invention, the inhibitory nucleic acid molecule can be against human beta-2 microglobulin, a component of the MEW class I molecule, or CD52.

[0239] Further disclosed herein are recombinant DNA constructs and viral vectors comprising the nucleic acid molecule, genetically-modified cells comprising the nucleic acid molecule, and pharmaceutical compositions comprising such cells. Also disclosed are genetically-modified cells expressing an engineered antigen receptor (e.g., a CAR or exogenous TCR) which have reduced cell-surface expression of beta-2 microglobulin, MEW class I molecules, or CD52, and may or may not express the particular nucleic acid molecule of the invention.

[0240] In some embodiments, administration of genetically-modified cells of the invention reduces the symptoms or severity of diseases, such cancers, which can be targeted by genetically-modified cells of the present disclosure.

[0241] Also disclosed herein are methods of immunotherapy for treating cancer in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising a genetically-modified cell disclosed herein and a pharmaceutically acceptable carrier. In such methods, wherein a CAR is expressed and cell surface beta-2 microglobulin and/or MHC class I molecules is reduced, incomplete knockout leads to a reduction in both allogenicity of the cells and killing of the cells by NK cells.

[0242] Further disclosed are methods for producing an enriched population of genetically-modified cells, wherein a CAR is expressed and cell surface CD52 is reduced by RNA interference, allowing for negative selection of CAR-positive cells having reduced CD52 expression.

2.2 Nucleic Acid Molecules

[0243] In certain embodiments, the invention provides a nucleic acid molecule comprising: (a) a first expression cassette comprising a nucleic acid sequence encoding an engineered antigen receptor; (b) a second expression cassette comprising a nucleic acid sequence encoding an inhibitory nucleic acid molecule; (c) a 5' homology arm; and (d) a 3' homology arm. The 5' homology arm and the 3' homology arm can be engineered at any suitable length to have homology to chromosomal regions flanking a nuclease recognition sequence in a gene of interest, which can be any desired gene in a target cell in which a suitable recognition sequence is present.

[0244] The nucleic acid molecule of the invention can have any number of orientations. Some non-limiting examples illustrated in FIG. 1. In particular embodiments, the first and second expression cassettes can be in the same orientation. This orientation can be either 5' to 3' relative to the homology arms or, alternatively, 3' to 5'. In either case, the first expression cassette may be 5' to the second cassette, or the second cassette may be 5' to the first cassette. In other embodiments, the first and second expression cassettes can be in different orientations in the nucleic acid molecule. For example, the first expression cassette may be oriented 5' to 3', whereas the second expression cassette may be oriented 3' to 5'. Alternatively, the first expression cassette may be oriented 3' to 5' and the second expression cassette may be oriented 5' to 3'.

[0245] In embodiments wherein the expression cassettes are in opposite orientations, they may be oriented in a "tail-to-tail" configuration, such that the first expression cassette is oriented 3' to 5' and is positioned 5' to the second expression cassette, which is oriented 5' to 3'. In a similar tail-to-tail embodiment, the second expression cassette is oriented 3' to 5' and is positioned 5' to the first expression cassette, which is oriented 5' to 3'.

[0246] In other embodiments wherein the expression cassettes are in opposite orientations, they may be oriented in a "head-to-head" configuration, such that the first expression cassette is oriented 5' to 3' and is positioned 5' to the second expression cassette, which is oriented 3' to 5'. In a similar head-to-head embodiment, the second expression cassette is oriented 5' to 3' and is positioned 5' to the first expression cassette, which is oriented 3' to 5'.

[0247] In some embodiments, the nucleic acid molecule can comprise multiple copies of the second expression cassette. The copies of the second expression cassette can be identical or vary from one another. In some cases, the copies can include a promoter, a coding sequence for the inhibitory nucleic acid molecule, and a sequence, such as a (cPPT/CTS) sequence, to terminate translation of the inhibitory nucleic acid molecule. The copies of the second expression cassette can be in tandem to one another in the nucleic acid molecule, and can be in the same orientation, or in opposite orientations. Alternatively, the copies may not be in tandem, and can be in the same orientation, or in opposite orientations.

[0248] The expression cassettes of the nucleic acid molecule can include various promoters which drive expression of the engineered antigen receptor and/or the inhibitory nucleic acid molecule. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-1.alpha. (EF-1.alpha.). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0249] Synthetic promoters are also contemplated as part of the present disclosure. For example, in particular embodiments, the promoter driving expression of the engineered antigen receptor is a JeT promoter (see, WO/2002/012514).

[0250] In some embodiments, the promoters are selected based on the desired outcome. It is recognized that different applications can be enhanced by the use of different promoters in the expression cassettes to modulate the timing, location and/or level of expression of the polynucleotides disclosed herein. Such expression constructs may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

[0251] Promoters particularly useful for driving expression of an RNA interference molecule are well known in the art and can include, without limitation, pol III promoters, such as U6 or H1.

[0252] The 5' and 3' homology arms of the nucleic acid molecule have sequence homology to corresponding sequences 5' upstream and 3' downstream of the nuclease recognition sequence in the genome. The homology arms promote insertion of the nucleic acid molecule into the cleavage site generated by the nuclease. In general, homology arms can have a length of at least 50 base pairs, preferably at least 100 base pairs, and up to 2000 base pairs or more, and can have at least 90%, preferably at least 95%, or more, sequence homology to their corresponding sequences in the genome.

[0253] In order to assess the expression of an engineered antigen receptor (e.g. a CAR or exogenous T cell receptor) in a genetically-modified cell, the nucleic acid molecule of the invention can optionally comprise an epitope which can be used to detect the presence of the encoded cell surface protein. In some examples described herein, a CAR coding sequence may include a QBend10 epitope which allows for detection using an anti-CD34 antibody (see, WO2013/153391).

[0254] In other examples, an expression cassette can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes and fluorescent marker genes.

[0255] Expression may also be assessed by determining protein expression of the polypeptide targeted by the inhibitory nucleic acid sequence. For example, expression of beta-2 microglobulin and CD52 can be detected on the cell surface by a number of techniques known in the art. Expression can also be determined by positive or negative selection procedures which purify particular populations of cells expressing, or lacking expression, of the cell surface polypeptides.

[0256] Also provided herein are vectors comprising the nucleic acid molecules of the present disclosure. In some embodiments, the nucleic acid molecule is cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, or a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0257] In other embodiments, nucleic acid molecules of the invention are provided on viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193). Where the nucleic acid of the invention is provided in a viral vector that promotes random integration into the genome, and does not require the presence of 5' and 3' homology arms for homologous recombination, the nucleic acid of the invention can be provided without 5' and 3' homology arms.

2.3 Chimeric Antigen Receptors (CARs)

[0258] Provided herein are genetically-modified cells expressing an engineered antigen receptor. In some embodiments, the engineered antigen receptor is a chimeric antigen receptor (CAR). Generally, a CAR of the present disclosure will comprise at least an extracellular domain and an intracellular domain. In some embodiments, the extracellular domain comprises a target-specific binding element otherwise referred to as a ligand-binding domain or moiety. In some embodiments, the intracellular domain, or cytoplasmic domain, comprises at least one co-stimulatory domain and one or more signaling domains. In other embodiments, the CAR may only comprise a signaling domain, such as CD3.quadrature., and the cell may comprise one or more co-stimulatory domains on another construct expressed in the cell.

[0259] In some embodiments, a CAR comprises an extracellular, target-specific binding element otherwise referred to as a ligand-binding domain or moiety. The choice of ligand-binding domain depends upon the type and number of ligands that define the surface of a target cell. For example, the ligand-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as ligands for the ligand-binding domain in the CAR of the present disclosure can include those associated with viral, bacterial and parasitic infections, autoimmune disease, and cancer cells. In some embodiments, the CAR of the present disclosure is engineered to target a tumor antigen of interest by way of engineering a desired ligand-binding moiety that specifically binds to an antigen on a tumor cell. In the context of the present disclosure, "tumor antigen" refers to antigens that are common to specific hyperproliferative disorders such as cancer.

[0260] In some embodiments, the extracellular ligand-binding domain of the CAR is specific for any antigen or epitope of interest, particularly any tumor antigen or epitope of interest. As non-limiting examples, in some embodiments the antigen of the target is a tumor-associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD22, CD30, CD40, CLL-1, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, B-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-1a, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGFl)-l, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the Al domain of tenascin-C(TnC Al) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD38, CD123, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), CS1, or a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gp120); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen such as the E6 or E7 oncoproteins, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen, as well as any derivate or variant of these surface markers. In a particular embodiment of the present disclosure, the ligand-binding domain is specific for CD19.

[0261] In some embodiments, the extracellular domain of a chimeric antigen receptor further comprises an autoantigen (see, Payne et al. (2016) Science, Vol. 353 (6295): 179-184), which can be recognized by autoantigen-specific B cell receptors on B lymphocytes, thus directing T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases. Such CARs can be referred to as chimeric autoantibody receptors (CAARs), and the incorporation of one or more co-stimulatory domains described herein into such CAARs is encompassed by the present disclosure.

[0262] In some embodiments, the extracellular domain of a chimeric antigen receptor can comprise a naturally-occurring ligand for an antigen of interest, or a fragment of a naturally-occurring ligand which retains the ability to bind the antigen of interest.

[0263] In some embodiments, a CAR comprises a transmembrane domain which links the extracellular ligand-binding domain or autoantigen with the intracellular signaling and co-stimulatory domains via a hinge or spacer sequence. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. For example, the transmembrane polypeptide can be a subunit of the T-cell receptor (i.e., an .alpha., .beta., .gamma. or .zeta., polypeptide constituting CD3 complex), IL2 receptor p55 (a chain), p75 (.beta. chain) or .gamma. chain, subunit chain of Fc receptors (e.g., Fcy receptor III) or CD proteins such as the CD8 alpha chain. Alternatively the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. In particular examples, the transmembrane domain is a CD8.quadrature. transmembrane polypeptide.

[0264] The hinge region refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. For example, a hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. Hinge regions may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence. In particular examples, a hinge domain can comprise a part of a human CD8 alpha chain, Fc.gamma.Rllla receptor or IgGl.

[0265] The intracellular signaling domain of a CAR of the present disclosure is responsible for activation of at least one of the normal effector functions of the cell in which the CAR has been placed and/or activation of proliferative and cell survival pathways. The term "effector function" refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. An intracellular signaling domain, such as CD3.quadrature., can provide an activation signal to the cell in response to binding of the extracellular domain. As discussed, the activation signal can induce an effector function of the cell such as, for example, cytolytic activity or cytokine secretion.

[0266] The intracellular domain of the CAR can include one or more intracellular co-stimulatory domains which transmit a co-stimulatory signal to promote cell proliferation, cell survival, and/or cytokine secretion after binding of the extracellular domain. Such intracellular co-stimulatory domains include those known in the art such as, without limitation, CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83, N1, or N6.

[0267] The CAR can be specific for any type of cancer cell. Such cancers can include, without limitation, carcinoma, lymphoma, sarcoma, blastomas, leukemia, cancers of B cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma. In certain embodiments, cancers of B cell origin include, without limitation, B lineage acute lymphoblastic leukemia, B cell chronic lymphocytic leukemia, B cell non-Hodgkin's lymphoma, and multiple myeloma.

2.4 Methods for Producing Recombinant Viral Vectors

[0268] In some embodiments, the present disclosure provides recombinant AAV vectors for use in the methods of the present disclosure. Recombinant AAV vectors are typically produced in mammalian cell lines such as HEK-293. Because the viral cap and rep genes are removed from the vector to prevent its self-replication to make room for the therapeutic gene(s) to be delivered (e.g. the endonuclease gene), it is necessary to provide these in trans in the packaging cell line. In addition, it is necessary to provide the "helper" (e.g. adenoviral) components necessary to support replication (Cots D, Bosch A, Chillon M (2013) Curr. Gene Ther. 13(5): 370-81). Frequently, recombinant AAV vectors are produced using a triple-transfection in which a cell line is transfected with a first plasmid encoding the "helper" components, a second plasmid comprising the cap and rep genes, and a third plasmid comprising the viral ITRs containing the intervening DNA sequence to be packaged into the virus. Viral particles comprising a genome (ITRs and intervening gene(s) of interest) encased in a capsid are then isolated from cells by freeze-thaw cycles, sonication, detergent, or other means known in the art. Particles are then purified using cesium-chloride density gradient centrifugation or affinity chromatography and subsequently delivered to the gene(s) of interest to cells, tissues, or an organism such as a human patient. Accordingly, methods are provided herein for producing recombinant AAV vectors comprising the nucleic acid molecules of the invention described herein.

[0269] In some embodiments, genetic transfer is accomplished via lentiviral vectors. Lentiviruses, in contrast to other retroviruses, in some contexts may be used for transducing certain non-dividing cells. Non-limiting examples of lentiviral vectors include those derived from a lentivirus, such as Human Immunodeficiency Virus 1 (HIV-1), HIV-2, an Simian Immunodeficiency Virus (SIV), Human T-lymphotropic virus 1 (HTLV-1), HTLV-2 or equine infection anemia virus (E1AV). For example, lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted, making the vector safer for therapeutic purposes. Lentiviral vectors are known in the art, see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection, and for transfer of the nucleic acid into a host cell. Known lentiviruses can be readily obtained from depositories or collections such as the American Type Culture Collection ("ATCC"; 10801 University Blvd., Manassas, Va. 20110-2209), or isolated from known sources using commonly available techniques.

[0270] In specific embodiments, lentiviral vectors are prepared using a first plasmid encoding the gag, pol, tat, and rev genes cloned from human immunodeficiency virus (HIV) and a second plasmid encoding the envelope protein from vesicular stomatitis virus (VSV-G) used to pseudotype viral particles. A transfer vector, such as the pCDH-EF1-MCS vector, can be used with a suitable promoter. All three plasmids can then be transfected into lentivirus cells, such as the Lenti-X-293T cells, and lentivirus can then be harvested, concentrated and screened after a suitable incubation time. Accordingly, methods are provided herein for producing recombinant lentiviral vectors comprising the nucleic acid molecule of the invention described herein.

2.5 Genetically-Modified Cells

[0271] Provided herein are cells genetically-modified to comprise the nucleic acid molecule of the invention described herein. Further provided are genetically-modified cells (e.g., human T cells expressing a CAR or exogenous TCR) with reduced cell-surface expression of beta-2 microglobulin, MHC class I molecules, and or CD52, which do not necessarily comprise the particular nucleic acid molecule of the invention.

[0272] In different variations of the present disclosure, a nucleic acid molecule of the invention is present within the genome of the genetically-modified cell or, alternatively, is not integrated into the genome of the cell. In particular embodiments, the nucleic acid molecule of the invention is inserted into the genome of a cell by targeted insertion at a cleavage site produced by a double-strand break, such as that produced by an engineered nuclease. The presence of 5' and 3' homology arms flanking the first and second expression cassettes of the nucleic acid molecule promote homologous recombination of the nucleic acid molecule into the cleavage site, resulting in targeted insertion.

[0273] In some embodiments where the nucleic acid molecule is not integrated into the genome, the nucleic acid molecule can be present in the genetically-modified cell in a recombinant DNA construct, in an mRNA, in a viral genome, or other nucleic acid which is not integrated into the genome of the cell.

[0274] In specific embodiments, the cells comprising the nucleic acid molecule of the invention, and other genetically-modified cells of the invention, are eukaryotic cells. In particular embodiments, such cells are T cells or NK cells, particularly human T cells or NK cells. In some embodiments, the cells are primary T cells or primary NK cells.

[0275] T cells and NK cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure, any number of T cell and NK cell lines available in the art may be used. In some embodiments of the present disclosure, T cells and NK cells are obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis.

[0276] Genetically-modified cells comprising the nucleic acid molecule disclosed herein, and other genetically-modified cells of the invention, can exhibit a number of functional properties dependent upon which polypeptide is reduced in the cell and/or targeted by the inhibitory nucleic acid molecule. For example, in some genetically-modified cells of the invention, beta-2 microglobulin is reduced, or the inhibitory nucleic acid molecule is against human beta-2 microglobulin, and cell surface beta-2 microglobulin expression is reduced, to a small percentage of wild-type expression. Such genetically-modified cells can be less susceptible to endogenous NK cell killing, have extended persistence time in a subject, exhibit enhanced expansion in a subject, and/or have reduced allogenicity than cells with wild-type levels of B2M or cells which are completely B2M-negative. Reductions in beta-2 microglobulin consequently result in a reduction in cell surface expression of MHC class I molecules, because beta-2 microglobulin is necessary for their assembly and function. Therefore, the same properties are also applicable to genetically-modified cells of the invention which have reduced cell surface expression of MHC class I molecules.

[0277] Susceptibility to NK cell killing can be determined by methods known in the art such as those described further herein. Reductions in NK cell killing can be by about 5%, 10%, 20%, 30%, 40%, 50% 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96, 97%, 98%, 99%, or up to 100% when compared to a control cell.

[0278] The genetically-modified cells of the invention are capable of expansion in a subject following administration. Here, expansion is considered an increase in cell number resulting from proliferation and division in vivo. The degree of expansion depends, in part, on the subject's response to the cells; for example, if the cells are identified as allogeneic and/or non-self, the subject's immune system may reduce the ability of the cells to expand and further reduce persistence of the cells post-administration. Thus, in some examples, genetically-modified cells of the invention can exhibit an increase in expansion in a subject that is about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% 150%, 200%, 250%, 200%, 350%, 400%, 450%, 500%, up to 1000%, or more, when compared to a control cell. Expansion in vivo can be determined post-administration by any method known in the art. Persistence time of a genetically-modified cell in a subject can be considered, for example, as the amount of time post-administration of the cell that it can be detected in the subject by any method known in the art. In some examples, a genetically-modified cell of the invention will have an increase in persistence time that is up to about 1 week, 2 weeks, 3, weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more, longer than a control cell.

[0279] Allogenicity can be determine by any method known in the art, such as those methods described further herein. The genetically-modified cells of the invention can exhibit a reduction in allogenicity of about 5%, 10%, 20%, 30%, 40%, 50% 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% 94%, 95%, 96, 97%, 98%, 99%, or up to 100% when compared to a control cell.

2.6 Methods for Producing Genetically-Modified Cells

[0280] The present disclosure provides methods for producing genetically-modified cells comprising the nucleic acid molecule of the invention described herein. In specific embodiments, methods are provided for modifying the cell to comprise the nucleic acid molecule. In different aspects of the present disclosure, the nucleic acid molecule is integrated into the genome of the cell or, alternatively, is not integrated into the genome of the cell.

[0281] In some embodiments, the nucleic acid molecule is introduced into a cell using any technology known in the art. In specific embodiments, vectors or expression cassettes comprising the nucleic acid molecule disclosed herein is introduced into a cell using a viral vector. Such vectors are known in the art and include lentiviral vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors (reviewed in Vannucci, et al. (2013 New Microbiol. 36:1-22). Recombinant AAV vectors useful in the present disclosure can have any serotype that allows for transduction of the virus into the cell and insertion of the nuclease gene into the cell and, in particular embodiments, into the cell genome. In particular embodiments, recombinant AAV vectors have a serotype of AAV2, AAV6, or AAV8. Recombinant AAV vectors can also be self-complementary such that they do not require second-strand DNA synthesis in the host cell (McCarty, et al. (2001) Gene Ther. 8:1248-54).

[0282] In some embodiments, nucleic acid molecules disclosed herein are delivered into a cell in the form of DNA (e.g., circular or linearized plasmid DNA or PCR products) and/or via a viral vector. In some embodiments, the nucleic acid molecule disclosed herein is coupled covalently or non-covalently to a nanoparticle or encapsulated within such a nanoparticle using methods known in the art (Sharma, et al. (2014) Biomed Res Int. 2014). A nanoparticle is a nanoscale delivery system whose length scale is <1 .quadrature.m, preferably <100 nm. Such nanoparticles may be designed using a core composed of metal, lipid, polymer, or biological macromolecule, and multiple copies of the nucleic acid molecules can be attached to or encapsulated with the nanoparticle core. This increases the copy number of the DNA that is delivered to each cell and, so, increases the intracellular expression to maximize the likelihood that the encoded products will be expressed. The surface of such nanoparticles may be further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) to form a core-shell nanoparticle whose surface confers additional functionalities to enhance cellular delivery and uptake of the payload (Jian et al. (2012) Biomaterials. 33(30): 7621-30). Nanoparticles may additionally be advantageously coupled to targeting molecules to direct the nanoparticle to the appropriate cell type and/or increase the likelihood of cellular uptake. Examples of such targeting molecules include antibodies specific for cell surface receptors and the natural ligands (or portions of the natural ligands) for cell surface receptors.

[0283] In some embodiments, the nucleic acid molecule disclosed herein can be encapsulated within liposomes or complexed using cationic lipids (see, e.g., LIPOFECTAMINE, Life Technologies Corp., Carlsbad, Calif.; Zuris et al. (2015) Nat Biotechnol. 33: 73-80; Mishra et al. (2011) J Drug Deliv. 2011:863734). The liposome and lipoplex formulations can protect the payload from degradation, and facilitate cellular uptake and delivery efficiency through fusion with and/or disruption of the cellular membranes of the cells.

[0284] In some embodiments, the nucleic acid molecule disclosed herein can be encapsulated within polymeric scaffolds (e.g., PLGA) or complexed using cationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv. 2(4): 523-536). In some embodiments, the nucleic acid molecule disclosed herein can be combined with amphiphilic molecules that self-assemble into micelles (Tong et al. (2007) J Gene Med. 9(11): 956-66). Polymeric micelles may include a micellar shell formed with a hydrophilic polymer (e.g., polyethyleneglycol) that can prevent aggregation, mask charge interactions, and reduce nonspecific interactions outside of the cell.

[0285] In some embodiments, the nucleic acid molecule disclosed herein can be formulated as an emulsion for delivery to the cell. The term "emulsion" refers to, without limitation, any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets, including lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water, when a water immiscible phase is mixed with an aqueous phase. These other lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases. Emulsions are composed of an aqueous phase and a lipophilic phase (typically containing an oil and an organic solvent). Emulsions also frequently contain one or more surfactants. Nanoemulsion formulations are well known, e.g., as described in US Patent Application Nos. 2002/0045667 and 2004/0043041, and U.S. Pat. Nos. 6,015,832, 6,506,803, 6,635,676, and 6,559,189, each of which is incorporated herein by reference in its entirety.

[0286] In some embodiments, the nucleic acid molecule disclosed herein can be covalently attached to, or non-covalently associated with, multifunctional polymer conjugates, DNA dendrimers, and polymeric dendrimers (Mastorakos et al. (2015) Nanoscale. 7(9): 3845-56; Cheng et al. (2008) J Pharm Sci. 97(1): 123-43). The dendrimer generation can control the payload capacity and size, and can provide a high payload capacity. Moreover, display of multiple surface groups can be leveraged to improve stability and reduce nonspecific interactions.

[0287] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means. Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection. Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362. Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

2.7 Pharmaceutical Compositions

[0288] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a genetically-modified cell, or a population of genetically-modified cells, of the present disclosure and a pharmaceutically-acceptable carrier. Such pharmaceutical compositions can be prepared in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (21st ed. 2005). In the manufacture of a pharmaceutical formulation according to the present disclosure, cells are typically admixed with a pharmaceutically acceptable carrier and the resulting composition is administered to a subject. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. In some embodiments, pharmaceutical compositions of the present disclosure further comprises one or more additional agents useful in the treatment of a disease in the subject. In additional embodiments, where the genetically-modified cell is a genetically-modified human T cell or NK cell (or a cell derived therefrom), pharmaceutical compositions of the present disclosure further include biological molecules, such as cytokines (e.g., IL-2, IL-7, IL-15, and/or IL-21), which promote in vivo cell proliferation and engraftment. Pharmaceutical compositions comprising genetically-modified cells of the present disclosure can be administered in the same composition as an additional agent or biological molecule or, alternatively, can be co-administered in separate compositions.

[0289] In some embodiments, the pharmaceutical compositions of the present disclosure are useful for treating any disease state that can be targeted by T cell adoptive immunotherapy. In a particular embodiment, the pharmaceutical compositions of the present disclosure are useful as immunotherapy in the treatment of cancer. Such cancers can include, without limitation, carcinoma, lymphoma, sarcoma, blastomas, leukemia, cancers of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma, leukemia, and Hodgkin's lymphoma. In certain embodiments, cancers of B-cell origin include, without limitation, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma. Non-limiting examples of cancer which may be treated with the pharmaceutical compositions and medicaments of the present disclosure are carcinomas, lymphomas, sarcomas, melanomas, blastomas, leukemias, and germ cell tumors, including but not limited to cancers of B-cell origin, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, liver cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, multiple myeloma, Hodgkin lymphoma, non-Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, immunoblastic large cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-cell lymphoma, and any combinations of said cancers. In certain embodiments, cancers of B-cell origin include, without limitation, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell lymphoma, diffuse large B cell lymphoma, pre-B ALL (pediatric indication), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt's lymphoma, and B-cell non-Hodgkin's lymphoma.

[0290] In some of these embodiments wherein cancer is treated with the presently disclosed genetically-modified cells, the subject administered the genetically-modified cells is further administered an additional therapeutic, such as radiation, surgery, or a chemotherapeutic agent.

[0291] The invention further provides a population of genetically-modified cells comprising a plurality of genetically-modified cells described herein. Such genetically-modified cells can comprise in their genome a nucleic acid molecule encoding an engineered antigen receptor, such as a chimeric antigen receptor or exogenous T cell receptor, and an inhibitory nucleic acid molecule, such as an RNA interference molecule. Such genetically-modified cells can also comprise in their genome a nucleic acid molecule encoding an engineered antigen receptor, such as a chimeric antigen receptor or exogenous T cell receptor, and have reduced cell surface expression of beta-2 microglobulin, MHC class I molecules, or CD52, without necessarily comprising the particular nucleic acid molecule of the invention. Thus, in various embodiments of the invention, a population of genetically-modified cells is provided wherein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100%, of cells in the population are a genetically-modified cell described herein.

2.8 Methods of Administering Genetically-Modified Cells

[0292] Another aspect disclosed herein is the administration of the genetically-modified cells of the present disclosure to a subject in need thereof. In particular embodiments, the pharmaceutical compositions described herein are administered to a subject in need thereof.

[0293] For example, an effective amount of a genetically-modified cell or population of genetically-modified cells of the invention which express a cell surface chimeric antigen receptor or exogenous T cell receptor, can be administered to a subject having a disease. In particular embodiments, the disease can be cancer, such as a cancer of B-cell origin. Thus, the present disclosure also provides a method for providing a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the step of administering to the mammal a CAR T cell, wherein the CAR comprises an extracellular ligand-binding domain that specifically interacts with a predetermined target, such as a tumor antigen, and an intracellular domain that comprises at least one signaling domain, such as CD3, and optionally one or more co-stimulatory signaling domains. The administered CAR T cells are able to reduce the proliferation, reduce the number, or kill target cells in the recipient. Unlike antibody therapies, genetically-modified cells of the present disclosure are able to replicate and expand in vivo, resulting in long-term persistence that can lead to sustained control of a disease.

[0294] In examples wherein the inhibitory nucleic acid molecule is against human beta-2 microglobulin or a component of the MHC class I molecule, or wherein beta-2 microglobulin or MHC class I molecules are otherwise reduced, expansion and/or persistence of such CAR T cells can be enhanced in the subject when compared to a CAR T cell having wild-type levels of beta-2 microglobulin or MHC class I molecules, or no cell surface beta-2 microglobulin or MHC class I expression. Further, allogenicity of the CAR T cell can be reduced when compared to a CAR T cell having a wild-type level of cell surface expression of beta-2 microglobulin and MHC class I molecules. These advantageous characteristics result from the incomplete reduction of cell surface beta-2 microglobulin (and consequently MHC class I molecules) to a small percentage of wild-type expression, which allows for reduced allogenicity but avoidance of NK cells which would otherwise target a beta-2 microglobulin-negative cell.

[0295] Examples of possible routes of administration include parenteral, (e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), or infusion) administration. Moreover, the administration may be by continuous infusion or by single or multiple boluses. In specific embodiments, one or both of the agents is infused over a period of less than about 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, or 1 hour. In still other embodiments, the infusion occurs slowly at first and then is increased over time.

[0296] In some embodiments, a genetically-modified eukaryotic cell or population thereof of the present disclosure targets a tumor antigen for the purposes of treating cancer. Such cancers can include, without limitation, carcinoma, lymphoma, sarcoma, blastomas, leukemia, cancers of B cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin's lymphoma. In specific embodiments, cancers and disorders include but are not limited to pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B cell lymphoma, salvage post allogenic bone marrow transplantation, and the like. These cancers can be treated using a combination of CARs that target, for example, CD19, CD20, CD22, and/or ROR1. In some non-limiting examples, a genetically-modified eukaryotic cell or population thereof of the present disclosure targets carcinomas, lymphomas, sarcomas, melanomas, blastomas, leukemias, and germ cell tumors, including but not limited to cancers of B-cell origin, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, liver cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, environmentally induced cancers including those induced by asbestos, multiple myeloma, Hodgkin lymphoma, non-Hodgkin's lymphomas, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, immunoblastic large cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, and T-cell lymphoma, and any combinations of said cancers. In certain embodiments, cancers of B-cell origin include, without limitation, B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell lymphoma, diffuse large B cell lymphoma, pre-B ALL (pediatric indication), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt's lymphoma, multiple myeloma, and B-cell non-Hodgkin's lymphoma.

[0297] When an "effective amount" or "therapeutic amount" is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size (if present), extent of infection or metastasis, and condition of the patient (subject). In some embodiments, a pharmaceutical composition comprising the genetically-modified cells described herein is administered at a dosage of 104 to 109 cells/kg body weight, including all integer values within those ranges. In further embodiments, the dosage is 105 to 106 cells/kg body weight, including all integer values within those ranges. In some embodiments, cell compositions are administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0298] In some embodiments, administration of genetically-modified cells of the present disclosure reduce at least one symptom of a target disease or condition. For example, administration of genetically-modified cells of the present disclosure can reduce at least one symptom of a cancer, such as cancers of B-cell origin. Symptoms of cancers, such as cancers of B-cell origin, are well known in the art and can be determined by known techniques.

EXPERIMENTAL

[0299] This disclosure is further illustrated by the following examples, which should not be construed as limiting. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are intended to be encompassed in the scope of the claims that follow the examples below.

Example 1

NK Cell Killing of B2M Knockout Primary Human T Cells

1. Methods and Materials

[0300] Primary human T cells were stimulated for 3 days using ImmunoCult anti-CD2/CD3/CD28 (StemCell Technologies) in the presence of IL-2 (Gibco) in XVIVO-15 medium (Lonza) supplemented with 5% fetal bovine serum. RNA encoding B2M13-14x479 nuclease was introduced into the T cells using the 4D Nucleofector (Lonza). Cells were cultured in the presence of IL-2 for 6 days before magnetic depletion of remaining B2M.sup.+ cells using biotinylated anti-human B2M (BioLegend) and a Biotin Selection Kit (StemCell Technologies). NK cells were isolated from PBMC samples of the same donor using a CD56 positive selection kit (StemCell Technologies). Daudi cells were purchased from ATCC. Daudi cells are naturally B2M.sup.- and are reported to be highly sensitive to NK cytolysis. All target cells were labeled with 1 uM CellTrace Violet (LifeTechnologies) to distinguish them from effectors in mixed cultures. Isolated NK cells were mixed with either autologous B2M.sup.+ T cell targets (negative control for NK cytolysis), Daudi targets (positive control for NK cytolysis), or autologous B2M KO T cell targets (experimental sample) at effector:target ratios of 2:1, 1:1, 0.5:1, and 0:1. Killing was assessed after 2 h of co-culture. Killing by NK cells was measured by staining with CaspACE-VAD-FMK (Promega).

2. Results

[0301] NK cells elicited only dim VAD-FMK signals in autologous B2M.sup.+ targets, indicating low levels of apoptosis induction by active caspases (FIGS. 2A-2D). In comparison, high VAD-FMK signals were induced in large percentages of Class I--Daudi cells (71-83%, FIGS. 2I-2L), indicating extensive caspase cascade activation. Similarly, B2M.sup.- autologous T cells returned high VAD-FMK signals in response to NK encounter (19-47%, FIG. 2 E-H), indicative of caspase-mediated apoptosis induction. A graphical summary of these results appears in FIG. 3.

3. Conclusions

[0302] Complete knockout of cell surface B2M using engineered meganucleases sensitizes primary human T cells to NK cell attack and killing.

Example 2

Characterization of Candidate shRNAs Against B2M and Effect of B2M Knockdown on NK Cell Killing of Primary Human T Cells

1. Materials and Methods

[0303] Five Mission-shRNA lentiviral transfer plasmids encoding different B2M targeting sequences were purchased from Sigma-Aldrich. Second-generation lentiviral vectors were produced in-house using Lenti-X 293T cells (ClonTech) and a triple transfection method (Lipofectamine 2000--Thermo-Fisher). T cells were prepared for lentiviral transduction by stimulating for 3 days with ImmunoCult anti-CD2/CD3/CD28 as in Example 1. Transduction was carried out in the presence of 5 uM polybrene (Sigma-Aldritch) and transduced cells were selected with puromycin (InVivoGen) beginning at 48 h post-transduction and concluding 72 h following drug addition. Selected cells were expanded for 5 days in IL-2 supplemented medium before a flow cytometric analysis of B2M surface expression to determine the extent of knockdown. Cultures receiving B2M shRNAs were used as targets in NK and CTL killing assays. The NK killing assays were carried out as described in Example 1, but the K562 cell line was used as the positive control for NK cytolysis. For the CTL killing assay, CD8+ T cells from a donor unrelated to the donor of the target cells were isolated and used as effectors. The NK killing assay was carried out for 2 h and the CTL assay was carried out for 6 h. For both assays, target cells were labeled with 1 uM CellTrace Violet (Life Technologies), and killing was measured using CaspACE-VAD-FMK (Promega).

2. Results

[0304] Five shRNAs were screened in human T cells for interference with B2M expression. Two sequences did not reduce the mean fluorescence intensity of B2M in a cytometric analysis (not shown). Three shRNA sequences did reduce the MFI of B2M expression, with sequence 254 and 255 reducing MFI by approximately 50% and sequence 472 reducing the MFI by approximately 95% (FIG. 4).

[0305] CTL and NK killing of targets exhibiting altered B2M expression was next measured. NK cells, but not CTLs induced caspase activation (measured by VAD-FMK signal) in Class I deficient K562 cells (46% vs. 5%--FIGS. 5A and 5B). Conversely, CTLs induced caspase activation (32%) in mismatched B2M.sup.+ T cells while NK cells induced a signal in a lower frequency of mismatched T cells (14%) (FIGS. 5C and 5D). In T cell targets exhibiting a 50% reduction in B2M antigen density, NK cells elicited caspase activity in 17% of targets while mismatched CTLs did so in 36% of targets (FIGS. 6A and 6B). In T cell targets exhibiting a 95% knockdown of B2M levels, NK cells elicited caspase activation in 16% of targets, while mismatched CTLs did so in 20.8% of targets (FIGS. 5 C and D).

3. Conclusions

[0306] B2M expression can be effectively knocked down using shRNA delivered by a viral vector. Using caspase (VAD-FMK) activity to measure apoptosis induction in target cells by NK cells or CTLs, it was determined that B2M knockdown does not alter a target's susceptibility to NK cytolysis, as both B2M knockdown targets exhibited the same VAD-FMK frequency as un-manipulated targets, and less VAD-FMK signal than K562 targets. In addition, B2M knockdown confers some protection against CTL cytolysis, as the frequency of VAD-FMK+ targets in the shRNA 472 group was approximately half the frequency observed in the positive control. In fact, there was a direct relationship between the degree of knockdown and the degree of protection against CTL activity from NK cells.

Example 3

Production and Characterization of CAR T Cells Utilizing shRNA to Reduce Cell Surface Expression of B2M

1. Materials and Methods

[0307] A number of constructs were prepared comprising an anti-CD19 CAR coding sequence and an shRNA against B2M. These are illustrated in FIG. 7A-7F and are provided in SEQ ID NOs: 18-23. CAR constructs 7007 and 7217 (SEQ ID NOs: 18 and 19) comprise the CAR coding sequence and the shRNA472 sequence in the same 5' to 3' orientation. CAR constructs 7008 and 7218 (SEQ ID NOs: 20 and 21) comprise the CAR coding sequence in the 3' to 5' orientation, and shRNA472 sequence in the 5' to 3' orientation (i.e., tail-to-tail). CAR constructs 7009 and 7219 (SEQ ID NOs: 22 and 23) comprise both the CAR coding sequence and the shRNA472 sequence in the 3' to 5' orientation. The 5' and 3' homology arms flanking the CAR coding sequence and the shRNA472 sequence have homology to regions upstream and downstream of the TRC 1-2 recognition sequence in the TRAC locus.

[0308] CAR T cells will be prepared using primary donor human T cells transduced with recombinant AAV vectors comprising one of the CAR/shRNA constructs above, with simultaneous nucleofection of mRNA encoding the TRC 1-2x.87EE to induce a double-strand break at the TRC 1-2 recognition sequence and promote targeted insertion of the construct into the genome of the T cells. Beta-2 microglobulin expression will be determined as described above to determine which orientation of the first and second expression cassettes will result in the highest and/or the most consistent CAR expression, along with the most consistent level of B2M knockdown on the cell surface.

[0309] CAR T cells produced with certain constructs will be evaluated in both the allogenicity and NK cell killing assays previously described above. Further, CAR T cells produced using the disclosed constructs will be evaluated in various stress tests, in which the CAR T cells are repeatedly exposed to antigen in order to determine changes in cell proliferation/expansion and cytotoxic potential. CAR T cells produced using the disclosed constructs will also be utilized with in vivo tumor models to determine their ability to clear tumor cells in an animal and to evaluate their ability to persist in vivo. It is expected, based on the Examples described herein, that CAR T cells having a reduced but incomplete knockdown of cell surface beta-2 microglobulin will have greater persistence and/or enhanced expansion in vivo when compared to CAR T cells which are completely B2M-negative and may be susceptible to NK cell killing.

[0310] In a particular study, CAR T cells were prepared that are TCR-negative, CAR-positive, and have reduced cell surface expression of B2M. CAR T cells were prepared using donor templates that comprise a promoter-driven CAR coding sequence, a T2A element, and one or multiple promoter-driven B2M shRNA cassettes. In this study, an apheresis sample was drawn from a healthy, informed, and compensated donor, and the T cells were enriched using the CD3 positive selection kit II in accord with the manufacturer's instructions (Stem Cell Technologies). T cells were activated using ImmunoCult T cell stimulator (anti-CD2/CD3/CD28--Stem Cell Technologies) in X-VIVO 15 medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2 (Gibco). After 3 days of stimulation, cells were collected and samples of 1e6 cells were electroporated with the following mixture of nucleic acid species using the Lonza 4D NucleoFector.

[0311] 1 .mu.g mRNA encoding the TRC 1-2x.87EE meganuclease which produces a double-strand break in Exon 1 of the T cell receptor alpha constant region gene

[0312] 1.5 .mu.l of 100 mM siRNA specific for TMEM173 (STING)

[0313] 1 .mu.g of linearized plasmid DNA comprising a donor template In this experiment, three different CAR constructs were analyzed for their ability to knock down B2M surface expression. All three constructs use homology to genomic regions flanking the TRC 1-2x.87EE binding site (referred to as the TRC 1-2 recognition site) to direct targeted insertion into the T cell receptor alpha constant region locus, and they all express a CAR that comprises a CD34 epitope tag (for detection). Construct 7002 (SEQ ID NO: 11) does not encode an shRNA gene. Construct 7008 (SEQ ID NO: 20) encodes one copy of shRNA472. Construct 7029 (SEQ ID NO: 24) encodes two copies of this shRNA cassette. Expression from each shRNA cassette is driven by a U6 promoter.

[0314] Cell cultures were maintained for 10 additional days in X-VIVO15 medium supplemented with 5% FBS and 30 ng/ml of IL-2. On d 4, 7, and 10 post-nucleofection, the cultures were sampled and analyzed for surface expression of CD3 (anti-CD3-BV711, BioLegend), CD34 (anti-CD34-PE, LifeTechnologies), and B2M (anti-B2M-APC, BioLegend). Flow cytometry data were acquired on a Beckman-Coulter CytoFLEX-LX.

2. Results

[0315] B2M surface levels were measured in samples nucleofected with a control CAR construct (7002) or with CAR constructs expressing one (7008) or two (7029) copies of B2M-specific shRNA (FIG. 12). When comparing the CD3-/CD34+ populations in 7002 (control) and 7008 (single shRNA) expressing cells, 7008 expressing cells were observed to display slightly lower levels of surface B2M (FIG. 12A). Notably, cells nucleofected with construct 7029 (two shRNA copies) displayed approximately half of the amount of B2M displayed on the surface of control cells (7002) (FIG. 12B). This observation was specific to the CD3-/CD34+ population, but was not observed in the CD3-/CD34- population (FIG. 12C).

3. Conclusions

[0316] A pre-screened B2M-targeting shRNA can knock down B2M expression levels on the surface of cells into which the construct has been delivered (via targeted insertion into the T cell receptor alpha constant region locus). Due to the high abundance of B2M transcripts, these data suggest that a single shRNA copy can be sufficient for low levels of B2M knockdown, whereas multiple copies of the shRNA cassette may be required to achieve more significant knockdown.

Example 4

Production and Characterization of CAR T Cells Utilizing shRNA to Reduce Cell Surface Expression of B2M

1. Materials and Methods

[0317] A number of constructs were prepared comprising an anti-CD19 CAR coding sequence and an shRNA against B2M. These are illustrated in FIG. 7A-7F and are provided in SEQ ID NOs: 18-23. CAR constructs 7007 and 7217 (SEQ ID NOs: 18 and 19) comprise the CAR coding sequence and the shRNA472 sequence in the same 5' to 3' orientation. CAR constructs 7008 and 7218 (SEQ ID NOs: 20 and 21) comprise the CAR coding sequence in the 3' to 5' orientation, and shRNA472 sequence in the 5' to 3' orientation (i.e., tail-to-tail). CAR constructs 7009 and 7219 (SEQ ID NOs: 22 and 23) comprise both the CAR coding sequence and the shRNA472 sequence in the 3' to 5' orientation. CAR constructs 7056, 7059, and 7060 contain modified versions of the U6-shRNA gene cassette. A cloning site that was located between the U6 promoter and the hairpin sequence in constructs 7007-7009, and in 7217-7219 was removed. Construct 7056 comprises the CAR coding sequence and the shRNA472 sequence in the 3' to 5' orientation. Construct 7056 comprises the CAR coding sequence in 3' to 5' orientation, and the shRNA472 sequence in the 5' to 3' orientation (tail-to-tail). Construct 7060 comprises the CAR coding sequence in 3' to 5' orientation and two copies of the U6-shRNA472 sequence in 5' to 3' orientation. The 5' and 3' homology arms flanking the CAR coding sequence and the shRNA472 sequence have homology to regions upstream and downstream of the TRC 1-2 recognition sequence in the T cell receptor alpha constant locus.

[0318] In a particular study, CAR T cells were prepared that are TCR-negative, CAR-positive, and have reduced cell surface expression of B2M. CAR T cells were prepared using donor templates that comprise a promoter-driven CAR coding sequence, and one or multiple promoter-driven B2M shRNA cassettes. In this study, an apheresis sample was drawn from a healthy, informed, and compensated donor, and the T cells were enriched using the CD3 positive selection kit II in accord with the manufacturer's instructions (Stem Cell Technologies). T cells were activated using ImmunoCult T cell stimulator (anti-CD2/CD3/CD28--Stem Cell Technologies) in X-VIVO 15 medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2 (Gibco). After 3 days of stimulation, cells were collected and samples of 1e6 cells were electroporated with the following mixture of nucleic acid species using the Lonza 4D NucleoFector.

[0319] 1 .mu.g mRNA encoding the TRC 1-2x.87EE meganuclease which produces a double-strand break in Exon 1 of the T cell receptor alpha constant region gene

[0320] 1.5 .mu.l of 100 mM siRNA specific for TMEM173 (STING)

[0321] 1 .mu.g of linearized plasmid DNA comprising a donor template In this experiment, four different CAR constructs were analyzed for their ability to knock down B2M surface expression. All four constructs use homology to genomic regions flanking the TRC 1-2x.87EE recognition site to direct targeted insertion into the T cell receptor alpha constant region locus, and they all express a CAR that comprises a CD34 epitope tag (for detection). Construct 7002 (SEQ ID NO: 11) does not encode an shRNA gene. Construct 7056 (SEQ ID NO: 25) encodes one copy of shRNA472, and both cassettes are in the 3' to 5' orientation (head-to-tail). Construct 7059 (SEQ ID NO: 26) encodes one copy of this shRNA cassette, with the CAR expression cassette in a 3' to 5' orientation, and the shRNA472 cassette in a 5' to 3' orientation (tail-to-tail). Construct 7060 (SEQ ID NO: 27) is in the same orientation as construct 7059 but encodes two copies of the shRNA472 cassette (tail-to-tail). Expression from each shRNA cassette is driven by a U6 promoter. Cell cultures were maintained for up to 10 additional days in X-VIVO15 medium supplemented with 5% FBS and 30 ng/ml of IL-2. On d4, 7, and/or 10 post-nucleofection, the cultures were sampled and analyzed for surface expression of CD3 (anti-CD3-BV711, BioLegend), CD34 (anti-CD34-PE, or APC, LifeTechnologies), B2M (anti-B2M-APC, or PE, BioLegend), and/or HLA-A, B, and C (clone W6/32, BV605). Flow cytometry data were acquired on a Beckman-Coulter CytoFLEX-LX.

2. Results

[0322] B2M surface levels were measured in samples nucleofected with a control CAR construct (7002) or with CAR constructs expressing one (7056 or 7059) or two (7060) copies of B2M-specific shRNA in either head-to-tail (7056) or tail-to-tail (7059, 7060) configurations. A restriction digest site that was present in previous constructs between the U6 promoter and the shRNA sequence was been removed from these shRNA472 vectors. It was hypothesized that the palindromic restriction digest site interfered with the efficacy of the constructs and the ability of the shRNA to knock down B2M.

[0323] When comparing the CD3-/CD34+ populations in 7002 (control) and 7056 (single shRNA) expressing cells, 7056 expressing cells were observed to display lower levels of surface B2M (77% knockdown) (FIG. 13A). Cells nucleofected with 7059 (single copy, tail-to-tail) displayed a 90.1% knockdown of B2M relative to 7002 control cells (FIG. 13B), while 7060 nucleofection (two copies, tail-to-tail) resulted in a 92% knockdown relative to 7002 controls (FIG. 13C).

3. Conclusions

[0324] A pre-screened B2M-targeting shRNA can knock down B2M expression levels on the surface of cells into which the construct has been delivered (via targeted insertion into the T cell receptor alpha constant region locus). Removing the cloning site between the U6 promoter and the hairpin sequence improves the efficiency with which B2M is knocked down. 7008 (tail-to-tail, one shRNA472 cassette--FIG. 12A) supports minimal B2M knockdown while 7059 (one cassette, tail-to-tail--FIG. 13B) supports greater than 90% knockdown. As was observed using a CD52-specific shRNA, superior knockdown was observed when the CAR promoter and the shRNA promoter were oriented in different directions (tail-to-tail configuration). Adding a second shRNA sequence did not provide any noticeable benefit (92% versus 90.1% knockdown).

Example 5

Production and Characterization of CAR T Cells Utilizing shRNA to Reduce Cell Surface Expression of B2M

1. Materials and Methods

[0325] In this study, an apheresis sample was drawn from a healthy, informed, and compensated donor, and the T cells were enriched using the CD3 positive selection kit II in accord with the manufacturer's instructions (Stem Cell Technologies). T cells were activated using ImmunoCult T cell stimulator (anti-CD2/CD3/CD28--Stem Cell Technologies) in X-VIVO 15 medium (Lonza) supplemented with 5% fetal bovine serum and 10 ng/ml IL-2 (Gibco). After 3 days of stimulation, cells were collected and samples of 1e6 cells were electroporated with 1 ug of RNA encoding the TRC 1-2L.1592 meganuclease, which recognizes and cleaves the TRC 1-2 recognition sequence in the T cell receptor alpha constant locus, and were transduced with AAV packaged with construct 7056 at an MOI of 25000 viral genomes/cell.

[0326] Cell cultures were maintained for up to 10 additional days in X-VIVO15 medium supplemented with 5% FBS and 30 ng/ml of IL-2. On day 4, 7, and/or 10 post-nucleofection, the cultures were sampled and analyzed for surface expression of CD3 (anti-CD3-PE, BioLegend), (anti-FMC63 anti-CAR clone VM16 conjugated to AlexaFluor488), B2M (anti-B2M-APC, or PE, BioLegend), and HLA-A, B, and C (clone W6/32, BV605). Flow cytometry data were acquired on a Beckman-Coulter CytoFLEX-LX.

2. Results

[0327] B2M and HLA-ABC levels were measured in samples expressing construct 7056 and control populations. FIG. 14A shows the B2M surface levels in CD3-/CAR+ cells compared to TRAC-edited cells expressing no shRNA from a control culture. FIG. 14B shows B2M levels on CD3-/CAR+ versus CD3+/CAR- populations in the same culture. FIGS. 14C and 14D make the same respective comparisons in displays of HLA-ABC surface levels. The CD3-/CAR+ fraction of cells transduced with AAV-7056 displayed levels of B2M and HLA-ABC that are reduced by greater than 90% compared to control populations.

3. Conclusions

[0328] A pre-screened B2M-targeting shRNA can knock down B2M expression levels on the surface of cells into which the construct has been delivered (via targeted insertion into the T cell receptor alpha constant region locus). This effect is specific to CAR+ populations (i.e., cells in which targeted integration into the TRAC locus has occurred). This experiment demonstrates that B2M can be efficiently knocked down using a single copy of shRNA472 co-delivered to the TRAC locus with the CAR gene on the same AAV template.

Example 6

Characterization of Candidate shRNAs Against CD52 in Primary Human T Cells

1. Materials and Methods

[0329] Five Mission-shRNA lentiviral transfer plasmids encoding different CD52 targeting sequences were purchased from Sigma-Aldrich. Second-generation lentiviral vectors were produced in-house using Lenti-X 293T cells (ClonTech) and a triple transfection method (Lipofectamine 2000--Thermo-Fisher). T cells were prepared for lentiviral transduction by stimulating for 3 days with ImmunoCult anti-CD2/CD3/CD28 as in Example 1. Transduction was carried out in the presence of 5 uM polybrene (Sigma-Aldritch) and transduced cells were expanded for 5 days in IL-2 supplemented medium before a flow cytometric analysis of CD52 surface levels. Cells were not selected with puromycin because a heterogeneous population was desired for downstream attempts at magnetic depletion of CD52Hi cells. Cells transduced with a lentivirus encoding shRNA 568 were labeled with biotinylated anti-CD52 (Miltenyi Biotec), and magnetic separation was performed using a Biotin Positive Selection Kit (StemCell Technologies). A post-separation analysis of surface CD52 was performed.

2. Results

[0330] Of the 5 shRNA sequences screened, 3 (shRNA568, shRNA572, and shRNA876) interfered with CD52 expression. CD52 surface expression profiles are displayed in FIG. 8. Levels of CD52 displayed on the surface of T cells are shown in FIG. 9 for mock transduced T cells (9A), T cells transduced with an shRNA-568 lentivirus (9B), and LV-shRNA568 transduced cells that have undergone a CD52 magnetic depletion (9C).

3. Conclusions

[0331] CD52 antigen density on the surface of cells can be reduced using shRNA delivered by a viral vector. Sequence 568 exhibited the highest degree of CD52 knockdown. Knockdown of CD52 using this shRNA sequence was sufficient to allow for magnetic depletion of non-transduced CD52 Hi cells.

Example 7

CD52 Knockdown Profiles Using CAR/CD52 Constructs with Different Orientations

1. Materials and Methods

[0332] T cells were stimulated for 3 days using ImmunoCult anti-CD2/CD3/CD28 as described in EXAMPLE 1. After 3 days, TRC 1-2x.87EE mRNA, STING siRNA, and linearized AAV transfer vector encoding different CAR constructs (FIG. 10) were delivered to the T cells using the 4-D Nucleofector (Lonza). Cultures of nucleofected T cells were carried for 10 days in medium supplemented with IL-2 prior to flow cytometric analyses of CD3, CAR (CD34 epitope-tagged), and CD52.

2. Results

[0333] To demonstrate un-manipulated levels of CD52 surface display on TRAC-edited CAR T cells, a TRC 1-2x.87EE nuclease and a CD34-tagged CAR construct encoding no shRNA sequence were delivered. CD52 levels on TCR KO cells, TCR KO CAR+ cells and nonedited cells are overlaid in the histogram in FIG. 11A. Three CAR constructs encoding a U6 promoter-controlled CD52 shRNA were evaluated for ability to knock down CD52 when integrated into the TRAC locus. When the CAR gene and the shRNA cassette are both in forward orientation, CD52 antigen density is reduced by approximately 50% (FIG. 11C; construct 7004). Reversing the transcriptional orientation of the CAR gene alone (i.e., a tail-to-tail configuration) reduces the amount of CD52 displayed on the surface by approximately 95% (FIG. 11B; construct 7013), while reversing the orientation of both the CAR gene and the U6-shRNA element reduces the CD52 signal by approximately 90% (FIG. 11D; construct 7014).

3. Conclusions

[0334] The CD52 specific shRNA sequence 568 can interfere with CD52 expression when only one copy is delivered by targeted insertion into the TRAC locus. Altering the transcriptional orientation of either the CAR gene only (i.e., tail-to-tail configuration) or both the CAR and shRNA genes can influence the efficiency of target gene knockdown. Reversing only the CAR gene's orientation resulted in the most efficient knockdown.

4. Further Studies

[0335] A number of constructs were prepared comprising an anti-CD19 CAR coding sequence and an shRNA against CD52. These are illustrated in FIG. 10A-10H and are provided in SEQ ID NOs: 10-17. As described above, CAR constructs 7004 (SEQ ID NO: 12), 7013 (SEQ ID NO: 14), and 7014 (SEQ ID NO: 16) were previously evaluated for their ability to reduce CD52 expression while expressing a CAR. The 5' and 3' homology arms flanking the CAR coding sequence and the shRNA sequence have homology to regions upstream and downstream of the TRC 1-2 recognition sequence in the TRAC locus.

[0336] In additional studies, CAR T cells will be prepared using primary donor human T cells transduced with recombinant AAV vectors comprising one of the CAR/shRNA constructs above, with simultaneous nucleofection of mRNA encoding the TRC 1-2x.87EE to induce a double-strand break at the TRC 1-2 recognition sequence and promote targeted insertion of the construct into the genome of the T cells. CD52 expression will be determined as described above to determine which orientation of the first and second expression cassettes will result in the highest and/or the most consistent CAR expression, along with the most consistent level of CD52 knockdown on the cell surface.

[0337] CAR T cells produced with certain constructs will be evaluated in both the allogenicity and NK cell killing assays previously described above. Further, CAR T cells produced using the disclosed constructs will be evaluated in various stress tests, in which the CAR T cells are repeatedly exposed to antigen in order to determine changes in cell proliferation/expansion and cytotoxic potential. CAR T cells produced using the disclosed constructs will also be utilized with in vivo tumor models to determine their ability to clear tumor cells in an animal and to evaluate their ability to persist in vivo. It is expected, based on the Examples described herein, that enriched populations of CAR T cells can be produced for in vivo use by an advantageous negative-selection for CAR T cells having reduced cell surface expression of CD52.

Sequence CWU 1

1

28122DNAHomo sapiens 1tggcctggag caacaaatct ga 22262DNAArtificial SequenceSynthesized 2gtaccggagg tttgaagatg ccgcatttct cgagaaatgc ggcatcttca aacctttttt 60tg 62358DNAArtificial SequenceSynthesized 3ccggctggtc tttctatctc ttgtactcga gtacaagaga tagaaagacc agtttttg 58458DNAArtificial SequenceSynthesized 4ccggcagcag agaatggaaa gtcaactcga gttgactttc cattctctgc tgtttttg 58558DNAArtificial SequenceSynthesized 5ccggcagata caaactggac tctcactcga gtgagagtcc agtttgtatc tgtttttg 58659DNAArtificial SequenceSynthesized 6ccggccacca tcactcgcaa gagaactcga gttctcttgc gagtgatggt ggttttttg 59758DNAArtificial SequenceSynthesized 7ccggcctcct ggttatggta cagatctcga gatctgtacc ataaccagga ggtttttg 58858DNAArtificial SequenceSynthesized 8ccggcaatgc cataatccac ctcttctcga gaagaggtgg attatggcat tgtttttg 58958DNAArtificial SequenceSynthesized 9ccgggcagca tgagcggagg catttctcga gaaatgcctc cgctcatgct gctttttg 58107524DNAArtificial SequenceSynthesized 10cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgggcggagt tagggcggag ccaatcagcg tgcgccgttc 2160cgaaagttgc cttttatggc tgggcggaga atgggcggtg aacgccgatg attatataag 2220gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg 2280tttgttccgg aaagccacca tggcgctccc agtgacagcc ttacttttac ctctggcgtt 2340attattgcac gcggctcgtc ctgacataca gatgactcag actacctctt ccctatctgc 2400ttctttaggc gaccgagtaa caatatcttg ccgggccagc caggacatct caaaatactt 2460aaactggtat cagcagaagc cggacggaac agttaagttg ctcatttacc acacgtcgag 2520attacactca ggcgttccta gccgattttc gggttccggt tccggtacgg actacagcct 2580gacaatcagt aaccttgagc aggaggacat cgccacctac ttctgtcagc agggcaacac 2640gctcccgtac acattcggtg ggggaactaa gctggagatt accggaggcg gtggcagcgg 2700tggcggcggc agcgggggtg gcggctcgga ggtcaagtta caggagagcg gaccgggctt 2760ggtcgcacct agccagagcc tctcagtcac gtgcactgtg tctggagtca gtctcccaga 2820ctacggggta tcatggatac gacagccgcc tagaaagggc ttagagtggc tgggggttat 2880ctggggaagt gaaaccacat actacaactc agctctcaag agccgcctca ccatcattaa 2940ggacaacagt aagtcgcagg ttttcttaaa gatgaactct ctccagactg acgacaccgc 3000tatttactac tgcgcgaagc actactacta cggcgggagt tacgcaatgg actactgggg 3060tcagggcact tctgtgaccg tatccagcga gttacctacc cagggaacat tttcaaatgt 3120ttctacaaat gtatccccag cgaagcccac tactacccca gccccacgtc cccccacgcc 3180agctccaacg atagcaagtc agcccttatc tcttcgccct gaggcttgca ggcccgcggc 3240gggcggcgcc gttcacacgc gaggactaga cttcgcctgc gacatctaca tctgggcacc 3300actagccggg acttgcggag tgttgttgtt gagcttggta ataacgctct actgcaaagc 3360gagccgcaaa aaagcggcgg cggcggctaa aagcccgttt gcgagcccgg cgagcagcgc 3420gcaggaagaa gatgcgagca gctgccgcgc gccgagcgaa gaagaaggca gctgcgaact 3480gagagtgaag ttctctcgct ccgcggacgc acccgcttac cagcagggtc agaaccagct 3540atacaacgag ttaaacctgg ggcgccggga ggagtacgac gtgttagaca agcgtagagg 3600tagggacccg gagatgggag gcaagcctcg gagaaagaac ccccaggagg gcctgtacaa 3660cgaactccag aaggacaaga tggctgaggc gtactcggag attggtatga agggcgagag 3720acgtcgcgga aagggacacg acggcttata ccaggggctt tccaccgcga ccaaggacac 3780atacgacgcg ctgcacatgc aagccttacc acctcgatga ggtaccagcg gccgcttcga 3840gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa 3900aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc 3960aataaacaag ttaacaacaa caattcgaat ttaaatcgga tccgcaacaa atctgacttt 4020gcatgtgcaa acgccttcaa caacagcatt attccagaag acaccttctt ccccagccca 4080ggtaagggca gctttggtgc cttcgcaggc tgtttccttg cttcaggaat ggccaggttc 4140tgcccagagc tctggtcaat gatgtctaaa actcctctga ttggtggtct cggccttatc 4200cattgccacc aaaaccctct ttttactaag aaacagtgag ccttgttctg gcagtccaga 4260gaatgacacg ggaaaaaagc agatgaagag aaggtggcag gagagggcac gtggcccagc 4320ctcagtctct ccaactgagt tcctgcctgc ctgcctttgc tcagactgtt tgccccttac 4380tgctcttcta ggcctcattc taagcccctt ctccaagttg cctctcctta tttctccctg 4440tctgccaaaa aatctttccc agctcactaa gtcagtctca cgcagtcact cattaaccca 4500ccaatcactg attgtgccgg cacatgaatg caccaggtgt tgaagtggag gaattaaaaa 4560gtcagatgag gggtgtgccc agaggaagca ccattctagt tgggggagcc catctgtcag 4620ctgggaaaag tccaaataac ttcagattgg aatgtgtttt aactcagggt tgagaaaaca 4680gccaccttca ggacaaaagt cagggaaggg ctctctgaag aaatgctact tgaagatacc 4740agccctacca agggcaggga gaggaccaat tgatggagtt ggccactccc tctctgcgcg 4800ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc 4860cggcctcagt gagcgagcga gcgcgcagag agggagtggc caacggcgcg cctgcaggtc 4920tcaaaaatag ctaccctctc cggcatgaat ttatcagcta gaacggttga atatcatatt 4980gatggtgatt tgactgtctc cggcctttct cacccgtttg aatctttacc tacacattac 5040tcaggcattg catttaaaat atatgagggt tctaaaaatt tttatccttg cgttgaaata 5100aaggcttctc ccgcaaaagt attacagggt cataatgttt ttggtacaac cgatttagct 5160ttatgctctg aggctttatt gcttaatttt gctaattctt tgccttgcct gtatgattta 5220ttggatgttg gaattcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca 5280ccgcatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg 5340acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta 5400cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc 5460gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat 5520aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat 5580ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 5640aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 5700tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 5760agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 5820cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 5880taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 5940tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 6000tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 6060cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 6120gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 6180cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 6240actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 6300ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 6360tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 6420tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 6480acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 6540ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 6600ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 6660ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 6720gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 6780ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 6840aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 6900gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 6960gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 7020aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 7080cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 7140tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 7200ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 7260atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 7320cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 7380ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 7440gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 7500cgcgcgttgg ccgattcatt aatg 7524117739DNAArtificial SequenceSynthesized 11cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgatccagac atgataagat acattgatga gtttggacaa 2160accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaac tggtacctca 2340tcgaggtggt aaggcttgca tgtgcagcgc gtcgtatgtg tccttggtcg cggtggaaag 2400cccctggtat aagccgtcgt gtccctttcc gcgacgtctc tcgcccttca taccaatctc 2460cgagtacgcc tcagccatct tgtccttctg gagttcgttg tacaggccct cctgggggtt 2520ctttctccga ggcttgcctc ccatctccgg gtccctacct ctacgcttgt ctaacacgtc 2580gtactcctcc cggcgcccca ggtttaactc gttgtatagc tggttctgac cctgctggta 2640agcgggtgcg tccgcggagc gagagaactt cactctcagt tcgcagctgc cttcttcttc 2700gctcggcgcg cggcagctgc tcgcatcttc ttcctgcgcg ctgctcgccg ggctcgcaaa 2760cgggctttta gccgccgccg ccgctttttt gcggctcgct ttgcagtaga gcgttattac 2820caagctcaac aacaacactc cgcaagtccc ggctagtggt gcccagatgt agatgtcgca 2880ggcgaagtct agtcctcgcg tgtgaacggc gccgcccgcc gcgggcctgc aagcctcagg 2940gcgaagagat aagggctgac ttgctatcgt tggagctggc gtggggggac gtggggctgg 3000ggtagtagtg ggcttcgctg gggatacatt tgtagaaaca tttgaaaatg ttccctgggt 3060aggtaactcg ctggatacgg tcacagaagt gccctgaccc cagtagtcca ttgcgtaact 3120cccgccgtag tagtagtgct tcgcgcagta gtaaatagcg gtgtcgtcag tctggagaga 3180gttcatcttt aagaaaacct gcgacttact gttgtcctta atgatggtga ggcggctctt 3240gagagctgag ttgtagtatg tggtttcact tccccagata acccccagcc actctaagcc 3300ctttctaggc ggctgtcgta tccatgatac cccgtagtct gggagactga ctccagacac 3360agtgcacgtg actgagaggc tctggctagg tgcgaccaag cccggtccgc tctcctgtaa 3420cttgacctcc gagccgccac ccccgctgcc gccgccaccg ctgccaccgc ctccggtaat 3480ctccagctta gttcccccac cgaatgtgta cgggagcgtg ttgccctgct gacagaagta 3540ggtggcgatg tcctcctgct caaggttact gattgtcagg ctgtagtccg taccggaacc 3600ggaacccgaa aatcggctag gaacgcctga gtgtaatctc gacgtgtggt aaatgagcaa 3660cttaactgtt ccgtccggct tctgctgata ccagtttaag tattttgaga tgtcctggct 3720ggcccggcaa gatattgtta ctcggtcgcc taaagaagca gatagggaag aggtagtctg 3780agtcatctgt atgtcaggac gagccgcgtg caataataac gccagaggta aaagtaaggc 3840tgtcactggg agcgccatgg tggctttccg gaacaaacaa gaaccgcgac ccaaatcccg 3900gctgcgacgg aactagctgt gccacacccg gcgcgtcctt atataatcat cggcgttcac 3960cgcccattct ccgcccagcc ataaaaggca actttcggaa cggcgcacgc tgattggctc 4020cgccctaact ccgcccacta gtgcggccgc ttcgagcaga catgataaga tacattgatg 4080agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg 4140atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt 4200cgaatttaaa tcggatccgc aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca 4260gcattattcc agaagacacc ttcttcccca gcccaggtaa gggcagcttt ggtgccttcg 4320caggctgttt ccttgcttca ggaatggcca ggttctgccc agagctctgg tcaatgatgt 4380ctaaaactcc tctgattggt ggtctcggcc ttatccattg ccaccaaaac cctcttttta 4440ctaagaaaca gtgagccttg ttctggcagt ccagagaatg acacgggaaa aaagcagatg 4500aagagaaggt ggcaggagag ggcacgtggc ccagcctcag tctctccaac tgagttcctg 4560cctgcctgcc tttgctcaga ctgtttgccc cttactgctc ttctaggcct cattctaagc 4620cccttctcca agttgcctct ccttatttct ccctgtctgc caaaaaatct ttcccagctc 4680actaagtcag tctcacgcag tcactcatta acccaccaat cactgattgt gccggcacat 4740gaatgcacca ggtgttgaag tggaggaatt aaaaagtcag atgaggggtg tgcccagagg 4800aagcaccatt ctagttgggg gagcccatct gtcagctggg aaaagtccaa ataacttcag 4860attggaatgt gttttaactc agggttgaga aaacagccac cttcaggaca aaagtcaggg 4920aagggctctc tgaagaaatg ctacttgaag ataccagccc taccaagggc agggagagga 4980ccaattgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgcccg 5040ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg 5100cagagaggga gtggccaacg gcgcgcctgc aggtctcaaa aatagctacc ctctccggca 5160tgaatttatc agctagaacg gttgaatatc atattgatgg tgatttgact gtctccggcc 5220tttctcaccc gtttgaatct ttacctacac attactcagg cattgcattt aaaatatatg 5280agggttctaa aaatttttat ccttgcgttg aaataaaggc ttctcccgca aaagtattac 5340agggtcataa tgtttttggt acaaccgatt tagctttatg ctctgaggct ttattgctta 5400attttgctaa ttctttgcct tgcctgtatg atttattgga tgttggaatt cctgatgcgg 5460tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca 5520atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 5580ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 5640agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 5700gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 5760ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 5820aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 5880aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 5940cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 6000ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 6060cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 6120ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 6180gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 6240gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 6300acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 6360cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 6420acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact

6480ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 6540ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 6600gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 6660atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 6720ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 6780attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 6840ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 6900aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 6960aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 7020ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 7080tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 7140ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 7200cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 7260agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 7320gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 7380ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 7440tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 7500tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 7560cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 7620tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 7680gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatg 7739128073DNAArtificial SequenceSynthesized 12cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgggcggagt tagggcggag ccaatcagcg tgcgccgttc 2160cgaaagttgc cttttatggc tgggcggaga atgggcggtg aacgccgatg attatataag 2220gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg 2280tttgttccgg aaagccacca tggcgctccc agtgacagcc ttacttttac ctctggcgtt 2340attattgcac gcggctcgtc ctgacataca gatgactcag actacctctt ccctatctgc 2400ttctttaggc gaccgagtaa caatatcttg ccgggccagc caggacatct caaaatactt 2460aaactggtat cagcagaagc cggacggaac agttaagttg ctcatttacc acacgtcgag 2520attacactca ggcgttccta gccgattttc gggttccggt tccggtacgg actacagcct 2580gacaatcagt aaccttgagc aggaggacat cgccacctac ttctgtcagc agggcaacac 2640gctcccgtac acattcggtg ggggaactaa gctggagatt accggaggcg gtggcagcgg 2700tggcggcggc agcgggggtg gcggctcgga ggtcaagtta caggagagcg gaccgggctt 2760ggtcgcacct agccagagcc tctcagtcac gtgcactgtg tctggagtca gtctcccaga 2820ctacggggta tcatggatac gacagccgcc tagaaagggc ttagagtggc tgggggttat 2880ctggggaagt gaaaccacat actacaactc agctctcaag agccgcctca ccatcattaa 2940ggacaacagt aagtcgcagg ttttcttaaa gatgaactct ctccagactg acgacaccgc 3000tatttactac tgcgcgaagc actactacta cggcgggagt tacgcaatgg actactgggg 3060tcagggcact tctgtgaccg tatccagcga gttacctacc cagggaacat tttcaaatgt 3120ttctacaaat gtatccccag cgaagcccac tactacccca gccccacgtc cccccacgcc 3180agctccaacg atagcaagtc agcccttatc tcttcgccct gaggcttgca ggcccgcggc 3240gggcggcgcc gttcacacgc gaggactaga cttcgcctgc gacatctaca tctgggcacc 3300actagccggg acttgcggag tgttgttgtt gagcttggta ataacgctct actgcaaagc 3360gagccgcaaa aaagcggcgg cggcggctaa aagcccgttt gcgagcccgg cgagcagcgc 3420gcaggaagaa gatgcgagca gctgccgcgc gccgagcgaa gaagaaggca gctgcgaact 3480gagagtgaag ttctctcgct ccgcggacgc acccgcttac cagcagggtc agaaccagct 3540atacaacgag ttaaacctgg ggcgccggga ggagtacgac gtgttagaca agcgtagagg 3600tagggacccg gagatgggag gcaagcctcg gagaaagaac ccccaggagg gcctgtacaa 3660cgaactccag aaggacaaga tggctgaggc gtactcggag attggtatga agggcgagag 3720acgtcgcgga aagggacacg acggcttata ccaggggctt tccaccgcga ccaaggacac 3780atacgacgcg ctgcacatgc aagccttacc acctcgatga ggtaccagcg gccgcttcga 3840gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa 3900aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc 3960aataaacaag ttaacaacaa caattcgaag gatctcgacg gtatcgatca cgagactagc 4020ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata 4080tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat 4140tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat 4200tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg 4260ctttatatat cttgtggaaa ggacgaaaca ccggcctcct ggttatggta cagatctcga 4320gatctgtacc ataaccagga ggtttttgaa ttctcgacct cgagacaaat ggcagtattc 4380atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta 4440gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa 4500aattttcggg tttattacag ggacagcaga gatccacttt ggccgcggat ccgcaacaaa 4560tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 4620cccagcccag gtaagggcag ctttggtgcc ttcgcaggct gtttccttgc ttcaggaatg 4680gccaggttct gcccagagct ctggtcaatg atgtctaaaa ctcctctgat tggtggtctc 4740ggccttatcc attgccacca aaaccctctt tttactaaga aacagtgagc cttgttctgg 4800cagtccagag aatgacacgg gaaaaaagca gatgaagaga aggtggcagg agagggcacg 4860tggcccagcc tcagtctctc caactgagtt cctgcctgcc tgcctttgct cagactgttt 4920gccccttact gctcttctag gcctcattct aagccccttc tccaagttgc ctctccttat 4980ttctccctgt ctgccaaaaa atctttccca gctcactaag tcagtctcac gcagtcactc 5040attaacccac caatcactga ttgtgccggc acatgaatgc accaggtgtt gaagtggagg 5100aattaaaaag tcagatgagg ggtgtgccca gaggaagcac cattctagtt gggggagccc 5160atctgtcagc tgggaaaagt ccaaataact tcagattgga atgtgtttta actcagggtt 5220gagaaaacag ccaccttcag gacaaaagtc agggaagggc tctctgaaga aatgctactt 5280gaagatacca gccctaccaa gggcagggag aggaccaatt gatggagttg gccactccct 5340ctctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct 5400ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aacggcgcgc 5460ctgcaggtct caaaaatagc taccctctcc ggcatgaatt tatcagctag aacggttgaa 5520tatcatattg atggtgattt gactgtctcc ggcctttctc acccgtttga atctttacct 5580acacattact caggcattgc atttaaaata tatgagggtt ctaaaaattt ttatccttgc 5640gttgaaataa aggcttctcc cgcaaaagta ttacagggtc ataatgtttt tggtacaacc 5700gatttagctt tatgctctga ggctttattg cttaattttg ctaattcttt gccttgcctg 5760tatgatttat tggatgttgg aattcctgat gcggtatttt ctccttacgc atctgtgcgg 5820tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag 5880ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 5940atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 6000gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa 6060tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg 6120aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 6180accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 6240tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 6300gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 6360ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 6420gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 6480gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 6540agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 6600gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 6660cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 6720gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 6780gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 6840ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 6900gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 6960ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 7020tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 7080actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 7140taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 7200gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 7260tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 7320ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 7380gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 7440tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 7500cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 7560gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 7620actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 7680ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 7740gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 7800atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 7860tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 7920tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 7980aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 8040gcctctcccc gcgcgttggc cgattcatta atg 8073138013DNAArtificial SequenceSynthesized 13cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgggcggagt tagggcggag ccaatcagcg tgcgccgttc 2160cgaaagttgc cttttatggc tgggcggaga atgggcggtg aacgccgatg attatataag 2220gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg 2280tttgttccgg aaagccacca tggcgctccc agtgacagcc ttacttttac ctctggcgtt 2340attattgcac gcggctcgtc ctgacataca gatgactcag actacctctt ccctatctgc 2400ttctttaggc gaccgagtaa caatatcttg ccgggccagc caggacatct caaaatactt 2460aaactggtat cagcagaagc cggacggaac agttaagttg ctcatttacc acacgtcgag 2520attacactca ggcgttccta gccgattttc gggttccggt tccggtacgg actacagcct 2580gacaatcagt aaccttgagc aggaggacat cgccacctac ttctgtcagc agggcaacac 2640gctcccgtac acattcggtg ggggaactaa gctggagatt accggaggcg gtggcagcgg 2700tggcggcggc agcgggggtg gcggctcgga ggtcaagtta caggagagcg gaccgggctt 2760ggtcgcacct agccagagcc tctcagtcac gtgcactgtg tctggagtca gtctcccaga 2820ctacggggta tcatggatac gacagccgcc tagaaagggc ttagagtggc tgggggttat 2880ctggggaagt gaaaccacat actacaactc agctctcaag agccgcctca ccatcattaa 2940ggacaacagt aagtcgcagg ttttcttaaa gatgaactct ctccagactg acgacaccgc 3000tatttactac tgcgcgaagc actactacta cggcgggagt tacgcaatgg actactgggg 3060tcagggcact tctgtgaccg tatccagcac tactacccca gccccacgtc cccccacgcc 3120agctccaacg atagcaagtc agcccttatc tcttcgccct gaggcttgca ggcccgcggc 3180gggcggcgcc gttcacacgc gaggactaga cttcgcctgc gacatctaca tctgggcacc 3240actagccggg acttgcggag tgttgttgtt gagcttggta ataacgctct actgcaaagc 3300gagccgcaaa aaagcggcgg cggcggcgaa aagcccgttt gcgagcccgg cgagcagcgc 3360gcaggaagaa gatgcgagca gctgccgcgc gccgagcgaa gaagaaggca gctgcgaact 3420gagagtgaag ttctctcgct ccgcggacgc acccgcttac cagcagggtc agaaccagct 3480atacaacgag ttaaacctgg ggcgccggga ggagtacgac gtgttagaca agcgtagagg 3540tagggacccg gagatgggag gcaagcctcg gagaaagaac ccccaggagg gcctgtacaa 3600cgaactccag aaggacaaga tggctgaggc gtactcggag attggtatga agggcgagag 3660acgtcgcgga aagggacacg acggcttata ccaggggctt tccaccgcga ccaaggacac 3720atacgacgcg ctgcacatgc aagccttacc acctcgatga ggtaccagcg gccgcttcga 3780gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa 3840aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc 3900aataaacaag ttaacaacaa caattcgaag gatctcgacg gtatcgatca cgagactagc 3960ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata 4020tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat 4080tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat 4140tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg 4200ctttatatat cttgtggaaa ggacgaaaca ccggcctcct ggttatggta cagatctcga 4260gatctgtacc ataaccagga ggtttttgaa ttctcgacct cgagacaaat ggcagtattc 4320atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta 4380gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa 4440aattttcggg tttattacag ggacagcaga gatccacttt ggccgcggat ccgcaacaaa 4500tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 4560cccagcccag gtaagggcag ctttggtgcc ttcgcaggct gtttccttgc ttcaggaatg 4620gccaggttct gcccagagct ctggtcaatg atgtctaaaa ctcctctgat tggtggtctc 4680ggccttatcc attgccacca aaaccctctt tttactaaga aacagtgagc cttgttctgg 4740cagtccagag aatgacacgg gaaaaaagca gatgaagaga aggtggcagg agagggcacg 4800tggcccagcc tcagtctctc caactgagtt cctgcctgcc tgcctttgct cagactgttt 4860gccccttact gctcttctag gcctcattct aagccccttc tccaagttgc ctctccttat 4920ttctccctgt ctgccaaaaa atctttccca gctcactaag tcagtctcac gcagtcactc 4980attaacccac caatcactga ttgtgccggc acatgaatgc accaggtgtt gaagtggagg 5040aattaaaaag tcagatgagg ggtgtgccca gaggaagcac cattctagtt gggggagccc 5100atctgtcagc tgggaaaagt ccaaataact tcagattgga atgtgtttta actcagggtt 5160gagaaaacag ccaccttcag gacaaaagtc agggaagggc tctctgaaga aatgctactt 5220gaagatacca gccctaccaa gggcagggag aggaccaatt gatggagttg gccactccct 5280ctctgcgcgc tcgctcgctc actgaggccg cccgggcaaa gcccgggcgt cgggcgacct 5340ttggtcgccc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aacggcgcgc 5400ctgcaggtct caaaaatagc taccctctcc ggcatgaatt tatcagctag aacggttgaa 5460tatcatattg atggtgattt gactgtctcc ggcctttctc acccgtttga atctttacct 5520acacattact caggcattgc atttaaaata tatgagggtt ctaaaaattt ttatccttgc 5580gttgaaataa aggcttctcc cgcaaaagta ttacagggtc

ataatgtttt tggtacaacc 5640gatttagctt tatgctctga ggctttattg cttaattttg ctaattcttt gccttgcctg 5700tatgatttat tggatgttgg aattcctgat gcggtatttt ctccttacgc atctgtgcgg 5760tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag 5820ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 5880atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 5940gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa 6000tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg 6060aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 6120accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 6180tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 6240gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 6300ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 6360gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 6420gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 6480agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 6540gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 6600cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 6660gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 6720gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 6780ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 6840gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 6900ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 6960tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 7020actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 7080taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 7140gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 7200tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 7260ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 7320gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 7380tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 7440cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 7500gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 7560actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 7620ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 7680gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 7740atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 7800tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 7860tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 7920aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 7980gcctctcccc gcgcgttggc cgattcatta atg 8013148090DNAArtificial SequenceSynthesized 14cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgatccagac atgataagat acattgatga gtttggacaa 2160accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaac tggtacctca 2340tcgaggtggt aaggcttgca tgtgcagcgc gtcgtatgtg tccttggtcg cggtggaaag 2400cccctggtat aagccgtcgt gtccctttcc gcgacgtctc tcgcccttca taccaatctc 2460cgagtacgcc tcagccatct tgtccttctg gagttcgttg tacaggccct cctgggggtt 2520ctttctccga ggcttgcctc ccatctccgg gtccctacct ctacgcttgt ctaacacgtc 2580gtactcctcc cggcgcccca ggtttaactc gttgtatagc tggttctgac cctgctggta 2640agcgggtgcg tccgcggagc gagagaactt cactctcagt tcgcagctgc cttcttcttc 2700gctcggcgcg cggcagctgc tcgcatcttc ttcctgcgcg ctgctcgccg ggctcgcaaa 2760cgggctttta gccgccgccg ccgctttttt gcggctcgct ttgcagtaga gcgttattac 2820caagctcaac aacaacactc cgcaagtccc ggctagtggt gcccagatgt agatgtcgca 2880ggcgaagtct agtcctcgcg tgtgaacggc gccgcccgcc gcgggcctgc aagcctcagg 2940gcgaagagat aagggctgac ttgctatcgt tggagctggc gtggggggac gtggggctgg 3000ggtagtagtg ggcttcgctg gggatacatt tgtagaaaca tttgaaaatg ttccctgggt 3060aggtaactcg ctggatacgg tcacagaagt gccctgaccc cagtagtcca ttgcgtaact 3120cccgccgtag tagtagtgct tcgcgcagta gtaaatagcg gtgtcgtcag tctggagaga 3180gttcatcttt aagaaaacct gcgacttact gttgtcctta atgatggtga ggcggctctt 3240gagagctgag ttgtagtatg tggtttcact tccccagata acccccagcc actctaagcc 3300ctttctaggc ggctgtcgta tccatgatac cccgtagtct gggagactga ctccagacac 3360agtgcacgtg actgagaggc tctggctagg tgcgaccaag cccggtccgc tctcctgtaa 3420cttgacctcc gagccgccac ccccgctgcc gccgccaccg ctgccaccgc ctccggtaat 3480ctccagctta gttcccccac cgaatgtgta cgggagcgtg ttgccctgct gacagaagta 3540ggtggcgatg tcctcctgct caaggttact gattgtcagg ctgtagtccg taccggaacc 3600ggaacccgaa aatcggctag gaacgcctga gtgtaatctc gacgtgtggt aaatgagcaa 3660cttaactgtt ccgtccggct tctgctgata ccagtttaag tattttgaga tgtcctggct 3720ggcccggcaa gatattgtta ctcggtcgcc taaagaagca gatagggaag aggtagtctg 3780agtcatctgt atgtcaggac gagccgcgtg caataataac gccagaggta aaagtaaggc 3840tgtcactggg agcgccatgg tggctttccg gaacaaacaa gaaccgcgac ccaaatcccg 3900gctgcgacgg aactagctgt gccacacccg gcgcgtcctt atataatcat cggcgttcac 3960cgcccattct ccgcccagcc ataaaaggca actttcggaa cggcgcacgc tgattggctc 4020cgccctaact ccgcccacta gtgcggccgc ccccttcacc gagggcctat ttcccatgat 4080tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 4140tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 4200gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 4260gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gcctcctggt 4320tatggtacag atctcgagat ctgtaccata accaggaggt ttttgaattc tcgacctcga 4380gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg 4440caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac 4500aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc 4560cgcggatccg caacaaatct gactttgcat gtgcaaacgc cttcaacaac agcattattc 4620cagaagacac cttcttcccc agcccaggta agggcagctt tggtgccttc gcaggctgtt 4680tccttgcttc aggaatggcc aggttctgcc cagagctctg gtcaatgatg tctaaaactc 4740ctctgattgg tggtctcggc cttatccatt gccaccaaaa ccctcttttt actaagaaac 4800agtgagcctt gttctggcag tccagagaat gacacgggaa aaaagcagat gaagagaagg 4860tggcaggaga gggcacgtgg cccagcctca gtctctccaa ctgagttcct gcctgcctgc 4920ctttgctcag actgtttgcc ccttactgct cttctaggcc tcattctaag ccccttctcc 4980aagttgcctc tccttatttc tccctgtctg ccaaaaaatc tttcccagct cactaagtca 5040gtctcacgca gtcactcatt aacccaccaa tcactgattg tgccggcaca tgaatgcacc 5100aggtgttgaa gtggaggaat taaaaagtca gatgaggggt gtgcccagag gaagcaccat 5160tctagttggg ggagcccatc tgtcagctgg gaaaagtcca aataacttca gattggaatg 5220tgttttaact cagggttgag aaaacagcca ccttcaggac aaaagtcagg gaagggctct 5280ctgaagaaat gctacttgaa gataccagcc ctaccaaggg cagggagagg accaattgat 5340ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc 5400cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg 5460agtggccaac ggcgcgcctg caggtctcaa aaatagctac cctctccggc atgaatttat 5520cagctagaac ggttgaatat catattgatg gtgatttgac tgtctccggc ctttctcacc 5580cgtttgaatc tttacctaca cattactcag gcattgcatt taaaatatat gagggttcta 5640aaaattttta tccttgcgtt gaaataaagg cttctcccgc aaaagtatta cagggtcata 5700atgtttttgg tacaaccgat ttagctttat gctctgaggc tttattgctt aattttgcta 5760attctttgcc ttgcctgtat gatttattgg atgttggaat tcctgatgcg gtattttctc 5820cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 5880gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg 5940gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6000tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc 6060ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt 6120cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 6180ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 6240agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 6300tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 6360gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 6420gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 6480attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 6540gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 6600agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 6660ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 6720cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 6780gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 6840cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 6900gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 6960ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 7020acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 7080ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 7140aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 7200aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 7260ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 7320ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 7380actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 7440caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 7500gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 7560ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 7620cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 7680cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 7740acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 7800ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 7860gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 7920tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 7980accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 8040cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg 8090158030DNAArtificial SequenceSynthesized 15cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgatccagac atgataagat acattgatga gtttggacaa 2160accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaac tggtacctca 2340tcgaggtggt aaggcttgca tgtgcagcgc gtcgtatgtg tccttggtcg cggtggaaag 2400cccctggtat aagccgtcgt gtccctttcc gcgacgtctc tcgcccttca taccaatctc 2460cgagtacgcc tcagccatct tgtccttctg gagttcgttg tacaggccct cctgggggtt 2520ctttctccga ggcttgcctc ccatctccgg gtccctacct ctacgcttgt ctaacacgtc 2580gtactcctcc cggcgcccca ggtttaactc gttgtatagc tggttctgac cctgctggta 2640agcgggtgcg tccgcggagc gagagaactt cactctcagt tcgcagctgc cttcttcttc 2700gctcggcgcg cggcagctgc tcgcatcttc ttcctgcgcg ctgctcgccg ggctcgcaaa 2760cgggcttttc gccgccgccg ccgctttttt gcggctcgct ttgcagtaga gcgttattac 2820caagctcaac aacaacactc cgcaagtccc ggctagtggt gcccagatgt agatgtcgca 2880ggcgaagtct agtcctcgcg tgtgaacggc gccgcccgcc gcgggcctgc aagcctcagg 2940gcgaagagat aagggctgac ttgctatcgt tggagctggc gtggggggac gtggggctgg 3000ggtagtagtg ctggatacgg tcacagaagt gccctgaccc cagtagtcca ttgcgtaact 3060cccgccgtag tagtagtgct tcgcgcagta gtaaatagcg gtgtcgtcag tctggagaga 3120gttcatcttt aagaaaacct gcgacttact gttgtcctta atgatggtga ggcggctctt 3180gagagctgag ttgtagtatg tggtttcact tccccagata acccccagcc actctaagcc 3240ctttctaggc ggctgtcgta tccatgatac cccgtagtct gggagactga ctccagacac 3300agtgcacgtg actgagaggc tctggctagg tgcgaccaag cccggtccgc tctcctgtaa 3360cttgacctcc gagccgccac ccccgctgcc gccgccaccg ctgccaccgc ctccggtaat 3420ctccagctta gttcccccac cgaatgtgta cgggagcgtg ttgccctgct gacagaagta 3480ggtggcgatg tcctcctgct caaggttact gattgtcagg ctgtagtccg taccggaacc 3540ggaacccgaa aatcggctag gaacgcctga gtgtaatctc gacgtgtggt aaatgagcaa 3600cttaactgtt ccgtccggct tctgctgata ccagtttaag tattttgaga tgtcctggct 3660ggcccggcaa gatattgtta ctcggtcgcc taaagaagca gatagggaag aggtagtctg 3720agtcatctgt atgtcaggac gagccgcgtg caataataac gccagaggta aaagtaaggc 3780tgtcactggg agcgccatgg tggctttccg gaacaaacaa gaaccgcgac ccaaatcccg 3840gctgcgacgg aactagctgt gccacacccg gcgcgtcctt atataatcat cggcgttcac 3900cgcccattct ccgcccagcc ataaaaggca actttcggaa cggcgcacgc tgattggctc 3960cgccctaact ccgcccacta gtgcggccgc ccccttcacc gagggcctat ttcccatgat 4020tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 4080tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 4140gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 4200gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gcctcctggt 4260tatggtacag atctcgagat ctgtaccata accaggaggt ttttgaattc tcgacctcga 4320gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg 4380caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac 4440aaattacaaa aattcaaaat

tttcgggttt attacaggga cagcagagat ccactttggc 4500cgcggatccg caacaaatct gactttgcat gtgcaaacgc cttcaacaac agcattattc 4560cagaagacac cttcttcccc agcccaggta agggcagctt tggtgccttc gcaggctgtt 4620tccttgcttc aggaatggcc aggttctgcc cagagctctg gtcaatgatg tctaaaactc 4680ctctgattgg tggtctcggc cttatccatt gccaccaaaa ccctcttttt actaagaaac 4740agtgagcctt gttctggcag tccagagaat gacacgggaa aaaagcagat gaagagaagg 4800tggcaggaga gggcacgtgg cccagcctca gtctctccaa ctgagttcct gcctgcctgc 4860ctttgctcag actgtttgcc ccttactgct cttctaggcc tcattctaag ccccttctcc 4920aagttgcctc tccttatttc tccctgtctg ccaaaaaatc tttcccagct cactaagtca 4980gtctcacgca gtcactcatt aacccaccaa tcactgattg tgccggcaca tgaatgcacc 5040aggtgttgaa gtggaggaat taaaaagtca gatgaggggt gtgcccagag gaagcaccat 5100tctagttggg ggagcccatc tgtcagctgg gaaaagtcca aataacttca gattggaatg 5160tgttttaact cagggttgag aaaacagcca ccttcaggac aaaagtcagg gaagggctct 5220ctgaagaaat gctacttgaa gataccagcc ctaccaaggg cagggagagg accaattgat 5280ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc 5340cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg 5400agtggccaac ggcgcgcctg caggtctcaa aaatagctac cctctccggc atgaatttat 5460cagctagaac ggttgaatat catattgatg gtgatttgac tgtctccggc ctttctcacc 5520cgtttgaatc tttacctaca cattactcag gcattgcatt taaaatatat gagggttcta 5580aaaattttta tccttgcgtt gaaataaagg cttctcccgc aaaagtatta cagggtcata 5640atgtttttgg tacaaccgat ttagctttat gctctgaggc tttattgctt aattttgcta 5700attctttgcc ttgcctgtat gatttattgg atgttggaat tcctgatgcg gtattttctc 5760cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct 5820gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg 5880gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 5940tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc 6000ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt 6060cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat 6120ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg 6180agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt 6240tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga 6300gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa 6360gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt 6420attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 6480gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 6540agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga 6600ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat 6660cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct 6720gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc 6780cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 6840gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 6900ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 6960acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca 7020ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta 7080aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc 7140aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa 7200ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca 7260ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta 7320actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc 7380caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca 7440gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta 7500ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag 7560cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt 7620cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc 7680acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac 7740ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac 7800gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc 7860tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat 7920accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag 7980cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg 8030168089DNAArtificial SequenceSynthesized 16cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgatccagac atgataagat acattgatga gtttggacaa 2160accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaac tggtacctca 2340tcgaggtggt aaggcttgca tgtgcagcgc gtcgtatgtg tccttggtcg cggtggaaag 2400cccctggtat aagccgtcgt gtccctttcc gcgacgtctc tcgcccttca taccaatctc 2460cgagtacgcc tcagccatct tgtccttctg gagttcgttg tacaggccct cctgggggtt 2520ctttctccga ggcttgcctc ccatctccgg gtccctacct ctacgcttgt ctaacacgtc 2580gtactcctcc cggcgcccca ggtttaactc gttgtatagc tggttctgac cctgctggta 2640agcgggtgcg tccgcggagc gagagaactt cactctcagt tcgcagctgc cttcttcttc 2700gctcggcgcg cggcagctgc tcgcatcttc ttcctgcgcg ctgctcgccg ggctcgcaaa 2760cgggctttta gccgccgccg ccgctttttt gcggctcgct ttgcagtaga gcgttattac 2820caagctcaac aacaacactc cgcaagtccc ggctagtggt gcccagatgt agatgtcgca 2880ggcgaagtct agtcctcgcg tgtgaacggc gccgcccgcc gcgggcctgc aagcctcagg 2940gcgaagagat aagggctgac ttgctatcgt tggagctggc gtggggggac gtggggctgg 3000ggtagtagtg ggcttcgctg gggatacatt tgtagaaaca tttgaaaatg ttccctgggt 3060aggtaactcg ctggatacgg tcacagaagt gccctgaccc cagtagtcca ttgcgtaact 3120cccgccgtag tagtagtgct tcgcgcagta gtaaatagcg gtgtcgtcag tctggagaga 3180gttcatcttt aagaaaacct gcgacttact gttgtcctta atgatggtga ggcggctctt 3240gagagctgag ttgtagtatg tggtttcact tccccagata acccccagcc actctaagcc 3300ctttctaggc ggctgtcgta tccatgatac cccgtagtct gggagactga ctccagacac 3360agtgcacgtg actgagaggc tctggctagg tgcgaccaag cccggtccgc tctcctgtaa 3420cttgacctcc gagccgccac ccccgctgcc gccgccaccg ctgccaccgc ctccggtaat 3480ctccagctta gttcccccac cgaatgtgta cgggagcgtg ttgccctgct gacagaagta 3540ggtggcgatg tcctcctgct caaggttact gattgtcagg ctgtagtccg taccggaacc 3600ggaacccgaa aatcggctag gaacgcctga gtgtaatctc gacgtgtggt aaatgagcaa 3660cttaactgtt ccgtccggct tctgctgata ccagtttaag tattttgaga tgtcctggct 3720ggcccggcaa gatattgtta ctcggtcgcc taaagaagca gatagggaag aggtagtctg 3780agtcatctgt atgtcaggac gagccgcgtg caataataac gccagaggta aaagtaaggc 3840tgtcactggg agcgccatgg tggctttccg gaacaaacaa gaaccgcgac ccaaatcccg 3900gctgcgacgg aactagctgt gccacacccg gcgcgtcctt atataatcat cggcgttcac 3960cgcccattct ccgcccagcc ataaaaggca actttcggaa cggcgcacgc tgattggctc 4020cgccctaact ccgcccacta gtgcggccgc gcggccaaag tggatctctg ctgtccctgt 4080aataaacccg aaaattttga atttttgtaa tttgtttttg taattcttta gtttgtatgt 4140ctgttgctat tatgtctact attctttccc ctgcactgta ccccccaatc cccccttttc 4200ttttaaaatt gtggatgaat actgccattt gtctcgaggt cgagaattca aaaacctcct 4260ggttatggta cagatctcga gatctgtacc ataaccagga ggccggtgtt tcgtcctttc 4320cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag 4380tccattttaa aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt 4440cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattccaat tatctctcta 4500acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctcggtgaag 4560ggggatccgc aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc 4620agaagacacc ttcttcccca gcccaggtaa gggcagcttt ggtgccttcg caggctgttt 4680ccttgcttca ggaatggcca ggttctgccc agagctctgg tcaatgatgt ctaaaactcc 4740tctgattggt ggtctcggcc ttatccattg ccaccaaaac cctcttttta ctaagaaaca 4800gtgagccttg ttctggcagt ccagagaatg acacgggaaa aaagcagatg aagagaaggt 4860ggcaggagag ggcacgtggc ccagcctcag tctctccaac tgagttcctg cctgcctgcc 4920tttgctcaga ctgtttgccc cttactgctc ttctaggcct cattctaagc cccttctcca 4980agttgcctct ccttatttct ccctgtctgc caaaaaatct ttcccagctc actaagtcag 5040tctcacgcag tcactcatta acccaccaat cactgattgt gccggcacat gaatgcacca 5100ggtgttgaag tggaggaatt aaaaagtcag atgaggggtg tgcccagagg aagcaccatt 5160ctagttgggg gagcccatct gtcagctggg aaaagtccaa ataacttcag attggaatgt 5220gttttaactc agggttgaga aaacagccac cttcaggaca aaagtcaggg aagggctctc 5280tgaagaaatg ctacttgaag ataccagccc taccaagggc agggagagga ccaattgatg 5340gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc 5400gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga 5460gtggccaacg gcgcgcctgc aggtctcaaa aatagctacc ctctccggca tgaatttatc 5520agctagaacg gttgaatatc atattgatgg tgatttgact gtctccggcc tttctcaccc 5580gtttgaatct ttacctacac attactcagg cattgcattt aaaatatatg agggttctaa 5640aaatttttat ccttgcgttg aaataaaggc ttctcccgca aaagtattac agggtcataa 5700tgtttttggt acaaccgatt tagctttatg ctctgaggct ttattgctta attttgctaa 5760ttctttgcct tgcctgtatg atttattgga tgttggaatt cctgatgcgg tattttctcc 5820ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg 5880atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 5940cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 6000gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 6060tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 6120ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 6180cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 6240gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 6300ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 6360tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 6420aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 6480ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 6540agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 6600gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 6660gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 6720gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 6780tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 6840ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 6900cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 6960gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 7020cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 7080tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 7140aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 7200aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 7260gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 7320cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 7380ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 7440accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 7500tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 7560cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 7620gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 7680ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 7740cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 7800tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 7860ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 7920ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 7980ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 8040gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatg 8089178029DNAArtificial SequenceSynthesized 17cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgatccagac atgataagat acattgatga gtttggacaa 2160accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaac tggtacctca 2340tcgaggtggt aaggcttgca tgtgcagcgc gtcgtatgtg tccttggtcg cggtggaaag 2400cccctggtat aagccgtcgt gtccctttcc gcgacgtctc tcgcccttca taccaatctc 2460cgagtacgcc tcagccatct tgtccttctg gagttcgttg tacaggccct cctgggggtt 2520ctttctccga ggcttgcctc ccatctccgg gtccctacct ctacgcttgt ctaacacgtc 2580gtactcctcc cggcgcccca ggtttaactc gttgtatagc tggttctgac cctgctggta 2640agcgggtgcg tccgcggagc gagagaactt cactctcagt tcgcagctgc cttcttcttc 2700gctcggcgcg cggcagctgc tcgcatcttc ttcctgcgcg ctgctcgccg ggctcgcaaa 2760cgggcttttc gccgccgccg ccgctttttt gcggctcgct ttgcagtaga gcgttattac 2820caagctcaac aacaacactc cgcaagtccc ggctagtggt gcccagatgt agatgtcgca 2880ggcgaagtct agtcctcgcg tgtgaacggc gccgcccgcc gcgggcctgc aagcctcagg 2940gcgaagagat aagggctgac ttgctatcgt tggagctggc gtggggggac gtggggctgg 3000ggtagtagtg ctggatacgg tcacagaagt gccctgaccc cagtagtcca ttgcgtaact 3060cccgccgtag tagtagtgct tcgcgcagta gtaaatagcg gtgtcgtcag tctggagaga 3120gttcatcttt aagaaaacct gcgacttact gttgtcctta atgatggtga ggcggctctt 3180gagagctgag ttgtagtatg tggtttcact tccccagata acccccagcc actctaagcc 3240ctttctaggc ggctgtcgta tccatgatac cccgtagtct gggagactga ctccagacac

3300agtgcacgtg actgagaggc tctggctagg tgcgaccaag cccggtccgc tctcctgtaa 3360cttgacctcc gagccgccac ccccgctgcc gccgccaccg ctgccaccgc ctccggtaat 3420ctccagctta gttcccccac cgaatgtgta cgggagcgtg ttgccctgct gacagaagta 3480ggtggcgatg tcctcctgct caaggttact gattgtcagg ctgtagtccg taccggaacc 3540ggaacccgaa aatcggctag gaacgcctga gtgtaatctc gacgtgtggt aaatgagcaa 3600cttaactgtt ccgtccggct tctgctgata ccagtttaag tattttgaga tgtcctggct 3660ggcccggcaa gatattgtta ctcggtcgcc taaagaagca gatagggaag aggtagtctg 3720agtcatctgt atgtcaggac gagccgcgtg caataataac gccagaggta aaagtaaggc 3780tgtcactggg agcgccatgg tggctttccg gaacaaacaa gaaccgcgac ccaaatcccg 3840gctgcgacgg aactagctgt gccacacccg gcgcgtcctt atataatcat cggcgttcac 3900cgcccattct ccgcccagcc ataaaaggca actttcggaa cggcgcacgc tgattggctc 3960cgccctaact ccgcccacta gtgcggccgc gcggccaaag tggatctctg ctgtccctgt 4020aataaacccg aaaattttga atttttgtaa tttgtttttg taattcttta gtttgtatgt 4080ctgttgctat tatgtctact attctttccc ctgcactgta ccccccaatc cccccttttc 4140ttttaaaatt gtggatgaat actgccattt gtctcgaggt cgagaattca aaaacctcct 4200ggttatggta cagatctcga gatctgtacc ataaccagga ggccggtgtt tcgtcctttc 4260cacaagatat ataaagccaa gaaatcgaaa tactttcaag ttacggtaag catatgatag 4320tccattttaa aacataattt taaaactgca aactacccaa gaaattatta ctttctacgt 4380cacgtatttt gtactaatat ctttgtgttt acagtcaaat taattccaat tatctctcta 4440acagccttgt atcgtatatg caaatatgaa ggaatcatgg gaaataggcc ctcggtgaag 4500ggggatccgc aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc 4560agaagacacc ttcttcccca gcccaggtaa gggcagcttt ggtgccttcg caggctgttt 4620ccttgcttca ggaatggcca ggttctgccc agagctctgg tcaatgatgt ctaaaactcc 4680tctgattggt ggtctcggcc ttatccattg ccaccaaaac cctcttttta ctaagaaaca 4740gtgagccttg ttctggcagt ccagagaatg acacgggaaa aaagcagatg aagagaaggt 4800ggcaggagag ggcacgtggc ccagcctcag tctctccaac tgagttcctg cctgcctgcc 4860tttgctcaga ctgtttgccc cttactgctc ttctaggcct cattctaagc cccttctcca 4920agttgcctct ccttatttct ccctgtctgc caaaaaatct ttcccagctc actaagtcag 4980tctcacgcag tcactcatta acccaccaat cactgattgt gccggcacat gaatgcacca 5040ggtgttgaag tggaggaatt aaaaagtcag atgaggggtg tgcccagagg aagcaccatt 5100ctagttgggg gagcccatct gtcagctggg aaaagtccaa ataacttcag attggaatgt 5160gttttaactc agggttgaga aaacagccac cttcaggaca aaagtcaggg aagggctctc 5220tgaagaaatg ctacttgaag ataccagccc taccaagggc agggagagga ccaattgatg 5280gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc 5340gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga 5400gtggccaacg gcgcgcctgc aggtctcaaa aatagctacc ctctccggca tgaatttatc 5460agctagaacg gttgaatatc atattgatgg tgatttgact gtctccggcc tttctcaccc 5520gtttgaatct ttacctacac attactcagg cattgcattt aaaatatatg agggttctaa 5580aaatttttat ccttgcgttg aaataaaggc ttctcccgca aaagtattac agggtcataa 5640tgtttttggt acaaccgatt tagctttatg ctctgaggct ttattgctta attttgctaa 5700ttctttgcct tgcctgtatg atttattgga tgttggaatt cctgatgcgg tattttctcc 5760ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg 5820atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 5880cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 5940gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 6000tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 6060ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 6120cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 6180gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 6240ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 6300tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 6360aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 6420ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 6480agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 6540gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 6600gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 6660gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 6720tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 6780ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 6840cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 6900gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 6960cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 7020tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 7080aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 7140aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 7200gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 7260cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 7320ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc 7380accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 7440tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 7500cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 7560gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 7620ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 7680cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 7740tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 7800ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 7860ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 7920ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 7980gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatg 8029188082DNAArtificial SequenceSynthesized 18cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgggcggagt tagggcggag ccaatcagcg tgcgccgttc 2160cgaaagttgc cttttatggc tgggcggaga atgggcggtg aacgccgatg attatataag 2220gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg 2280tttgttccgg aaagccacca tggcgctccc agtgacagcc ttacttttac ctctggcgtt 2340attattgcac gcggctcgtc ctgacataca gatgactcag actacctctt ccctatctgc 2400ttctttaggc gaccgagtaa caatatcttg ccgggccagc caggacatct caaaatactt 2460aaactggtat cagcagaagc cggacggaac agttaagttg ctcatttacc acacgtcgag 2520attacactca ggcgttccta gccgattttc gggttccggt tccggtacgg actacagcct 2580gacaatcagt aaccttgagc aggaggacat cgccacctac ttctgtcagc agggcaacac 2640gctcccgtac acattcggtg ggggaactaa gctggagatt accggaggcg gtggcagcgg 2700tggcggcggc agcgggggtg gcggctcgga ggtcaagtta caggagagcg gaccgggctt 2760ggtcgcacct agccagagcc tctcagtcac gtgcactgtg tctggagtca gtctcccaga 2820ctacggggta tcatggatac gacagccgcc tagaaagggc ttagagtggc tgggggttat 2880ctggggaagt gaaaccacat actacaactc agctctcaag agccgcctca ccatcattaa 2940ggacaacagt aagtcgcagg ttttcttaaa gatgaactct ctccagactg acgacaccgc 3000tatttactac tgcgcgaagc actactacta cggcgggagt tacgcaatgg actactgggg 3060tcagggcact tctgtgaccg tatccagcga gttacctacc cagggaacat tttcaaatgt 3120ttctacaaat gtatccccag cgaagcccac tactacccca gccccacgtc cccccacgcc 3180agctccaacg atagcaagtc agcccttatc tcttcgccct gaggcttgca ggcccgcggc 3240gggcggcgcc gttcacacgc gaggactaga cttcgcctgc gacatctaca tctgggcacc 3300actagccggg acttgcggag tgttgttgtt gagcttggta ataacgctct actgcaaagc 3360gagccgcaaa aaagcggcgg cggcggctaa aagcccgttt gcgagcccgg cgagcagcgc 3420gcaggaagaa gatgcgagca gctgccgcgc gccgagcgaa gaagaaggca gctgcgaact 3480gagagtgaag ttctctcgct ccgcggacgc acccgcttac cagcagggtc agaaccagct 3540atacaacgag ttaaacctgg ggcgccggga ggagtacgac gtgttagaca agcgtagagg 3600tagggacccg gagatgggag gcaagcctcg gagaaagaac ccccaggagg gcctgtacaa 3660cgaactccag aaggacaaga tggctgaggc gtactcggag attggtatga agggcgagag 3720acgtcgcgga aagggacacg acggcttata ccaggggctt tccaccgcga ccaaggacac 3780atacgacgcg ctgcacatgc aagccttacc acctcgatga ggtaccagcg gccgcttcga 3840gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa 3900aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc 3960aataaacaag ttaacaacaa caattcgaag gatctcgacg gtatcgatca cgagactagc 4020ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata 4080tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat 4140tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat 4200tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg 4260ctttatatat cttgtggaaa ggacgaaaca ccggtgtacc ggaggtttga agatgccgca 4320tttctcgaga aatgcggcat cttcaaacct ttttttgaat tctcgaccta gggacaaatg 4380gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 4440agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 4500aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gccgcggatc 4560cgcaacaaat ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac 4620accttcttcc ccagcccagg taagggcagc tttggtgcct tcgcaggctg tttccttgct 4680tcaggaatgg ccaggttctg cccagagctc tggtcaatga tgtctaaaac tcctctgatt 4740ggtggtctcg gccttatcca ttgccaccaa aaccctcttt ttactaagaa acagtgagcc 4800ttgttctggc agtccagaga atgacacggg aaaaaagcag atgaagagaa ggtggcagga 4860gagggcacgt ggcccagcct cagtctctcc aactgagttc ctgcctgcct gcctttgctc 4920agactgtttg ccccttactg ctcttctagg cctcattcta agccccttct ccaagttgcc 4980tctccttatt tctccctgtc tgccaaaaaa tctttcccag ctcactaagt cagtctcacg 5040cagtcactca ttaacccacc aatcactgat tgtgccggca catgaatgca ccaggtgttg 5100aagtggagga attaaaaagt cagatgaggg gtgtgcccag aggaagcacc attctagttg 5160ggggagccca tctgtcagct gggaaaagtc caaataactt cagattggaa tgtgttttaa 5220ctcagggttg agaaaacagc caccttcagg acaaaagtca gggaagggct ctctgaagaa 5280atgctacttg aagataccag ccctaccaag ggcagggaga ggaccaattg atggagttgg 5340ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 5400gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 5460acggcgcgcc tgcaggtctc aaaaatagct accctctccg gcatgaattt atcagctaga 5520acggttgaat atcatattga tggtgatttg actgtctccg gcctttctca cccgtttgaa 5580tctttaccta cacattactc aggcattgca tttaaaatat atgagggttc taaaaatttt 5640tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat tacagggtca taatgttttt 5700ggtacaaccg atttagcttt atgctctgag gctttattgc ttaattttgc taattctttg 5760ccttgcctgt atgatttatt ggatgttgga attcctgatg cggtattttc tccttacgca 5820tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc 5880atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 5940gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 6000gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 6060ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 6120tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 6180gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 6240acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 6300cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 6360catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 6420tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 6480cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 6540accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 6600cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 6660ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 6720accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 6780ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 6840attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 6900ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 6960tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 7020tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 7080gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 7140tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 7200ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 7260ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 7320agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 7380cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 7440caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 7500tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 7560ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 7620ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 7680gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 7740gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 7800tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 7860cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 7920gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 7980ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 8040acgcaaaccg cctctccccg cgcgttggcc gattcattaa tg 8082198022DNAArtificial SequenceSynthesized 19cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgggcggagt tagggcggag

ccaatcagcg tgcgccgttc 2160cgaaagttgc cttttatggc tgggcggaga atgggcggtg aacgccgatg attatataag 2220gacgcgccgg gtgtggcaca gctagttccg tcgcagccgg gatttgggtc gcggttcttg 2280tttgttccgg aaagccacca tggcgctccc agtgacagcc ttacttttac ctctggcgtt 2340attattgcac gcggctcgtc ctgacataca gatgactcag actacctctt ccctatctgc 2400ttctttaggc gaccgagtaa caatatcttg ccgggccagc caggacatct caaaatactt 2460aaactggtat cagcagaagc cggacggaac agttaagttg ctcatttacc acacgtcgag 2520attacactca ggcgttccta gccgattttc gggttccggt tccggtacgg actacagcct 2580gacaatcagt aaccttgagc aggaggacat cgccacctac ttctgtcagc agggcaacac 2640gctcccgtac acattcggtg ggggaactaa gctggagatt accggaggcg gtggcagcgg 2700tggcggcggc agcgggggtg gcggctcgga ggtcaagtta caggagagcg gaccgggctt 2760ggtcgcacct agccagagcc tctcagtcac gtgcactgtg tctggagtca gtctcccaga 2820ctacggggta tcatggatac gacagccgcc tagaaagggc ttagagtggc tgggggttat 2880ctggggaagt gaaaccacat actacaactc agctctcaag agccgcctca ccatcattaa 2940ggacaacagt aagtcgcagg ttttcttaaa gatgaactct ctccagactg acgacaccgc 3000tatttactac tgcgcgaagc actactacta cggcgggagt tacgcaatgg actactgggg 3060tcagggcact tctgtgaccg tatccagcac tactacccca gccccacgtc cccccacgcc 3120agctccaacg atagcaagtc agcccttatc tcttcgccct gaggcttgca ggcccgcggc 3180gggcggcgcc gttcacacgc gaggactaga cttcgcctgc gacatctaca tctgggcacc 3240actagccggg acttgcggag tgttgttgtt gagcttggta ataacgctct actgcaaagc 3300gagccgcaaa aaagcggcgg cggcggcgaa aagcccgttt gcgagcccgg cgagcagcgc 3360gcaggaagaa gatgcgagca gctgccgcgc gccgagcgaa gaagaaggca gctgcgaact 3420gagagtgaag ttctctcgct ccgcggacgc acccgcttac cagcagggtc agaaccagct 3480atacaacgag ttaaacctgg ggcgccggga ggagtacgac gtgttagaca agcgtagagg 3540tagggacccg gagatgggag gcaagcctcg gagaaagaac ccccaggagg gcctgtacaa 3600cgaactccag aaggacaaga tggctgaggc gtactcggag attggtatga agggcgagag 3660acgtcgcgga aagggacacg acggcttata ccaggggctt tccaccgcga ccaaggacac 3720atacgacgcg ctgcacatgc aagccttacc acctcgatga ggtaccagcg gccgcttcga 3780gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa 3840aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc 3900aataaacaag ttaacaacaa caattcgaag gatctcgacg gtatcgatca cgagactagc 3960ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata 4020tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat 4080tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat 4140tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg 4200ctttatatat cttgtggaaa ggacgaaaca ccggtgtacc ggaggtttga agatgccgca 4260tttctcgaga aatgcggcat cttcaaacct ttttttgaat tctcgaccta gggacaaatg 4320gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa 4380agaatagtag acataatagc aacagacata caaactaaag aattacaaaa acaaattaca 4440aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg gccgcggatc 4500cgcaacaaat ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac 4560accttcttcc ccagcccagg taagggcagc tttggtgcct tcgcaggctg tttccttgct 4620tcaggaatgg ccaggttctg cccagagctc tggtcaatga tgtctaaaac tcctctgatt 4680ggtggtctcg gccttatcca ttgccaccaa aaccctcttt ttactaagaa acagtgagcc 4740ttgttctggc agtccagaga atgacacggg aaaaaagcag atgaagagaa ggtggcagga 4800gagggcacgt ggcccagcct cagtctctcc aactgagttc ctgcctgcct gcctttgctc 4860agactgtttg ccccttactg ctcttctagg cctcattcta agccccttct ccaagttgcc 4920tctccttatt tctccctgtc tgccaaaaaa tctttcccag ctcactaagt cagtctcacg 4980cagtcactca ttaacccacc aatcactgat tgtgccggca catgaatgca ccaggtgttg 5040aagtggagga attaaaaagt cagatgaggg gtgtgcccag aggaagcacc attctagttg 5100ggggagccca tctgtcagct gggaaaagtc caaataactt cagattggaa tgtgttttaa 5160ctcagggttg agaaaacagc caccttcagg acaaaagtca gggaagggct ctctgaagaa 5220atgctacttg aagataccag ccctaccaag ggcagggaga ggaccaattg atggagttgg 5280ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 5340gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 5400acggcgcgcc tgcaggtctc aaaaatagct accctctccg gcatgaattt atcagctaga 5460acggttgaat atcatattga tggtgatttg actgtctccg gcctttctca cccgtttgaa 5520tctttaccta cacattactc aggcattgca tttaaaatat atgagggttc taaaaatttt 5580tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat tacagggtca taatgttttt 5640ggtacaaccg atttagcttt atgctctgag gctttattgc ttaattttgc taattctttg 5700ccttgcctgt atgatttatt ggatgttgga attcctgatg cggtattttc tccttacgca 5760tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc 5820atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 5880gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 5940gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 6000ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 6060tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 6120gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 6180acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 6240cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 6300catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 6360tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 6420cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 6480accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 6540cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 6600ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 6660accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 6720ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 6780attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 6840ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 6900tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 6960tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 7020gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 7080tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 7140ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 7200ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 7260agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 7320cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 7380caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 7440tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 7500ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 7560ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 7620gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 7680gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 7740tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 7800cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 7860gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 7920ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 7980acgcaaaccg cctctccccg cgcgttggcc gattcattaa tg 8022208099DNAArtificial SequenceSynthesized 20cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgatccagac atgataagat acattgatga gtttggacaa 2160accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaac tggtacctca 2340tcgaggtggt aaggcttgca tgtgcagcgc gtcgtatgtg tccttggtcg cggtggaaag 2400cccctggtat aagccgtcgt gtccctttcc gcgacgtctc tcgcccttca taccaatctc 2460cgagtacgcc tcagccatct tgtccttctg gagttcgttg tacaggccct cctgggggtt 2520ctttctccga ggcttgcctc ccatctccgg gtccctacct ctacgcttgt ctaacacgtc 2580gtactcctcc cggcgcccca ggtttaactc gttgtatagc tggttctgac cctgctggta 2640agcgggtgcg tccgcggagc gagagaactt cactctcagt tcgcagctgc cttcttcttc 2700gctcggcgcg cggcagctgc tcgcatcttc ttcctgcgcg ctgctcgccg ggctcgcaaa 2760cgggctttta gccgccgccg ccgctttttt gcggctcgct ttgcagtaga gcgttattac 2820caagctcaac aacaacactc cgcaagtccc ggctagtggt gcccagatgt agatgtcgca 2880ggcgaagtct agtcctcgcg tgtgaacggc gccgcccgcc gcgggcctgc aagcctcagg 2940gcgaagagat aagggctgac ttgctatcgt tggagctggc gtggggggac gtggggctgg 3000ggtagtagtg ggcttcgctg gggatacatt tgtagaaaca tttgaaaatg ttccctgggt 3060aggtaactcg ctggatacgg tcacagaagt gccctgaccc cagtagtcca ttgcgtaact 3120cccgccgtag tagtagtgct tcgcgcagta gtaaatagcg gtgtcgtcag tctggagaga 3180gttcatcttt aagaaaacct gcgacttact gttgtcctta atgatggtga ggcggctctt 3240gagagctgag ttgtagtatg tggtttcact tccccagata acccccagcc actctaagcc 3300ctttctaggc ggctgtcgta tccatgatac cccgtagtct gggagactga ctccagacac 3360agtgcacgtg actgagaggc tctggctagg tgcgaccaag cccggtccgc tctcctgtaa 3420cttgacctcc gagccgccac ccccgctgcc gccgccaccg ctgccaccgc ctccggtaat 3480ctccagctta gttcccccac cgaatgtgta cgggagcgtg ttgccctgct gacagaagta 3540ggtggcgatg tcctcctgct caaggttact gattgtcagg ctgtagtccg taccggaacc 3600ggaacccgaa aatcggctag gaacgcctga gtgtaatctc gacgtgtggt aaatgagcaa 3660cttaactgtt ccgtccggct tctgctgata ccagtttaag tattttgaga tgtcctggct 3720ggcccggcaa gatattgtta ctcggtcgcc taaagaagca gatagggaag aggtagtctg 3780agtcatctgt atgtcaggac gagccgcgtg caataataac gccagaggta aaagtaaggc 3840tgtcactggg agcgccatgg tggctttccg gaacaaacaa gaaccgcgac ccaaatcccg 3900gctgcgacgg aactagctgt gccacacccg gcgcgtcctt atataatcat cggcgttcac 3960cgcccattct ccgcccagcc ataaaaggca actttcggaa cggcgcacgc tgattggctc 4020cgccctaact ccgcccacta gtgcggccgc ccccttcacc gagggcctat ttcccatgat 4080tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 4140tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 4200gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 4260gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gtgtaccgga 4320ggtttgaaga tgccgcattt ctcgagaaat gcggcatctt caaacctttt tttgaattct 4380cgacctaggg acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg 4440ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat 4500tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc 4560cactttggcc gcggatccgc aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca 4620gcattattcc agaagacacc ttcttcccca gcccaggtaa gggcagcttt ggtgccttcg 4680caggctgttt ccttgcttca ggaatggcca ggttctgccc agagctctgg tcaatgatgt 4740ctaaaactcc tctgattggt ggtctcggcc ttatccattg ccaccaaaac cctcttttta 4800ctaagaaaca gtgagccttg ttctggcagt ccagagaatg acacgggaaa aaagcagatg 4860aagagaaggt ggcaggagag ggcacgtggc ccagcctcag tctctccaac tgagttcctg 4920cctgcctgcc tttgctcaga ctgtttgccc cttactgctc ttctaggcct cattctaagc 4980cccttctcca agttgcctct ccttatttct ccctgtctgc caaaaaatct ttcccagctc 5040actaagtcag tctcacgcag tcactcatta acccaccaat cactgattgt gccggcacat 5100gaatgcacca ggtgttgaag tggaggaatt aaaaagtcag atgaggggtg tgcccagagg 5160aagcaccatt ctagttgggg gagcccatct gtcagctggg aaaagtccaa ataacttcag 5220attggaatgt gttttaactc agggttgaga aaacagccac cttcaggaca aaagtcaggg 5280aagggctctc tgaagaaatg ctacttgaag ataccagccc taccaagggc agggagagga 5340ccaattgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgcccg 5400ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg 5460cagagaggga gtggccaacg gcgcgcctgc aggtctcaaa aatagctacc ctctccggca 5520tgaatttatc agctagaacg gttgaatatc atattgatgg tgatttgact gtctccggcc 5580tttctcaccc gtttgaatct ttacctacac attactcagg cattgcattt aaaatatatg 5640agggttctaa aaatttttat ccttgcgttg aaataaaggc ttctcccgca aaagtattac 5700agggtcataa tgtttttggt acaaccgatt tagctttatg ctctgaggct ttattgctta 5760attttgctaa ttctttgcct tgcctgtatg atttattgga tgttggaatt cctgatgcgg 5820tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca 5880atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 5940ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 6000agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 6060gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 6120ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 6180aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 6240aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 6300cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 6360ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 6420cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 6480ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 6540gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 6600gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 6660acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 6720cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 6780acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 6840ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 6900ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 6960gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 7020atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 7080ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 7140attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 7200ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 7260aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 7320aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 7380ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 7440tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 7500ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 7560cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 7620agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 7680gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 7740ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 7800tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 7860tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 7920cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 7980tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 8040gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatg 8099218039DNAArtificial SequenceSynthesized 21cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca

ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgatccagac atgataagat acattgatga gtttggacaa 2160accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaac tggtacctca 2340tcgaggtggt aaggcttgca tgtgcagcgc gtcgtatgtg tccttggtcg cggtggaaag 2400cccctggtat aagccgtcgt gtccctttcc gcgacgtctc tcgcccttca taccaatctc 2460cgagtacgcc tcagccatct tgtccttctg gagttcgttg tacaggccct cctgggggtt 2520ctttctccga ggcttgcctc ccatctccgg gtccctacct ctacgcttgt ctaacacgtc 2580gtactcctcc cggcgcccca ggtttaactc gttgtatagc tggttctgac cctgctggta 2640agcgggtgcg tccgcggagc gagagaactt cactctcagt tcgcagctgc cttcttcttc 2700gctcggcgcg cggcagctgc tcgcatcttc ttcctgcgcg ctgctcgccg ggctcgcaaa 2760cgggcttttc gccgccgccg ccgctttttt gcggctcgct ttgcagtaga gcgttattac 2820caagctcaac aacaacactc cgcaagtccc ggctagtggt gcccagatgt agatgtcgca 2880ggcgaagtct agtcctcgcg tgtgaacggc gccgcccgcc gcgggcctgc aagcctcagg 2940gcgaagagat aagggctgac ttgctatcgt tggagctggc gtggggggac gtggggctgg 3000ggtagtagtg ctggatacgg tcacagaagt gccctgaccc cagtagtcca ttgcgtaact 3060cccgccgtag tagtagtgct tcgcgcagta gtaaatagcg gtgtcgtcag tctggagaga 3120gttcatcttt aagaaaacct gcgacttact gttgtcctta atgatggtga ggcggctctt 3180gagagctgag ttgtagtatg tggtttcact tccccagata acccccagcc actctaagcc 3240ctttctaggc ggctgtcgta tccatgatac cccgtagtct gggagactga ctccagacac 3300agtgcacgtg actgagaggc tctggctagg tgcgaccaag cccggtccgc tctcctgtaa 3360cttgacctcc gagccgccac ccccgctgcc gccgccaccg ctgccaccgc ctccggtaat 3420ctccagctta gttcccccac cgaatgtgta cgggagcgtg ttgccctgct gacagaagta 3480ggtggcgatg tcctcctgct caaggttact gattgtcagg ctgtagtccg taccggaacc 3540ggaacccgaa aatcggctag gaacgcctga gtgtaatctc gacgtgtggt aaatgagcaa 3600cttaactgtt ccgtccggct tctgctgata ccagtttaag tattttgaga tgtcctggct 3660ggcccggcaa gatattgtta ctcggtcgcc taaagaagca gatagggaag aggtagtctg 3720agtcatctgt atgtcaggac gagccgcgtg caataataac gccagaggta aaagtaaggc 3780tgtcactggg agcgccatgg tggctttccg gaacaaacaa gaaccgcgac ccaaatcccg 3840gctgcgacgg aactagctgt gccacacccg gcgcgtcctt atataatcat cggcgttcac 3900cgcccattct ccgcccagcc ataaaaggca actttcggaa cggcgcacgc tgattggctc 3960cgccctaact ccgcccacta gtgcggccgc ccccttcacc gagggcctat ttcccatgat 4020tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 4080tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 4140gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 4200gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gtgtaccgga 4260ggtttgaaga tgccgcattt ctcgagaaat gcggcatctt caaacctttt tttgaattct 4320cgacctaggg acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg 4380ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat 4440tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc 4500cactttggcc gcggatccgc aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca 4560gcattattcc agaagacacc ttcttcccca gcccaggtaa gggcagcttt ggtgccttcg 4620caggctgttt ccttgcttca ggaatggcca ggttctgccc agagctctgg tcaatgatgt 4680ctaaaactcc tctgattggt ggtctcggcc ttatccattg ccaccaaaac cctcttttta 4740ctaagaaaca gtgagccttg ttctggcagt ccagagaatg acacgggaaa aaagcagatg 4800aagagaaggt ggcaggagag ggcacgtggc ccagcctcag tctctccaac tgagttcctg 4860cctgcctgcc tttgctcaga ctgtttgccc cttactgctc ttctaggcct cattctaagc 4920cccttctcca agttgcctct ccttatttct ccctgtctgc caaaaaatct ttcccagctc 4980actaagtcag tctcacgcag tcactcatta acccaccaat cactgattgt gccggcacat 5040gaatgcacca ggtgttgaag tggaggaatt aaaaagtcag atgaggggtg tgcccagagg 5100aagcaccatt ctagttgggg gagcccatct gtcagctggg aaaagtccaa ataacttcag 5160attggaatgt gttttaactc agggttgaga aaacagccac cttcaggaca aaagtcaggg 5220aagggctctc tgaagaaatg ctacttgaag ataccagccc taccaagggc agggagagga 5280ccaattgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgcccg 5340ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg 5400cagagaggga gtggccaacg gcgcgcctgc aggtctcaaa aatagctacc ctctccggca 5460tgaatttatc agctagaacg gttgaatatc atattgatgg tgatttgact gtctccggcc 5520tttctcaccc gtttgaatct ttacctacac attactcagg cattgcattt aaaatatatg 5580agggttctaa aaatttttat ccttgcgttg aaataaaggc ttctcccgca aaagtattac 5640agggtcataa tgtttttggt acaaccgatt tagctttatg ctctgaggct ttattgctta 5700attttgctaa ttctttgcct tgcctgtatg atttattgga tgttggaatt cctgatgcgg 5760tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca 5820atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg 5880ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 5940agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc 6000gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt 6060ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 6120aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 6180aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 6240cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 6300ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt 6360cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 6420ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 6480gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 6540gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 6600acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 6660cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 6720acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact 6780ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 6840ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 6900gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 6960atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 7020ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 7080attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 7140ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 7200aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 7260aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 7320ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 7380tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 7440ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 7500cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 7560agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 7620gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 7680ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 7740tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 7800tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct 7860cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 7920tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 7980gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatg 8039228098DNAArtificial SequenceSynthesized 22cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgatccagac atgataagat acattgatga gtttggacaa 2160accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaac tggtacctca 2340tcgaggtggt aaggcttgca tgtgcagcgc gtcgtatgtg tccttggtcg cggtggaaag 2400cccctggtat aagccgtcgt gtccctttcc gcgacgtctc tcgcccttca taccaatctc 2460cgagtacgcc tcagccatct tgtccttctg gagttcgttg tacaggccct cctgggggtt 2520ctttctccga ggcttgcctc ccatctccgg gtccctacct ctacgcttgt ctaacacgtc 2580gtactcctcc cggcgcccca ggtttaactc gttgtatagc tggttctgac cctgctggta 2640agcgggtgcg tccgcggagc gagagaactt cactctcagt tcgcagctgc cttcttcttc 2700gctcggcgcg cggcagctgc tcgcatcttc ttcctgcgcg ctgctcgccg ggctcgcaaa 2760cgggctttta gccgccgccg ccgctttttt gcggctcgct ttgcagtaga gcgttattac 2820caagctcaac aacaacactc cgcaagtccc ggctagtggt gcccagatgt agatgtcgca 2880ggcgaagtct agtcctcgcg tgtgaacggc gccgcccgcc gcgggcctgc aagcctcagg 2940gcgaagagat aagggctgac ttgctatcgt tggagctggc gtggggggac gtggggctgg 3000ggtagtagtg ggcttcgctg gggatacatt tgtagaaaca tttgaaaatg ttccctgggt 3060aggtaactcg ctggatacgg tcacagaagt gccctgaccc cagtagtcca ttgcgtaact 3120cccgccgtag tagtagtgct tcgcgcagta gtaaatagcg gtgtcgtcag tctggagaga 3180gttcatcttt aagaaaacct gcgacttact gttgtcctta atgatggtga ggcggctctt 3240gagagctgag ttgtagtatg tggtttcact tccccagata acccccagcc actctaagcc 3300ctttctaggc ggctgtcgta tccatgatac cccgtagtct gggagactga ctccagacac 3360agtgcacgtg actgagaggc tctggctagg tgcgaccaag cccggtccgc tctcctgtaa 3420cttgacctcc gagccgccac ccccgctgcc gccgccaccg ctgccaccgc ctccggtaat 3480ctccagctta gttcccccac cgaatgtgta cgggagcgtg ttgccctgct gacagaagta 3540ggtggcgatg tcctcctgct caaggttact gattgtcagg ctgtagtccg taccggaacc 3600ggaacccgaa aatcggctag gaacgcctga gtgtaatctc gacgtgtggt aaatgagcaa 3660cttaactgtt ccgtccggct tctgctgata ccagtttaag tattttgaga tgtcctggct 3720ggcccggcaa gatattgtta ctcggtcgcc taaagaagca gatagggaag aggtagtctg 3780agtcatctgt atgtcaggac gagccgcgtg caataataac gccagaggta aaagtaaggc 3840tgtcactggg agcgccatgg tggctttccg gaacaaacaa gaaccgcgac ccaaatcccg 3900gctgcgacgg aactagctgt gccacacccg gcgcgtcctt atataatcat cggcgttcac 3960cgcccattct ccgcccagcc ataaaaggca actttcggaa cggcgcacgc tgattggctc 4020cgccctaact ccgcccacta gtgcggccgc gcggccaaag tggatctctg ctgtccctgt 4080aataaacccg aaaattttga atttttgtaa tttgtttttg taattcttta gtttgtatgt 4140ctgttgctat tatgtctact attctttccc ctgcactgta ccccccaatc cccccttttc 4200ttttaaaatt gtggatgaat actgccattt gtccctaggt cgagaattca aaaaaaggtt 4260tgaagatgcc gcatttctcg agaaatgcgg catcttcaaa cctccggtac accggtgttt 4320cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc 4380atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac 4440tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattccaatt 4500atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc 4560tcggtgaagg gggatccgca acaaatctga ctttgcatgt gcaaacgcct tcaacaacag 4620cattattcca gaagacacct tcttccccag cccaggtaag ggcagctttg gtgccttcgc 4680aggctgtttc cttgcttcag gaatggccag gttctgccca gagctctggt caatgatgtc 4740taaaactcct ctgattggtg gtctcggcct tatccattgc caccaaaacc ctctttttac 4800taagaaacag tgagccttgt tctggcagtc cagagaatga cacgggaaaa aagcagatga 4860agagaaggtg gcaggagagg gcacgtggcc cagcctcagt ctctccaact gagttcctgc 4920ctgcctgcct ttgctcagac tgtttgcccc ttactgctct tctaggcctc attctaagcc 4980ccttctccaa gttgcctctc cttatttctc cctgtctgcc aaaaaatctt tcccagctca 5040ctaagtcagt ctcacgcagt cactcattaa cccaccaatc actgattgtg ccggcacatg 5100aatgcaccag gtgttgaagt ggaggaatta aaaagtcaga tgaggggtgt gcccagagga 5160agcaccattc tagttggggg agcccatctg tcagctggga aaagtccaaa taacttcaga 5220ttggaatgtg ttttaactca gggttgagaa aacagccacc ttcaggacaa aagtcaggga 5280agggctctct gaagaaatgc tacttgaaga taccagccct accaagggca gggagaggac 5340caattgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgcccgg 5400gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc 5460agagagggag tggccaacgg cgcgcctgca ggtctcaaaa atagctaccc tctccggcat 5520gaatttatca gctagaacgg ttgaatatca tattgatggt gatttgactg tctccggcct 5580ttctcacccg tttgaatctt tacctacaca ttactcaggc attgcattta aaatatatga 5640gggttctaaa aatttttatc cttgcgttga aataaaggct tctcccgcaa aagtattaca 5700gggtcataat gtttttggta caaccgattt agctttatgc tctgaggctt tattgcttaa 5760ttttgctaat tctttgcctt gcctgtatga tttattggat gttggaattc ctgatgcggt 5820attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 5880tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 5940cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 6000gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 6060tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 6120gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 6180atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 6240agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 6300ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 6360gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 6420gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 6480tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 6540acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 6600aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 6660cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 6720gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 6780cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 6840tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 6900tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 6960ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 7020tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 7080gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 7140ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 7200tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 7260agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 7320aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 7380cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 7440agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 7500tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 7560gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 7620gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 7680ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 7740gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 7800ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 7860ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 7920acatgttctt tcctgcgtta tcccctgatt ctgtggataa

ccgtattacc gcctttgagt 7980gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 8040cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatg 8098238038DNAArtificial SequenceSynthesized 23cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgatccagac atgataagat acattgatga gtttggacaa 2160accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaac tggtacctca 2340tcgaggtggt aaggcttgca tgtgcagcgc gtcgtatgtg tccttggtcg cggtggaaag 2400cccctggtat aagccgtcgt gtccctttcc gcgacgtctc tcgcccttca taccaatctc 2460cgagtacgcc tcagccatct tgtccttctg gagttcgttg tacaggccct cctgggggtt 2520ctttctccga ggcttgcctc ccatctccgg gtccctacct ctacgcttgt ctaacacgtc 2580gtactcctcc cggcgcccca ggtttaactc gttgtatagc tggttctgac cctgctggta 2640agcgggtgcg tccgcggagc gagagaactt cactctcagt tcgcagctgc cttcttcttc 2700gctcggcgcg cggcagctgc tcgcatcttc ttcctgcgcg ctgctcgccg ggctcgcaaa 2760cgggcttttc gccgccgccg ccgctttttt gcggctcgct ttgcagtaga gcgttattac 2820caagctcaac aacaacactc cgcaagtccc ggctagtggt gcccagatgt agatgtcgca 2880ggcgaagtct agtcctcgcg tgtgaacggc gccgcccgcc gcgggcctgc aagcctcagg 2940gcgaagagat aagggctgac ttgctatcgt tggagctggc gtggggggac gtggggctgg 3000ggtagtagtg ctggatacgg tcacagaagt gccctgaccc cagtagtcca ttgcgtaact 3060cccgccgtag tagtagtgct tcgcgcagta gtaaatagcg gtgtcgtcag tctggagaga 3120gttcatcttt aagaaaacct gcgacttact gttgtcctta atgatggtga ggcggctctt 3180gagagctgag ttgtagtatg tggtttcact tccccagata acccccagcc actctaagcc 3240ctttctaggc ggctgtcgta tccatgatac cccgtagtct gggagactga ctccagacac 3300agtgcacgtg actgagaggc tctggctagg tgcgaccaag cccggtccgc tctcctgtaa 3360cttgacctcc gagccgccac ccccgctgcc gccgccaccg ctgccaccgc ctccggtaat 3420ctccagctta gttcccccac cgaatgtgta cgggagcgtg ttgccctgct gacagaagta 3480ggtggcgatg tcctcctgct caaggttact gattgtcagg ctgtagtccg taccggaacc 3540ggaacccgaa aatcggctag gaacgcctga gtgtaatctc gacgtgtggt aaatgagcaa 3600cttaactgtt ccgtccggct tctgctgata ccagtttaag tattttgaga tgtcctggct 3660ggcccggcaa gatattgtta ctcggtcgcc taaagaagca gatagggaag aggtagtctg 3720agtcatctgt atgtcaggac gagccgcgtg caataataac gccagaggta aaagtaaggc 3780tgtcactggg agcgccatgg tggctttccg gaacaaacaa gaaccgcgac ccaaatcccg 3840gctgcgacgg aactagctgt gccacacccg gcgcgtcctt atataatcat cggcgttcac 3900cgcccattct ccgcccagcc ataaaaggca actttcggaa cggcgcacgc tgattggctc 3960cgccctaact ccgcccacta gtgcggccgc gcggccaaag tggatctctg ctgtccctgt 4020aataaacccg aaaattttga atttttgtaa tttgtttttg taattcttta gtttgtatgt 4080ctgttgctat tatgtctact attctttccc ctgcactgta ccccccaatc cccccttttc 4140ttttaaaatt gtggatgaat actgccattt gtccctaggt cgagaattca aaaaaaggtt 4200tgaagatgcc gcatttctcg agaaatgcgg catcttcaaa cctccggtac accggtgttt 4260cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc 4320atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac 4380tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattccaatt 4440atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc 4500tcggtgaagg gggatccgca acaaatctga ctttgcatgt gcaaacgcct tcaacaacag 4560cattattcca gaagacacct tcttccccag cccaggtaag ggcagctttg gtgccttcgc 4620aggctgtttc cttgcttcag gaatggccag gttctgccca gagctctggt caatgatgtc 4680taaaactcct ctgattggtg gtctcggcct tatccattgc caccaaaacc ctctttttac 4740taagaaacag tgagccttgt tctggcagtc cagagaatga cacgggaaaa aagcagatga 4800agagaaggtg gcaggagagg gcacgtggcc cagcctcagt ctctccaact gagttcctgc 4860ctgcctgcct ttgctcagac tgtttgcccc ttactgctct tctaggcctc attctaagcc 4920ccttctccaa gttgcctctc cttatttctc cctgtctgcc aaaaaatctt tcccagctca 4980ctaagtcagt ctcacgcagt cactcattaa cccaccaatc actgattgtg ccggcacatg 5040aatgcaccag gtgttgaagt ggaggaatta aaaagtcaga tgaggggtgt gcccagagga 5100agcaccattc tagttggggg agcccatctg tcagctggga aaagtccaaa taacttcaga 5160ttggaatgtg ttttaactca gggttgagaa aacagccacc ttcaggacaa aagtcaggga 5220agggctctct gaagaaatgc tacttgaaga taccagccct accaagggca gggagaggac 5280caattgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgcccgg 5340gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc 5400agagagggag tggccaacgg cgcgcctgca ggtctcaaaa atagctaccc tctccggcat 5460gaatttatca gctagaacgg ttgaatatca tattgatggt gatttgactg tctccggcct 5520ttctcacccg tttgaatctt tacctacaca ttactcaggc attgcattta aaatatatga 5580gggttctaaa aatttttatc cttgcgttga aataaaggct tctcccgcaa aagtattaca 5640gggtcataat gtttttggta caaccgattt agctttatgc tctgaggctt tattgcttaa 5700ttttgctaat tctttgcctt gcctgtatga tttattggat gttggaattc ctgatgcggt 5760attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 5820tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 5880cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 5940gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 6000tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 6060gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 6120atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 6180agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 6240ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 6300gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 6360gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 6420tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 6480acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 6540aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 6600cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 6660gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 6720cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 6780tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 6840tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 6900ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 6960tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 7020gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 7080ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 7140tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 7200agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 7260aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 7320cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 7380agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 7440tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 7500gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 7560gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 7620ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 7680gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 7740ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 7800ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 7860acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 7920gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 7980cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatg 8038248626DNAArtificial SequenceSynthesized 24cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtagatct catattctgg 1140cagggtcagt ggctccaact aacatttgtt tggtacttta cagtttatta aatagatgtt 1200tatatggaga agctctcatt tctttctcag aagagcctgg ctaggaaggt ggatgaggca 1260ccatattcat tttgcaggtg aaattcctga gatgtaagga gctgctgtga cttgctcaag 1320gccttatatc gagtaaacgg tagcgctggg gcttagacgc aggtgttctg atttatagtt 1380caaaacctct atcaatgaga gagcaatctc ctggtaatgt gatagatttc ccaacttaat 1440gccaacatac cataaacctc ccattctgct aatgcccagc ctaagttggg gagaccactc 1500cagattccaa gatgtacagt ttgctttgct gggccttttt cccatgcctg cctttactct 1560gccagagtta tattgctggg gttttgaaga agatcctatt aaataaaaga ataagcagta 1620ttattaagta gccctgcatt tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac 1680gttcactgaa atcatggcct cttggccaag attgatagct tgtgcctgtc cctgagtccc 1740agtccatcac gagcagctgg tttctaagat gctatttccc gtataaagca tgagaccgtg 1800acttgccagc cccacagagc cccgcccttg tccatcactg gcatctggac tccagcctgg 1860gttggggcaa agagggaaat gagatcatgt cctaaccctg atcctcttgt cccacagata 1920tccagaaccc tgaccctgcc gtgtaccagc tgagagactc taaatccagt gacaagtctg 1980tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2040tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2100ctgtggcctg gagcaactag tgatccagac atgataagat acattgatga gtttggacaa 2160accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2220ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2280atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaac tggtacctca 2340tcgaggtggt aaggcttgca tgtgcagcgc gtcgtatgtg tccttggtcg cggtggaaag 2400cccctggtat aagccgtcgt gtccctttcc gcgacgtctc tcgcccttca taccaatctc 2460cgagtacgcc tcagccatct tgtccttctg gagttcgttg tacaggccct cctgggggtt 2520ctttctccga ggcttgcctc ccatctccgg gtccctacct ctacgcttgt ctaacacgtc 2580gtactcctcc cggcgcccca ggtttaactc gttgtatagc tggttctgac cctgctggta 2640agcgggtgcg tccgcggagc gagagaactt cactctcagt tcgcagctgc cttcttcttc 2700gctcggcgcg cggcagctgc tcgcatcttc ttcctgcgcg ctgctcgccg ggctcgcaaa 2760cgggctttta gccgccgccg ccgctttttt gcggctcgct ttgcagtaga gcgttattac 2820caagctcaac aacaacactc cgcaagtccc ggctagtggt gcccagatgt agatgtcgca 2880ggcgaagtct agtcctcgcg tgtgaacggc gccgcccgcc gcgggcctgc aagcctcagg 2940gcgaagagat aagggctgac ttgctatcgt tggagctggc gtggggggac gtggggctgg 3000ggtagtagtg ggcttcgctg gggatacatt tgtagaaaca tttgaaaatg ttccctgggt 3060aggtaactcg ctggatacgg tcacagaagt gccctgaccc cagtagtcca ttgcgtaact 3120cccgccgtag tagtagtgct tcgcgcagta gtaaatagcg gtgtcgtcag tctggagaga 3180gttcatcttt aagaaaacct gcgacttact gttgtcctta atgatggtga ggcggctctt 3240gagagctgag ttgtagtatg tggtttcact tccccagata acccccagcc actctaagcc 3300ctttctaggc ggctgtcgta tccatgatac cccgtagtct gggagactga ctccagacac 3360agtgcacgtg actgagaggc tctggctagg tgcgaccaag cccggtccgc tctcctgtaa 3420cttgacctcc gagccgccac ccccgctgcc gccgccaccg ctgccaccgc ctccggtaat 3480ctccagctta gttcccccac cgaatgtgta cgggagcgtg ttgccctgct gacagaagta 3540ggtggcgatg tcctcctgct caaggttact gattgtcagg ctgtagtccg taccggaacc 3600ggaacccgaa aatcggctag gaacgcctga gtgtaatctc gacgtgtggt aaatgagcaa 3660cttaactgtt ccgtccggct tctgctgata ccagtttaag tattttgaga tgtcctggct 3720ggcccggcaa gatattgtta ctcggtcgcc taaagaagca gatagggaag aggtagtctg 3780agtcatctgt atgtcaggac gagccgcgtg caataataac gccagaggta aaagtaaggc 3840tgtcactggg agcgccatgg tggctttccg gaacaaacaa gaaccgcgac ccaaatcccg 3900gctgcgacgg aactagctgt gccacacccg gcgcgtcctt atataatcat cggcgttcac 3960cgcccattct ccgcccagcc ataaaaggca actttcggaa cggcgcacgc tgattggctc 4020cgccctaact ccgcccacta gtgcggccgc ccccttcacc gagggcctat ttcccatgat 4080tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac 4140tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta 4200gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa 4260gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg gtgtaccgga 4320ggtttgaaga tgccgcattt ctcgagaaat gcggcatctt caaacctttt tttgaattct 4380cgacctaggg acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg 4440ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat 4500tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc 4560cactttggcc gcggatcccc cttcaccgag ggcctatttc ccatgattcc ttcatatttg 4620catatacgat acaaggctgt tagagagata attggaatta atttgactgt aaacacaaag 4680atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta 4740aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta tttcgatttc 4800ttggctttat atatcttgtg gaaaggacga aacaccggtg taccggaggt ttgaagatgc 4860cgcatttctc gagaaatgcg gcatcttcaa accttttttt gaattctcga cctagggaca 4920aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 4980ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 5040tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac tttggccgcg 5100gatccgcaac aaatctgact ttgcatgtgc aaacgccttc aacaacagca ttattccaga 5160agacaccttc ttccccagcc caggtaaggg cagctttggt gccttcgcag gctgtttcct 5220tgcttcagga atggccaggt tctgcccaga gctctggtca atgatgtcta aaactcctct 5280gattggtggt ctcggcctta tccattgcca ccaaaaccct ctttttacta agaaacagtg 5340agccttgttc tggcagtcca gagaatgaca cgggaaaaaa gcagatgaag agaaggtggc 5400aggagagggc acgtggccca gcctcagtct ctccaactga gttcctgcct gcctgccttt 5460gctcagactg tttgcccctt actgctcttc taggcctcat tctaagcccc ttctccaagt 5520tgcctctcct tatttctccc tgtctgccaa aaaatctttc ccagctcact aagtcagtct 5580cacgcagtca ctcattaacc caccaatcac tgattgtgcc ggcacatgaa tgcaccaggt 5640gttgaagtgg aggaattaaa aagtcagatg aggggtgtgc ccagaggaag caccattcta 5700gttgggggag cccatctgtc agctgggaaa agtccaaata acttcagatt ggaatgtgtt 5760ttaactcagg gttgagaaaa cagccacctt caggacaaaa gtcagggaag ggctctctga 5820agaaatgcta cttgaagata ccagccctac caagggcagg gagaggacca attgatggag 5880ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg 5940cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg 6000gccaacggcg cgcctgcagg tctcaaaaat agctaccctc tccggcatga atttatcagc 6060tagaacggtt gaatatcata ttgatggtga tttgactgtc tccggccttt ctcacccgtt 6120tgaatcttta cctacacatt actcaggcat tgcatttaaa atatatgagg gttctaaaaa 6180tttttatcct tgcgttgaaa taaaggcttc tcccgcaaaa gtattacagg gtcataatgt 6240ttttggtaca accgatttag ctttatgctc tgaggcttta ttgcttaatt ttgctaattc 6300tttgccttgc ctgtatgatt tattggatgt tggaattcct gatgcggtat tttctcctta 6360cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg 6420ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt 6480gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc 6540agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat 6600ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 6660gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 6720tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 6780ttcaacattt ccgtgtcgcc

cttattccct tttttgcggc attttgcctt cctgtttttg 6840ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 6900gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 6960gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 7020acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 7080actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 7140ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 7200cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 7260gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag 7320caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 7380aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 7440ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 7500tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 7560ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 7620ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 7680ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 7740tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 7800cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 7860taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 7920gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 7980acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 8040ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 8100ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 8160cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 8220aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 8280gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 8340gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 8400gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 8460ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 8520ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 8580caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatg 8626256827DNAArtificial SequenceSynthesized 25cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtgggaaa tgagatcatg 1140tcctaaccct gatcctcttg tcccacagat atccagaacc ctgaccctgc cgtgtaccag 1200ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 1260acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 1320atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacta gtgatccaga 1380catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg 1440ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa 1500acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga 1560ggttttttaa agcaagtaaa ctggtacctc atcgaggtgg taaggcttgc atgtgcagcg 1620cgtcgtatgt gtccttggtc gcggtggaaa gcccctggta taagccgtcg tgtccctttc 1680cgcgacgtct ctcgcccttc ataccaatct ccgagtacgc ctcagccatc ttgtccttct 1740ggagttcgtt gtacaggccc tcctgggggt tctttctccg aggcttgcct cccatctccg 1800ggtccctacc tctacgcttg tctaacacgt cgtactcctc ccggcgcccc aggtttaact 1860cgttgtatag ctggttctga ccctgctggt aagcgggtgc gtccgcggag cgagagaact 1920tcactctcag ttcgcagctg ccttcttctt cgctcggcgc gcggcagctg ctcgcatctt 1980cttcctgcgc gctgctcgcc gggctcgcaa acgggctttt agccgccgcc gccgcttttt 2040tgcggctcgc tttgcagtag agcgttatta ccaagctcaa caacaacact ccgcaagtcc 2100cggctagtgg tgcccagatg tagatgtcgc aggcgaagtc tagtcctcgc gtgtgaacgg 2160cgccgcccgc cgcgggcctg caagcctcag ggcgaagaga taagggctga cttgctatcg 2220ttggagctgg cgtgggggga cgtggggctg gggtagtagt gggcttcgct ggggatacat 2280ttgtagaaac atttgaaaat gttccctggg taggtaactc gctggatacg gtcacagaag 2340tgccctgacc ccagtagtcc attgcgtaac tcccgccgta gtagtagtgc ttcgcgcagt 2400agtaaatagc ggtgtcgtca gtctggagag agttcatctt taagaaaacc tgcgacttac 2460tgttgtcctt aatgatggtg aggcggctct tgagagctga gttgtagtat gtggtttcac 2520ttccccagat aacccccagc cactctaagc cctttctagg cggctgtcgt atccatgata 2580ccccgtagtc tgggagactg actccagaca cagtgcacgt gactgagagg ctctggctag 2640gtgcgaccaa gcccggtccg ctctcctgta acttgacctc cgagccgcca cccccgctgc 2700cgccgccacc gctgccaccg cctccggtaa tctccagctt agttccccca ccgaatgtgt 2760acgggagcgt gttgccctgc tgacagaagt aggtggcgat gtcctcctgc tcaaggttac 2820tgattgtcag gctgtagtcc gtaccggaac cggaacccga aaatcggcta ggaacgcctg 2880agtgtaatct cgacgtgtgg taaatgagca acttaactgt tccgtccggc ttctgctgat 2940accagtttaa gtattttgag atgtcctggc tggcccggca agatattgtt actcggtcgc 3000ctaaagaagc agatagggaa gaggtagtct gagtcatctg tatgtcagga cgagccgcgt 3060gcaataataa cgccagaggt aaaagtaagg ctgtcactgg gagcgccatg gtggctttcc 3120ggaacaaaca agaaccgcga cccaaatccc ggctgcgacg gaactagctg tgccacaccc 3180ggcgcgtcct tatataatca tcggcgttca ccgcccattc tccgcccagc cataaaaggc 3240aactttcgga acggcgcacg ctgattggct ccgccctaac tccgcccact agtgcggccg 3300cactgcaaac ccagggctgc cttggaaaag gcgcaacccg ggccccctcg agccggcgcc 3360aaagtggatc tctgctgtcc ctgtaataaa cccgaaaatt ttgaattttt gtaatttgtt 3420tttgtaattc tttagtttgt atgtctgttg ctattatgtc tactattctt tcccctgcac 3480tgtacccccc aatcccccct tttcttttaa aattgtggat gaatactgcc atttgtctca 3540agatctagaa ttcaaaaaaa ggtttgaaga tgccgcattt ctcgagaaat gcggcatctt 3600caaacctccg gtacctcgtc ctttccacaa gatatataaa gccaagaaat cgaaatactt 3660tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa ctgcaaacta 3720cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg tgtttacagt 3780caaattaatt ctaattatct ctctaacagc cttgtatcgt atatgcaaat atgaaggaat 3840catgggaaat aggccctctc tgggtcccct ggatccgcaa caaatctgac tttgcatgtg 3900caaacgcctt caacaacagc attattccag aagacacctt cttccccagc ccaggtaagg 3960gcagctttgg tgccttcgca ggctgtttcc ttgcttcagg aatggccagg ttctgcccag 4020agctctggtc aatgatgtct aaaactcctc tgattggtgg tctcggcccc aattgatgga 4080gttggccact ccctctctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg 4140gcgtcgggcg acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt 4200ggccaacggc gcgcctgcag gtctcaaaaa tagctaccct ctccggcatg aatttatcag 4260ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt tctcacccgt 4320ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag ggttctaaaa 4380atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag ggtcataatg 4440tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat tttgctaatt 4500ctttgccttg cctgtatgat ttattggatg ttggaattcc tgatgcggta ttttctcctt 4560acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat 4620gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 4680tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 4740cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta 4800tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg 4860ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg 4920ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 4980attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 5040gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 5100ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 5160cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt 5220gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 5280tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 5340gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 5400ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 5460tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 5520gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 5580caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 5640cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 5700atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 5760gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 5820attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 5880cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 5940atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 6000tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 6060ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 6120ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 6180cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 6240gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 6300gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 6360acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 6420gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 6480agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 6540tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 6600agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 6660cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 6720gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 6780ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatg 6827266762DNAArtificial SequenceSynthesized 26cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtgggaaa tgagatcatg 1140tcctaaccct gatcctcttg tcccacagat atccagaacc ctgaccctgc cgtgtaccag 1200ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 1260acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 1320atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacta gtgatccaga 1380catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg 1440ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa 1500acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga 1560ggttttttaa agcaagtaaa ctggtacctc atcgaggtgg taaggcttgc atgtgcagcg 1620cgtcgtatgt gtccttggtc gcggtggaaa gcccctggta taagccgtcg tgtccctttc 1680cgcgacgtct ctcgcccttc ataccaatct ccgagtacgc ctcagccatc ttgtccttct 1740ggagttcgtt gtacaggccc tcctgggggt tctttctccg aggcttgcct cccatctccg 1800ggtccctacc tctacgcttg tctaacacgt cgtactcctc ccggcgcccc aggtttaact 1860cgttgtatag ctggttctga ccctgctggt aagcgggtgc gtccgcggag cgagagaact 1920tcactctcag ttcgcagctg ccttcttctt cgctcggcgc gcggcagctg ctcgcatctt 1980cttcctgcgc gctgctcgcc gggctcgcaa acgggctttt agccgccgcc gccgcttttt 2040tgcggctcgc tttgcagtag agcgttatta ccaagctcaa caacaacact ccgcaagtcc 2100cggctagtgg tgcccagatg tagatgtcgc aggcgaagtc tagtcctcgc gtgtgaacgg 2160cgccgcccgc cgcgggcctg caagcctcag ggcgaagaga taagggctga cttgctatcg 2220ttggagctgg cgtgggggga cgtggggctg gggtagtagt gggcttcgct ggggatacat 2280ttgtagaaac atttgaaaat gttccctggg taggtaactc gctggatacg gtcacagaag 2340tgccctgacc ccagtagtcc attgcgtaac tcccgccgta gtagtagtgc ttcgcgcagt 2400agtaaatagc ggtgtcgtca gtctggagag agttcatctt taagaaaacc tgcgacttac 2460tgttgtcctt aatgatggtg aggcggctct tgagagctga gttgtagtat gtggtttcac 2520ttccccagat aacccccagc cactctaagc cctttctagg cggctgtcgt atccatgata 2580ccccgtagtc tgggagactg actccagaca cagtgcacgt gactgagagg ctctggctag 2640gtgcgaccaa gcccggtccg ctctcctgta acttgacctc cgagccgcca cccccgctgc 2700cgccgccacc gctgccaccg cctccggtaa tctccagctt agttccccca ccgaatgtgt 2760acgggagcgt gttgccctgc tgacagaagt aggtggcgat gtcctcctgc tcaaggttac 2820tgattgtcag gctgtagtcc gtaccggaac cggaacccga aaatcggcta ggaacgcctg 2880agtgtaatct cgacgtgtgg taaatgagca acttaactgt tccgtccggc ttctgctgat 2940accagtttaa gtattttgag atgtcctggc tggcccggca agatattgtt actcggtcgc 3000ctaaagaagc agatagggaa gaggtagtct gagtcatctg tatgtcagga cgagccgcgt 3060gcaataataa cgccagaggt aaaagtaagg ctgtcactgg gagcgccatg gtggctttcc 3120ggaacaaaca agaaccgcga cccaaatccc ggctgcgacg gaactagctg tgccacaccc 3180ggcgcgtcct tatataatca tcggcgttca ccgcccattc tccgcccagc cataaaaggc 3240aactttcgga acggcgcacg ctgattggct ccgccctaac tccgcccact agtgcggccg 3300cgggcccgtt taaacgctag cgagagggcc tatttcccat gattccttca tatttgcata 3360tacgatacaa ggctgttaga gagataatta gaattaattt gactgtaaac acaaagatat 3420tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat 3480tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg 3540ctttatatat cttgtggaaa ggacgaggta ccggaggttt gaagatgccg catttctcga 3600gaaatgcggc atcttcaaac ctttttttga attctagatc ttgagacaaa tggcagtatt 3660catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt 3720agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca 3780aaattttcct aggtacgtat ctagtggatc cgcaacaaat ctgactttgc atgtgcaaac 3840gccttcaaca acagcattat tccagaagac accttcttcc ccagcccagg taagggcagc 3900tttggtgcct tcgcaggctg tttccttgct tcaggaatgg ccaggttctg cccagagctc 3960tggtcaatga tgtctaaaac tcctctgatt ggtggtctcg gccccaattg atggagttgg 4020ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 4080gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 4140acggcgcgcc tgcaggtctc aaaaatagct accctctccg gcatgaattt atcagctaga 4200acggttgaat atcatattga tggtgatttg actgtctccg gcctttctca cccgtttgaa 4260tctttaccta cacattactc aggcattgca tttaaaatat atgagggttc taaaaatttt 4320tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat tacagggtca taatgttttt 4380ggtacaaccg atttagcttt atgctctgag gctttattgc ttaattttgc taattctttg 4440ccttgcctgt atgatttatt ggatgttgga attcctgatg cggtattttc tccttacgca 4500tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc 4560atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 4620gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 4680gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 4740ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 4800tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 4860gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 4920acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 4980cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 5040catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 5100tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 5160cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 5220accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 5280cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 5340ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 5400accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 5460ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 5520attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 5580ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 5640tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 5700tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 5760gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 5820tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 5880ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 5940ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 6000agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 6060cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 6120caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 6180tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 6240ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac

6300ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 6360gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 6420gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 6480tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 6540cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc 6600gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg 6660ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat 6720acgcaaaccg cctctccccg cgcgttggcc gattcattaa tg 6762277227DNAArtificial SequenceSynthesized 27cagcagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag 60cctgaatggc gaatggaatt ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc 120gtttttcctg ttgcaatggc tggcggtaat attgttctgg atattaccag caaggccgat 180agtttgagtt cttctactca ggcaagtgat gttattacta atcaaagaag tattgcgaca 240acggttaatt tgcgtgatgg acagactctt ttactcggtg gcctcactga ttataaaaac 300acttctcagg attctggcgt accgttcctg tctaaaatcc ctttaatcgg cctcctgttt 360agctcccgct ctgattctaa cgaggaaagc acgttatacg tgctcgtcaa agcaaccata 420gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 480cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 540cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 600tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 660gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 720tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 780ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 840taacgcgaat tttaacaaaa tattaacgtt tacaatttaa atatttgctt atacaatctt 900cctgtttttg gggcttttct gattatcaac cggggtacat atgattgaca tgctagtttt 960acggcgcgcc gggttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 1020accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 1080cagagaggga gtggccaact ccatcactag gggttcctac gcgtgggaaa tgagatcatg 1140tcctaaccct gatcctcttg tcccacagat atccagaacc ctgaccctgc cgtgtaccag 1200ctgagagact ctaaatccag tgacaagtct gtctgcctat tcaccgattt tgattctcaa 1260acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac tgtgctagac 1320atgaggtcta tggacttcaa gagcaacagt gctgtggcct ggagcaacta gtgatccaga 1380catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg 1440ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa 1500acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga 1560ggttttttaa agcaagtaaa ctggtacctc atcgaggtgg taaggcttgc atgtgcagcg 1620cgtcgtatgt gtccttggtc gcggtggaaa gcccctggta taagccgtcg tgtccctttc 1680cgcgacgtct ctcgcccttc ataccaatct ccgagtacgc ctcagccatc ttgtccttct 1740ggagttcgtt gtacaggccc tcctgggggt tctttctccg aggcttgcct cccatctccg 1800ggtccctacc tctacgcttg tctaacacgt cgtactcctc ccggcgcccc aggtttaact 1860cgttgtatag ctggttctga ccctgctggt aagcgggtgc gtccgcggag cgagagaact 1920tcactctcag ttcgcagctg ccttcttctt cgctcggcgc gcggcagctg ctcgcatctt 1980cttcctgcgc gctgctcgcc gggctcgcaa acgggctttt agccgccgcc gccgcttttt 2040tgcggctcgc tttgcagtag agcgttatta ccaagctcaa caacaacact ccgcaagtcc 2100cggctagtgg tgcccagatg tagatgtcgc aggcgaagtc tagtcctcgc gtgtgaacgg 2160cgccgcccgc cgcgggcctg caagcctcag ggcgaagaga taagggctga cttgctatcg 2220ttggagctgg cgtgggggga cgtggggctg gggtagtagt gggcttcgct ggggatacat 2280ttgtagaaac atttgaaaat gttccctggg taggtaactc gctggatacg gtcacagaag 2340tgccctgacc ccagtagtcc attgcgtaac tcccgccgta gtagtagtgc ttcgcgcagt 2400agtaaatagc ggtgtcgtca gtctggagag agttcatctt taagaaaacc tgcgacttac 2460tgttgtcctt aatgatggtg aggcggctct tgagagctga gttgtagtat gtggtttcac 2520ttccccagat aacccccagc cactctaagc cctttctagg cggctgtcgt atccatgata 2580ccccgtagtc tgggagactg actccagaca cagtgcacgt gactgagagg ctctggctag 2640gtgcgaccaa gcccggtccg ctctcctgta acttgacctc cgagccgcca cccccgctgc 2700cgccgccacc gctgccaccg cctccggtaa tctccagctt agttccccca ccgaatgtgt 2760acgggagcgt gttgccctgc tgacagaagt aggtggcgat gtcctcctgc tcaaggttac 2820tgattgtcag gctgtagtcc gtaccggaac cggaacccga aaatcggcta ggaacgcctg 2880agtgtaatct cgacgtgtgg taaatgagca acttaactgt tccgtccggc ttctgctgat 2940accagtttaa gtattttgag atgtcctggc tggcccggca agatattgtt actcggtcgc 3000ctaaagaagc agatagggaa gaggtagtct gagtcatctg tatgtcagga cgagccgcgt 3060gcaataataa cgccagaggt aaaagtaagg ctgtcactgg gagcgccatg gtggctttcc 3120ggaacaaaca agaaccgcga cccaaatccc ggctgcgacg gaactagctg tgccacaccc 3180ggcgcgtcct tatataatca tcggcgttca ccgcccattc tccgcccagc cataaaaggc 3240aactttcgga acggcgcacg ctgattggct ccgccctaac tccgcccact agtgcggccc 3300gtttaaacgc tagcgagagg gcctatttcc catgattcct tcatatttgc atatacgata 3360caaggctgtt agagagataa ttagaattaa tttgactgta aacacaaaga tattagtaca 3420aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt 3480taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata 3540tatcttgtgg aaaggacgag gtaccggagg tttgaagatg ccgcatttct cgagaaatgc 3600ggcatcttca aacctttttt tgaattctag atcttgagac aaatggcagt attcatccac 3660aattttaaaa gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata 3720atagcaacag acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt 3780cctagcgaga gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg 3840ttagagagat aattagaatt aatttgactg taaacacaaa gatattagta caaaatacgt 3900gacgtagaaa gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg 3960actatcatat gcttaccgta acttgaaagt atttcgattt cttggcttta tatatcttgt 4020ggaaaggacg aggtaccgga ggtttgaaga tgccgcattt ctcgagaaat gcggcatctt 4080caaacctttt tttgaattct agatcttgag acaaatggca gtattcatcc acaattttaa 4140aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac 4200agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcctaggta 4260cgtatctagt ggatccgcaa caaatctgac tttgcatgtg caaacgcctt caacaacagc 4320attattccag aagacacctt cttccccagc ccaggtaagg gcagctttgg tgccttcgca 4380ggctgtttcc ttgcttcagg aatggccagg ttctgcccag agctctggtc aatgatgtct 4440aaaactcctc tgattggtgg tctcggcccc aattgatgga gttggccact ccctctctgc 4500gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg acctttggtc 4560gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggccaacggc gcgcctgcag 4620gtctcaaaaa tagctaccct ctccggcatg aatttatcag ctagaacggt tgaatatcat 4680attgatggtg atttgactgt ctccggcctt tctcacccgt ttgaatcttt acctacacat 4740tactcaggca ttgcatttaa aatatatgag ggttctaaaa atttttatcc ttgcgttgaa 4800ataaaggctt ctcccgcaaa agtattacag ggtcataatg tttttggtac aaccgattta 4860gctttatgct ctgaggcttt attgcttaat tttgctaatt ctttgccttg cctgtatgat 4920ttattggatg ttggaattcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 4980acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc 5040ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 5100ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 5160accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 5220gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 5280tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 5340ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 5400ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 5460gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 5520caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 5580ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact 5640cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 5700gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 5760taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 5820tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 5880agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 5940caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 6000ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 6060tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 6120agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 6180tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 6240agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 6300gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 6360gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 6420tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 6480gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 6540accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 6600accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 6660gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 6720ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 6780atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 6840gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 6900cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 6960gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 7020gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 7080tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 7140cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 7200ccccgcgcgt tggccgattc attaatg 722728118DNAArtificial SequenceSynthetic sequence 28ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 60agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaatttt 118

Claims

1. A method for producing a genetically-modified human T cell, said method comprising: (a) introducing into a human T cell a nucleic acid molecule comprising: (i) a first expression cassette comprising a nucleic acid sequence encoding a chimeric antigen receptor; (ii) a second expression cassette comprising a nucleic acid sequence encoding an RNA interference molecule that is inhibitory against beta-2 microglobulin; (iii) a 5' homology arm; and (iv) a 3' homology arm; wherein said 5' homology arm and said 3' homology arm have homology to chromosomal regions flanking a nuclease recognition sequence in a T cell receptor (TCR) alpha constant region gene; and (b) introducing into said human T cell an mRNA encoding an engineered nuclease having specificity for said nuclease recognition sequence, wherein said engineered nuclease is expressed in said cell; wherein said nucleic acid molecule is introduced into said human T cell using an adeno-associated virus (AAV) vector having a serotype of AAV6, wherein said engineered nuclease recognizes and cleaves said nuclease recognition sequence in the genome of said human T cell to generate a cleavage site, wherein said nucleic acid molecule is inserted into the genome of said human T cell at said cleavage site by homologous recombination, and wherein cell surface expression of beta-microglobulin on said genetically-modified human T cell is reduced by about 90% to about 95% compared to cell surface beta-2 microglobulin expression on a control human T cell that is not genetically-modified to reduce cell surface beta-2 microglobulin expression.

2. The method of claim 1, wherein said engineered nuclease is an engineered meganuclease, a TALEN, a zinc finger nuclease (ZFN), a CRISPR/Cas, a compact TALEN, or a megaTAL.

3. The method of claim 1, wherein said engineered nuclease is an engineered meganuclease.

4. The method of claim 1, wherein said nuclease recognition sequence consists of SEQ ID NO: 1.