Peptide-mediated Delivery Of Rna-guided Endonuclease Into Cells

Abstract

A composition is disclosed that comprises at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein the RGEN protein component and CPP are covalently or non-covalently linked to each other in an RGEN protein-CPP complex. The RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell. The RGEN protein component of an RGEN protein-CPP complex in certain embodiments can be associated with a suitable RNA component to provide an RGEN capable of specific DNA targeting. Further disclosed are compositions comprising at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one CPP, as well as methods of delivering RGEN proteins into microbial cells, as well as methods of targeting DNA with RGENs.

Description

[0001] This application is a Continuation of U.S. application Ser. No. 16/218,808 filed Dec. 13, 2018, which is a Divisional of U.S. application Ser. No. 15/523,741, filed May 2, 2017, which is a 371 of International Application No. PCT/US15/58760, filed Nov. 3, 2015, which claims the benefit of U.S. Provisional Application No. 62/075,999 filed Nov. 6, 2014, incorporated herein in its entirety by reference.

FIELD OF INVENTION

[0002] The invention is in the field of molecular biology. Specifically, this invention pertains to delivery of protein components of RNA-guided endonucleases into cells using cell-penetrating peptides.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0003] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 20200127_CL6273USPCN_SequenceListing_ST25 created Jan. 27, 2020, and having a size of 385 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII-formatted document is part of the specification and is herein incorporated by reference in its entirety.

BACKGROUND

[0004] A way to understand the function of a gene within an organism is to inhibit its expression. Inhibition of gene expression can be accomplished, for example, by interrupting or deleting the DNA sequence of the gene, resulting in "knock-out" of the gene (Austin et al., Nat. Genetics 36:921-924). Gene knock-outs mostly have been carried out through homologous recombination (HR), a technique applicable across a wide array of organisms from bacteria to mammals. Another way for studying gene function can be through genetic "knock-in", which is also usually performed by HR. HR for purposes of gene targeting (knock-out or knock-in) can employ the presence of an exogenously supplied DNA having homology with the target site ("donor DNA").

[0005] HR for gene targeting has been shown to be enhanced when the targeted DNA site contains a double-strand break (Rudin et al., Genetics 122:519-534; Smih et al., Nucl. Acids Res. 23:5012-5019). Strategies for introducing double-strand breaks to facilitate HR-mediated DNA targeting have therefore been developed. For example, zinc finger nucleases have been engineered to cleave specific DNA sites leading to enhanced levels of HR at the site when a donor DNA was present (Bibikova et al., Science 300:764; Bibikova et al., Mol. Cell. Biol. 21:289-297). Similarly, artificial meganucleases (homing endonucleases) and transcription activator-like effector (TALE) nucleases have also been developed for use in HR-mediated DNA targeting (Epinat et al., Nucleic Acids Res. 31: 2952-2962; Miller et al., Nat. Biotech. 29:143-148).

[0006] Loci encoding CRISPR (clustered regularly interspaced short palindromic repeats) DNA cleavage systems have been found exclusively in about 40% of bacterial genomes and most archaeal genomes (Horvath and Barrangou, Science 327:167-170; Karginov and Hannon, Mol. Cell 37:7-19). In particular, the CRISPR-associated (Cas) RNA-guided endonuclease (RGEN), Cas9, of the type II CRIPSR system has been developed as a means for introducing site-specific DNA strand breaks that stimulate HR with donor DNA (U.S. Provisional Appl. No. 61/868,706, filed Aug. 22, 2013). The sequence of the RNA component of Cas9 can be designed such that Cas9 recognizes and cleaves DNA containing (i) sequence complementary to a portion of the RNA component and (ii) a protospacer adjacent motif (PAM) sequence.

[0007] Native Cas9/RNA complexes comprise two RNA sequences, a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). A crRNA contains, in the 5'-to-3' direction, a unique sequence complementary to a target DNA site and a portion of a sequence encoded by a repeat region of the CRISPR locus from which the crRNA was derived. A tracrRNA contains, in the 5'-to-3' direction, a sequence that anneals with the repeat region of crRNA and a stem loop-containing portion. Recent work has led to the development of guide RNAs (gRNA), which are chimeric sequences containing, in the 5'-to-3' direction, a crRNA linked to a tracrRNA (U.S. Provisional Appl. No. 61/868,706, filed Aug. 22, 2013).

[0008] Protein and RNA components for performing Cas9-mediated DNA targeting in a cell have been provided in some studies through recombinant DNA expression strategies. For example, Cas9 protein has been expressed in cells using nucleic acid-based expression systems. Methods of expressing RNA components such as gRNA in certain cell types have included using RNA polymerase III (Pol III) promoters, which allow for transcription of RNA with precisely defined, unmodified, 5'- and 3'-ends (DiCarlo et al., Nucleic Acids Res. 41: 4336-4343; Ma et al., Mol. Ther. Nucleic Acids 3:e161). These protein and RNA expression techniques have been applied in cells of several different species including maize and soybean (U.S. Provisional Appl. No. 61/868,706, filed Aug. 22, 2013), as well as humans, mouse, zebrafish, Trichoderma and Saccharomyces cerevisiae.

[0009] Despite these advances, other means of providing protein and RNA components in a cell, such as a microbial cell, to mediate Cas9-mediated DNA targeting are of interest.

SUMMARY OF INVENTION

[0010] In one embodiment, the invention concerns a composition comprising at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein the protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, and wherein the RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a microbial cell.

[0011] In a second embodiment, the protein component of the RGEN is associated with at least one RNA component that comprises a sequence complementary to a target site sequence on a chromosome or episome in the microbial cell, wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence. In a third embodiment, the RNA component comprises a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) operably linked to a trans-activating CRISPR RNA (tracrRNA). In a fourth embodiment, the RGEN can cleave one or both DNA strands at the target site sequence.

[0012] In a fifth embodiment, the RGEN comprises a CRISPR-associated (Cas) protein-9 (Cas9) amino acid sequence.

[0013] In a sixth embodiment, the RGEN protein component and CPP are covalently linked.

[0014] In a seventh embodiment, the RGEN protein component and CPP are non-covalently linked.

[0015] In an eighth embodiment, the CPP is cationic or amphipathic.

[0016] In a ninth embodiment, the CPP comprises (i) a CPP from an Epstein-Barr virus Zebra trans-activator protein, (ii) a CPP having 6 or more contiguous arginine residues, (iii) a transportan-10 (TP10) CPP, or (iv) a CPP from a vascular endothelium cadherin protein.

[0017] In a tenth embodiment, the RGEN protein-CPP complex can traverse a cell wall and cell membrane of a microbial cell.

[0018] An eleventh embodiment concerns a microbial cell comprising a composition disclosed herein.

[0019] A twelfth embodiment concerns a method of delivering a protein component of an RNA-guided endonuclease (RGEN) into a microbial cell. This method comprises contacting a microbial cell with a composition comprising the RGEN protein component and at least one cell-penetrating peptide (CPP), wherein the RGEN protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex. As a result of this contacting step, the RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of the microbial cell, and thereby gain entry to the microbial cell.

[0020] In a thirteenth embodiment, with respect to the method, (i) the composition further comprises at least one RNA component that is associated with the protein component of the RGEN, or (ii) the microbial cell comprises the RNA component, wherein the RNA component associates with the protein component of the RGEN after the RGEN protein-CPP complex enters the microbial cell; wherein the RNA component in (i) or (ii) comprises a sequence complementary to a target site sequence on a chromosome or episome in the microbial cell, and wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence. In a fourteenth embodiment, the RGEN can cleave one or both DNA strands at the target site sequence. In a fifteenth embodiment, the microbial cell further comprises a donor polynucleotide comprising at least one sequence homologous to a sequence at or near the target site sequence, wherein the donor polynucleotide integrates at or near the target site sequence by homologous recombination.

[0021] A sixteenth embodiment concerns a polynucleotide sequence comprising a nucleotide sequence encoding an RGEN protein-CPP fusion protein that comprises a protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein optionally, the nucleotide sequence is operably linked to a promoter sequence.

[0022] A seventeenth embodiment concerns a method of producing an RGEN protein-CPP fusion protein. This method comprises: (a) providing a polynucleotide sequence comprising a nucleotide sequence encoding an RGEN protein-CPP fusion protein that comprises a protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein optionally, the nucleotide sequence is operably linked to a promoter sequence; (b) expressing the RGEN protein-CPP fusion protein from the polynucleotide sequence, thereby producing the RGEN protein-CPP fusion protein, wherein the expressing is optionally performed in a microbial cell; and (c) optionally, isolating the RGEN protein-CPP fusion protein produced in step (b).

[0023] An eighteenth embodiment concerns a composition comprising at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one cell-penetrating peptide (CPP), wherein the protein component and CPP are covalently, or non-covalently, linked to each other in a guide polynucleotide/Cas endonuclease-CPP complex, wherein the guide polynucleotide/Cas endonuclease-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a microbial cell.

[0024] A nineteenth embodiment concerns a method for modifying a target site in the genome of a microbial cell. This method comprises providing a guide polynucleotide, a cell-penetrating peptide (CPP) and a Cas endonuclease to a microbial cell, wherein the guide polynucleotide, Cas endonuclease and CPP are covalently, or non-covalently, linked to each other in a guide polynucleotide/Cas endonuclease-CPP complex, and wherein the guide polynucleotide/Cas endonuclease-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of the microbial cell

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

[0025] FIG. 1: pZUFCas9 plasmid (SEQ ID NO:6) contains the Yarrowia codon-optimized Cas9 expression cassette set forth in SEQ ID NO:5. Origins of replication (ARS 18, f1 ori, ColE1) are in cross-hatch, and selectable markers (Ura3, Amp) are in grey. Refer to Example 1.

[0026] FIG. 2A: pBAD/HisB plasmid (SEQ ID NO:10) for expressing heterologous proteins in E. coli. pBAD promoter is in white. Origin of replication is in cross-hatch. Refer to Example 1.

[0027] FIG. 2B: pRF48 plasmid (SEQ ID NO:11) for expressing Cas9-NLS ("Cas9" in figure) in E. coli. Origin of replication is in cross-hatch. Refer to Example 1.

[0028] FIG. 3A: pRF144 plasmid (SEQ ID NO:20) for expressing 6.times.His-Zebra CPP-Cas9-NLS fusion in E. coli. Origin of replication is in cross-hatch. Refer to Example 1.

[0029] FIG. 3B: pRF145 plasmid (SEQ ID NO:21) for expressing 6.times.His-PolyR CPP-Cas9-NLS fusion in E. coli. Origin of replication is in cross-hatch. Refer to Example 1.

[0030] FIG. 3C: pRF146 plasmid (SEQ ID NO:22) for expressing 6.times.His-TP10 CPP-Cas9-NLS fusion in E. coli. Origin of replication is in cross-hatch. Refer to Example 1.

[0031] FIG. 3D: pRF162 plasmid (SEQ ID NO:23) for expressing 6.times.His-pVEC CPP-Cas9-NLS fusion in E. coli. Origin of replication is in cross-hatch. Refer to Example 1.

[0032] FIG. 4: SDS-PAGE separation of purification fractions of 6.times.His-Zebra-Cas9-NLS. Lysates, washes, elution fractions, and molecular weight standards are indicated. Refer to Example 1.

[0033] FIG. 5: A structural model of a single guide polynucleotide such as a single guide RNA (sgRNA). A variable targeting (VT) domain is shown in gray. A Cas9 endonuclease recognition (CER) domain is shown in black.

[0034] FIG. 6: In vitro transcription of RGR sgRNA (targeting Can1-1 locus) off of template derived from plasmid pRF46 (SEQ ID NO:30). In vitro transcription reactions incubated for 2, 4, 6 and 18 hours produced similar levels of sgRNA. Ribozyme autocatalytic cleavage products were also produced. Refer to Example 2.

[0035] FIG. 7: In vitro cleavage assay using Zebra CPP-Cas9 complexed with sgRNA specific for Can1-1 target site. A DNA polynucleotide (982 bp) containing the Can1-1 target site was included in each reaction. Each reaction was electrophoretically resolved on a 1.2% gel. "Target only", "sgRNA only", "Zebra-Cas9 only", and "Zebra-Cas9 only (2.times.FT)" (FT, freeze-thaw) reactions did not cleave the target polynucleotide. "Zebra-Cas9/sgRNA", "Zebra-Cas9/sgRNA (2.times.FT)", and "Cas9/sgRNA" (wild type Cas9) reactions cleaved the target polynucleotide in a specific manner as indicated by the resulting cleavage products. Refer to Example 3.

[0036] FIG. 8: Measuring the genome-targeting efficiency of Zebra CPP-Cas9 (not associated with sgRNA) and Zebra CPP-Cas9/gRNA complexes after contact thereof with Yarrowia lipolytica cells. The final concentration of Zebra-Cas9 used alone was 5 .mu.M, while different final concentrations (1-5 .mu.M) of Zebra CPP-Cas9 were used in the sgRNA complexes. Mutation frequency is reported as the proportion of yeast colonies (grown on non-selective medium after contacting cells with either Zebra CPP-Cas9 or Zebra CPP-Cas9/gRNA) that scored as resistant to canavanine upon transfer to canavanine-containing medium. Refer to Example 4.

[0037] FIG. 9: Example of PAGE gel analysis of CPP-dsRED purification. 12.5% PAGE gel stained with Simply blue stain. Lane 1: Molecular weight standard, Lane 2: clarified cell extract tp10-dsREDexpress, Lane 3: clarified-cell extract post bead treatment tp10-dsREDexpress, lane 4: final protein solution tp10-dsREDexpress, Lane 5 clarified cell extract MPG-dsREDexpress, Lane 3: clarified-cell extract post bead treatment MPG-dsREDexpress, lane 4: final protein solution MPG-dsREDexpress.

TABLE-US-00001 TABLE 1 Summary of Nucleic Acid and Protein SEQ ID Numbers Nucleic acid Protein Description SEQ ID NO. SEQ ID NO. Streptococcus pyogenes Cas9 open reading frame 1 codon-optimized for expression in Y. lipolytica. (4107 bases) Streptococcus pyogenes Cas9 including C-terminal 2 3 linker and SV40 NLS ("Cas9-NLS"); open reading (4140 bases) (1379 aa) frame codon-optimized for expression in Y. lipolytica. Y. lipolytica FBA1 promoter. 4 (543 bases) Cas9-NLS expression cassette (FBA1 promoter 5 and Cas9-NLS open reading frame). (4683 bases) pZUFCas9 plasmid. 6 (10706 bases) Cas9-NLS forward PCR primer. 7 (35 bases) Cas9-NLS reverse PCR primer. 8 (31 bases) EcoRI-Cas9-NLS-HinDIII PCR product 9 (4166 bases) pBAD/HisB plasmid 10 (4092 bases) pRF48 plasmid 11 (8237 bases) Zebra cell-penetrating peptide (CPP), from Epstein- 12 Barr virus Zebra trans-activator protein (54 aa) pVEC CPP, from murine endothelial cadherin 13 protein (18 aa) TP10 CPP, from neuropeptide galanin protein 14 (21 aa) Poly-arginine (PolyR) CPP 15 (17 aa) Ncol-6xHis-Zebra CPP-EcoRI 16 (194 bases) Ncol-6xHis-pVEC CPP-EcoRI 17 (86 bases) Ncol-6xHis-TP10 CPP-EcoRI 18 (95 bases) Ncol-6xHis-PolyR CPP-EcoRI 19 (83 bases) pRF144 plasmid, encoding Zebra CPP-Cas9 fusion 20 protein (8294 bases) pRF145 plasmid, encoding PolyR CPP-Cas9 fusion 21 protein (8183 bases) pRF146 plasmid, encoding TP10 CPP-Cas9 fusion 22 protein (8195 bases) pRF162 plasmid, encoding pVEC CPP-Cas9 fusion 23 protein (8186 bases) Cas9 endonuclease recognition (CER) domain of a 24 gRNA. (80 bases) Y. lipolytica Can1-1 target site, or alternatively, 25 DNA encoding Can1-1 variable target domain of a (20 bases) gRNA. Hammerhead (HH) ribozyme. 26 (43 bases) HDV ribozyme. 27 (68 bases) HH-sgRNA-HDV (RGR) pre-sgRNA expression 28 cassette, or alternatively, "RGR" expression (211 bases) cassette (for targeting Can1-1 locus) T7 RNA polymerase promoter 29 (20 bases) pRF46 plasmid 30 (2875 bases) T7 forward primer 31 (20 bases) gRNArev1 reverse primer 32 (20 bases) IV-up primer 33 (21 bases) IV-down primer 34 (20 bases) Can1 cleavage assay DNA sequence 35 (982 bases) RNA loop-forming sequence (GAAA). 36 (4 bases) RNA loop-forming sequence (CAAA). 37 (4 bases) RNA loop-forming sequence (AAAG). 38 (4 bases) Zebra CPP-Cas9-NLS fusion protein 39 (1434 aa) PolyR CPP-Cas9-NLS fusion protein 40 (1397 aa) TP10 CPP-Cas9-NLS fusion protein 41 (1401 aa) pVEC CPP-Cas9-NLS fusion protein 42 (1398 aa) Example of a Cas9 target site: PAM sequence. 43 (23 bases) PAM sequence NGG. 44 (3 bases) PAM sequence NNAGAA. 45 (6 bases) PAM sequence NNAGAAW. 46 (7 bases) PAM sequence NGGNG. 47 (5 bases) PAM sequence NNNNGATT. 48 (8 bases) PAM sequence NAAAAC. 49 (6 bases) PAM sequence NG. 50 (2 bases) TracrRNA mate sequence example 1. 51 (22 bases) TracrRNA mate sequence example 2. 52 (15 bases) TracrRNA mate sequence example 3. 53 (12 bases) TracrRNA mate sequence example 4. 54 (13 bases) TracrRNA example 1. 55 (60 bases) TracrRNA example 2. 56 (45 bases) TracrRNA example 3. 57 (32 bases) TracrRNA example 4. 58 (85 bases) TracrRNA example 5. 59 (77 bases) TracrRNA example 6. 60 (65 bases) gRNA example 1. 61 (131 bases) gRNA example 2. 62 (117 bases) gRNA example 3. 63 (104 bases) gRNA example 4. 64 (99 bases) gRNA example 5. 65 (81 bases) gRNA example 6. 66 (68 bases) gRNA example 7. 67 (100 bases) Tat-derived CPP (GRKKRRQRRR) 68 (10 aa) Tat-derived CPP (RKKRRQRRR) 69 (9 aa) Tat-derived CPP (RKKRRQRR) 70 (8 aa) Penetratin CPP (RQIKIWFQNRRMKWKK) 71 (16 aa) Polyarginine CPP (THRLPRRRRRR) 72 (11 aa) Polyarginine CPP (GGRRARRRRRR) 73 (11 aa) pVEC CPP (shorter version), from murine 74 endothelial cadherin protein (17 aa) CPP comprising (KFF).sub.3K 75 (10 aa) MAP peptide CPP 76 (18 aa) CPP (RRQRRTSKLMKR) 77 (12 aa) CPP (KALAWEAKLAKALAKALAKHLAKALAKALKCEA) 78 (33 aa) Proline-rich CPP repeat VHLPPP 79 (6 aa) Proline-rich CPP repeat VHRPPP 80 (6 aa) MPG peptide CPP 81 (27 aa) Pep-1 peptide CPP 82 (21 aa) hCT CPP example 1 83 (24 aa) hCT CPP example 2 84 (18 aa) his tagged dsRED 85 E. coli codon optimized dsRED 86 pBAD/HisB 87 pRF161 88 TAT 89 TLM 90 MPG1 91 pep1 92 CFFKDEL 93 his-TAT E. coli optimized 94 his-TLM E. coli optimized 95 his-MPG1 E. coli optimized 96 his-pep1 E. coli optimized 97 his-CFFKDEL E. coli optimized 98 pRF224 99 pRF214 100 pRF213 101 pRF217 102 pRF216 103 oligo 36 104 His-Zebra PCR 105 His-tp10 PCR 106 His-pVEC PCR 107 pRF144 108 pRF162 109 pRF146 110 oligo 153 111 pRF186 112 pRF192 113 pRF190 114 his-CFFKDEL-Cas9 115 his-MPG1-Cas9 116 pRF48 117 pRF243 118 pRF238 119 galK gene 120 galE gene 121 galT gene 122 CER domain I 123 CER encoding DNA PCR 124 pRF291 125 CER forward 126 universal reverse 127 universal forward T7 primer 128 galK2-1 forward primer 129 galK2-1 reverse primer 130 galK2-1 sgRNA in vitro transcription template 131 T7 promoter 132 DNA encoding galK2-1 variable targeting domain 133 galK2-1 target site 134 galK2-1 sgRNA 135 his-MPG1-dsREDexpress; 136 pVEC-dsREDexpress 137 CFFKDEL-dsREDexpress 138 TLM-dsREDexpress 139 Zebra-dsREDexpress 140 pep1-dsREDexpress 141 tp10-dsREDexpress 142 Zebra-Cas9 143 pVEC-Cas9 144

DETAILED DESCRIPTION OF THE INVENTION

[0038] The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety.

[0039] As used herein, the term "invention" or "disclosed invention" is not meant to be limiting, but applies generally to any of the inventions defined in the claims or described herein. These terms are used interchangeably herein.

[0040] The term "cell" herein refers to any type of cell such as a prokaryotic or eukaryotic cell. A eukaryotic cell has a nucleus and other membrane-enclosed structures (organelles), whereas a prokaryotic cell lacks a nucleus. A cell in certain embodiments can be a mammalian cell or non-mammalian cell. Non-mammalian cells can be eukaryotic or prokaryotic. For example, a non-mammalian cell herein can refer to a microbial cell or cell of a non-mammalian multicellular organism such as a plant, insect, nematode, avian species, amphibian, reptile, or fish.

[0041] A microbial cell herein can refer to a fungal cell (e.g., yeast cell), prokaryotic cell, protist cell (e.g., algal cell), euglenoid cell, stramenopile cell, or oomycete cell, for example. A prokaryotic cell herein can refer to a bacterial cell or archaeal cell, for example. Fungal cells (e.g., yeast cells), protist cells (e.g., algal cells), euglenoid cells, stramenopile cells, and oomycete cells represent examples of eukaryotic microbial cells. A eukaryotic microbial cell has a nucleus and other membrane-enclosed structures (organelles), whereas a prokaryotic cell lacks a nucleus.

[0042] The term "yeast" herein refers to fungal species that predominantly exist in unicellular form. Yeast can alternatively be referred to as "yeast cells". A yeast herein can be characterized as either a conventional yeast or non-conventional yeast, for example.

[0043] The term "conventional yeast" ("model yeast") herein generally refers to Saccharomyces or Schizosaccharomyces yeast species. Conventional yeast in certain embodiments are yeast that favor homologous recombination (HR) DNA repair processes over repair processes mediated by non-homologous end-joining (NHEJ).

[0044] The term "non-conventional yeast" herein refers to any yeast that is not a Saccharomyces or Schizosaccharomyces yeast species. Non-conventional yeast are described in Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnoloqy: Practical Protocols (K. Wolf, K. D. Breunig, G. Barth, Eds., Springer-Verlag, Berlin, Germany, 2003) and Spencer et al. (Appl. Microbiol. Biotechnol. 58:147-156), which are incorporated herein by reference. Non-conventional yeast in certain embodiments may additionally (or alternatively) be yeast that favor NHEJ DNA repair processes over repair processes mediated by HR. Definition of a non-conventional yeast along these lines--preference of NHEJ over HR--is further disclosed by Chen et al. (PLoS ONE 8:e57952), which is incorporated herein by reference. Preferred non-conventional yeast herein are those of the genus Yarrowia (e.g., Yarrowia lipolytica).

[0045] The term "plant" herein refers to whole plants, plant organs, plant tissues, plant cells, seeds and progeny of the same. Plant cells include, without limitation, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. Plant parts include differentiated and undifferentiated tissues including, but not limited to roots, stems, shoots, leaves, pollens, seeds, tumor tissue and various forms of cells and culture (e.g., single cells, protoplasts, embryos, and callus tissue). The plant tissue may be in plant or in a plant organ, tissue or cell culture. The term "plant organ" refers to plant tissue or a group of tissues that constitute a morphologically and functionally distinct part of a plant. The term "genome" refers to the entire complement of genetic material (genes and non-coding sequences) that is present in each cell of an organism, or virus or organelle; and/or a complete set of chromosomes inherited as a (haploid) unit from one parent. "Progeny" comprises any subsequent generation of a plant.

[0046] A transgenic plant includes, for example, a plant which comprises within its genome a heterologous polynucleotide introduced by a transformation step. The heterologous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. A transgenic plant can also comprise more than one heterologous polynucleotide within its genome. Each heterologous polynucleotide may confer a different trait to the transgenic plant. A heterologous polynucleotide can include a sequence that originates from a foreign species, or, if from the same species, can be substantially modified from its native form. Transgenic plant material can include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The alterations of a plant genome (chromosomal or extra-chromosomal) by conventional plant breeding methods, by a genome editing procedure described herein that does not result in an insertion of a foreign polynucleotide, or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation are not intended to be regarded as transgenic.

[0047] A fertile plant is a plant that produces viable male and female gametes and is self-fertile. Such a self-fertile plant can produce a progeny plant without the contribution from any other plant of a gamete and the genetic material contained therein. Male-sterile plants include plants that do not produce male gametes that are viable or otherwise capable of fertilization. Female-sterile plants include plants that do not produce female gametes that are viable or otherwise capable of fertilization. It is recognized that male-sterile and female-sterile plants can be female-fertile and male-fertile, respectively. It is further recognized that a male-fertile (but female-sterile) plant can produce viable progeny when crossed with a female-fertile plant and that a female-fertile (but male-sterile) plant can produce viable progeny when crossed with a male-fertile plant.

[0048] The term "RNA-guided endonuclease" (RGEN) herein refers to a complex comprising at least one CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein and at least one RNA component. The terms "protein component of an RGEN" and "RGEN protein component" are used interchangeably herein and refer to a Cas protein, which is, or forms part of, the endonuclease component of an RGEN. A protein component in certain embodiments can be a complete endonuclease (e.g., Cas9); such a protein component can alternatively be referred to as "the endonuclease component" of an RGEN. An RGEN herein typically has specific DNA targeting activity, given its association with at least one RNA component.

[0049] Briefly, an RNA component of an RGEN contains sequence that is complementary to a DNA sequence in a target site sequence. Based on this complementarity, an RGEN can specifically recognize and cleave a particular DNA target site sequence. An RGEN herein can comprise Cas protein(s) and suitable RNA component(s) of any of the four known CRISPR systems (Horvath and Barrangou, Science 327:167-170) such as a type I, II, or III CRISPR system. An RGEN in preferred embodiments comprises a Cas9 endonuclease (CRISPR II system) and at least one RNA component (e.g., a crRNA and tracrRNA, or a gRNA).

[0050] The term "CRISPR" (clustered regularly interspaced short palindromic repeats) refers to certain genetic loci encoding factors of class I, II, or III DNA cleavage systems, for example, used by bacterial and archaeal cells to destroy foreign DNA (Horvath and Barrangou, Science 327:167-170). Components of CRISPR systems are taken advantage of herein in a heterologous manner for DNA targeting in cells.

[0051] The terms "type II CRISPR system" and "type II CRISPR-Cas system" are used interchangeably herein and refer to a DNA cleavage system utilizing a Cas9 endonuclease in complex with at least one RNA component. For example, a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In another example, a Cas9 can be in complex with a guide RNA. Thus, crRNA, tracrRNA, and guide RNA are non-limiting examples of RNA components herein.

[0052] The term CRISPR-associated ("Cas") endonuclease herein refers to a Cas protein encoded by a Cas gene. A Cas endonuclease, when in complex with a suitable RNA component, is capable of cleaving all or part of a specific DNA target sequence in certain embodiments. For example, it is can be capable of introducing a single- or double-strand break in a specific DNA target sequence; it can alternatively be characterized as being able to cleave one or both strands of a specific DNA target sequence. A Cas endonuclease can unwind the DNA duplex at the target sequence and cleaves at least one DNA strand, as mediated by recognition of the target sequence by a crRNA or guide RNA that is in complex with the Cas. Such recognition and cutting of a target sequence by a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. Alternatively, a Cas protein herein may lack DNA cleavage or nicking activity, but can still specifically bind to a DNA target sequence when complexed with a suitable RNA component. A preferred Cas protein herein is Cas9.

[0053] Any guided endonuclease can be used in the methods disclosed herein. Such endonucleases include, but are not limited to Cas9 and Cpfl endonucleases. Many endonucleases have been described to date that can recognize specific PAM sequences (see for example--U.S. patent applications 62/162,377 filed May 15, 2015 and 62/162,353 filed May 15, 2015 and Zetsche B et al. 2015. Cell 163, 1013) and cleave the target DNA at a specific positions. It is understood that based on the methods and embodiments described herein utilizing a guided Cas system, one can now tailor these methods such that they can utilize any guided endonuclease system.

[0054] "Cas9" (formerly referred to as Cas5, Csn1, or Csx12) herein refers to a Cas endonuclease of a type II CRISPR system that forms a complex with crRNA and tracrRNA, or with a guide RNA, for specifically recognizing and cleaving all or part of a DNA target sequence. Cas9 protein comprises an RuvC nuclease domain and an HNH (H-N-H) nuclease domain, each of which cleaves a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick). In general, the RuvC domain comprises subdomains I, II and III, where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu et al, Cell 157:1262-1278). "Apo-Cas9" refers to Cas9 that is not complexed with an RNA component. Apo-Cas9 can bind DNA, but does so in a non-specific manner, and cannot cleave DNA (Sternberg et al., Nature 507:62-67).

[0055] The term "RNA component" herein refers to an RNA component of an RGEN containing a ribonucleic acid sequence that is complementary to a strand of a DNA target sequence. This complementary sequence is referred to herein as a "guide sequence" or "variable targeting domain" sequence (FIG. 5). Examples of suitable RNA components herein include crRNA and guide RNA. RNA components in certain embodiments (e.g., guide RNA alone, crRNA+tracrRNA) can render an RGEN competent for specific DNA targeting.

[0056] The term "CRISPR RNA" (crRNA) herein refers to an RNA sequence that can form a complex with one or more Cas proteins (e.g., Cas9) and provides DNA binding specificity to the complex. A crRNA provides DNA binding specificity since it contains "guide sequence" ("variable targeting domain" [VT]) that is complementary to a strand of a DNA target sequence. A crRNA further comprises a "repeat sequence" ("tracr RNA mate sequence") encoded by a repeat region of the CRISPR locus from which the crRNA was derived. A repeat sequence of a crRNA can anneal to sequence at the 5'-end of a tracrRNA. crRNA in native CRISPR systems is derived from a "pre-crRNA" transcribed from a CRISPR locus. A pre-crRNA comprises spacer regions and repeat regions; spacer regions contain unique sequence complementary to a DNA target site sequence. Pre-crRNA in native systems is processed to multiple different crRNAs, each with a guide sequence along with a portion of repeat sequence. CRISPR systems utilize crRNA, for example, for DNA targeting specificity.

[0057] The term "trans-activating CRISPR RNA" (tracrRNA) herein refers to a non-coding RNA used in type II CRISPR systems, and contains, in the 5'-to-3' direction, (i) a sequence that anneals with the repeat region of CRISPR type II crRNA and (ii) a stem loop-containing portion (Deltcheva et al., Nature 471:602-607).

[0058] The terms "guide RNA" (gRNA) and "single guide RNA" (sgRNA) are used interchangeably herein. A gRNA herein can refer to a chimeric sequence containing a crRNA operably linked to a tracrRNA. Alternatively, a gRNA can refer to a synthetic fusion of a crRNA and a tracrRNA, for example. A gRNA can also be characterized in terms of having a guide sequence (variable targeting domain) followed by a Cas endonuclease recognition (CER) domain. A CER domain can comprise a tracrRNA mate sequence followed by a tracrRNA sequence.

[0059] A "CRISPR DNA" (crDNA) can optionally be used instead of an RNA component. A crDNA has a DNA sequence corresponding to the sequence of a crRNA as disclosed herein. A crDNA can be used with a tracrRNA in a crDNA/tracrRNA complex, which in turn can be associated with an RGEN protein component. U.S. Appl. No. 61/953,090 discloses crDNA and the methods of its use in RGEN-mediated DNA targeting. It is contemplated that any disclosure herein regarding a crRNA can similarly apply to using a crDNA, accordingly. Thus, in embodiments herein incorporating a crDNA, an "RNA-guided endonuclease" (RGEN) could instead be referred to as a complex comprising at least one Cas protein and at least one crDNA.

[0060] As used herein, the term "guide polynucleotide", relates to a polynucleotide sequence that can form a complex with a Cas endonuclease and enables the Cas endonuclease to recognize and optionally cleave a DNA target site. The guide polynucleotide can be a single molecule or a double molecule. The guide polynucleotide sequence can be a RNA sequence, a DNA sequence, or a combination thereof (an RNA-DNA combination sequence). Optionally, the guide polynucleotide can comprise at least one nucleotide, phosphodiester bond or linkage modification such as, but not limited, to Locked Nucleic Acid (LNA), 5-methyl dC, 2,6-Diaminopurine, 2'-Fluoro A, 2'-Fluoro U, 2'-O-Methyl RNA, Phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule, or 5' to 3' covalent linkage resulting in circularization. A guide polynucleotide that solely comprises ribonucleic acids is also referred to as a "guide RNA".

[0061] The guide polynucleotide can be a double molecule (also referred to as duplex guide polynucleotide) comprising a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that is complementary to a nucleotide sequence in a target DNA and a second nucleotide sequence domain (referred to as Cas endonuclease recognition domain or CER domain) that interacts with a Cas endonuclease polypeptide. The CER domain of the double molecule guide polynucleotide comprises two separate molecules that are hybridized along a region of complementarity. The two separate molecules can be RNA, DNA, and/or RNA-DNA-combination sequences. In some embodiments, the first molecule of the duplex guide polynucleotide comprising a VT domain linked to a CER domain ("crNucleotide") is referred to as "crDNA" (when composed of a contiguous stretch of DNA nucleotides) or "crRNA" (when composed of a contiguous stretch of RNA nucleotides), or "crDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). In some embodiments the second molecule of the duplex guide polynucleotide comprising a CER domain is referred to as "tracrRNA" (when composed of a contiguous stretch of RNA nucleotides) or "tracrDNA" (when composed of a contiguous stretch of DNA nucleotides) or "tracrDNA-RNA" (when composed of a combination of DNA and RNA nucleotides).

[0062] The guide polynucleotide can also be a single molecule comprising a first nucleotide sequence domain (referred to as Variable Targeting domain or VT domain) that is complementary to a nucleotide sequence in a target DNA and a second nucleotide domain (referred to as Cas endonuclease recognition domain or CER domain) that interacts with a Cas endonuclease polypeptide. By "domain" it is meant a contiguous stretch of nucleotides that can be RNA, DNA, and/or RNA-DNA-combination sequence. The VT domain and/or the CER domain of a single guide polynucleotide can comprise an RNA sequence, a DNA sequence, or a, RNA-DNA-combination sequence. In some embodiments the single guide polynucleotide comprises a crNucleotide (comprising a VT domain linked to a CER domain) linked to a tracrNucleotide (comprising a CER domain), wherein the linkage is a nucleotide sequence comprising an RNA sequence, a DNA sequence, or an RNA-DNA combination sequence. The single guide polynucleotide being comprised of sequences from the crNucleotide and tracrNucleotide may be referred to as "single guide RNA" (when composed of a contiguous stretch of RNA nucleotides) or "single guide DNA" (when composed of a contiguous stretch of DNA nucleotides) or "single guide RNA-DNA" (when composed of a combination of RNA and DNA nucleotides).

[0063] Thus, a guide polynucleotide and a type II Cas endonuclease in certain embodiments can form a complex with each other (referred to as a "guide polynucleotide/Cas endonuclease complex" or also referred to as "guide polynucleotide/Cas endonuclease system"), wherein the guide polynucleotide/Cas endonuclease complex can direct the Cas endonuclease to target a genomic target site in a cell (e.g., plant cell), optionally enabling the Cas endonuclease to introduce a single- or double-strand break into the genomic target site. A guide polynucleotide/Cas endonuclease complex can be linked to at least one CPP, wherein such complex is capable of binding to, and optionally creating a single- or double-strand break to, a target site of a cell (e.g., a plant cell).

[0064] The term "variable targeting domain" or "VT domain" is used interchangeably herein and refers to a nucleotide sequence that is complementary to one strand (nucleotide sequence) of a double strand DNA target site. The percent complementation between the first nucleotide sequence domain (VT domain) and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The variable target domain can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the variable targeting domain comprises a contiguous stretch of 12 to 30 nucleotides. The variable targeting domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., modifications described herein), or any combination thereof.

[0065] The term "Cas endonuclease recognition domain" or "CER domain" of a guide polynucleotide is used interchangeably herein and relates to a nucleotide sequence (such as a second nucleotide sequence domain of a guide polynucleotide), that interacts with a Cas endonuclease polypeptide. A CER domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., modifications described herein), or any combination thereof.

[0066] The terms "target site", "target sequence", "target DNA", "DNA target sequence", "target locus", "protospacer" and the like are used interchangeably herein. A target site sequence refers to a polynucleotide sequence on a chromosome, episome, or any other DNA molecule in the genome of a cell to which an RGEN herein can recognize, bind to, and optionally nick or cleave. A target site can be (i) an endogenous/native site in the cell, (ii) heterologous to the cell and therefore not be naturally occurring in the genome, or (iii) found in a heterologous genomic location compared to where it natively occurs.

[0067] A target site sequence herein is at least 13 nucleotides in length and has a strand with sufficient complementarity to a guide sequence (of a crRNA or gRNA) to be capable of hybridizing with the guide sequence and direct sequence-specific binding of a Cas protein or Cas protein complex to the target sequence (if a suitable PAM is adjacent to the target sequence in certain embodiments). A cleavage/nick site (applicable with a endonucleolytic or nicking Cas) can be within the target sequence (e.g., using a Cas9) or a cleavage/nick site could be outside of the target sequence (e.g., using a Cas9 fused to a heterologous endonuclease domain such as one derived from a Fokl enzyme). It is also possible for a target site sequence to be bound by an RGEN lacking cleavage or nicking activity.

[0068] An "artificial target site" or "artificial target sequence" herein refers to a target sequence that has been introduced into the genome of a cell. An artificial target sequence in some embodiments can be identical in sequence to a native target sequence in the genome of the cell, but be located at a different position (a heterologous position) in the genome, or it can different from the native target sequence if located at the same position in the genome of the cell.

[0069] An "episome" herein refers to a DNA molecule that can exist in a cell autonomously (can replicate and pass on to daughter cells) apart from the chromosomes of the cell. Episomal DNA can be either native or heterologous to a cell. Examples of native episomes herein include mitochondrial DNA (mtDNA) and chloroplast DNA. Examples of heterologous episomes herein include plasmids and yeast artificial chromosomes (YACs).

[0070] A "protospacer adjacent motif" (PAM) herein refers to a short sequence that is recognized by an RGEN herein. The sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used, but are typically 2, 3, 4, 5, 6, 7, or 8 nucleotides long, for example.

[0071] The terms "5'-cap" and "7-methylguanylate (m.sup.7G) cap" are used interchangeably herein. A 7-methylguanylate residue is located on the 5' terminus of RNA transcribed by RNA polymerase II (Pol II) in eukaryotes. A capped RNA herein has a 5'-cap, whereas an uncapped RNA does not have such a cap.

[0072] The terminology "uncapped", "not having a 5'-cap", and the like are used interchangeably herein to refer to RNA lacking a 5'-cap and optionally having, for example, a 5'-hydroxyl group instead of a 5'-cap. Uncapped RNA can better accumulate in the nucleus following transcription, since 5'-capped RNA is subject to nuclear export.

[0073] The terms "ribozyme", "ribonucleic acid enzyme" and "self-cleaving ribozyme" are used interchangeably herein. A ribozyme refers to one or more RNA sequences that form secondary, tertiary, and/or quaternary structure(s) that can cleave RNA at a specific site, particularly at a cis-site relative to the ribozyme sequence (i.e., auto-catalytic, or self-cleaving). The general nature of ribozyme nucleolytic activity has been described (e.g., Lilley, Biochem. Soc. Trans. 39:641-646). A "hammerhead ribozyme" (HHR) herein may comprise a small catalytic RNA motif made up of three base-paired stems and a core of highly conserved, non-complementary nucleotides that are involved in catalysis. Pley et al. (Nature 372:68-74) and Hammann et al. (RNA 18:871-885), which are incorporated herein by reference, disclose hammerhead ribozyme structure and activity. A hammerhead ribozyme herein may comprise a "minimal hammerhead" sequence as disclosed by Scott et al. (Cell 81:991-1002, incorporated herein by reference), for example.

[0074] The terms "targeting", "gene targeting", "DNA targeting", "editing", "gene editing" and "DNA editing" are used interchangeably herein. DNA targeting herein may be the specific introduction of an indel, knock-out, or knock-in at a particular DNA sequence, such as in a chromosome or episome of a cell. In general, DNA targeting can be performed herein by cleaving one or both strands at a specific DNA sequence in a cell with a Cas protein associated with a suitable RNA component. Such DNA cleavage, if a double-strand break (DSB), can prompt NHEJ processes which can lead to indel formation at the target site. Also, regardless of whether the cleavage is a single-strand break (SSB) or DSB, HR processes can be prompted if a suitable donor DNA polynucleotide is provided at the DNA nick or cleavage site. Such an HR process can be used to introduce a knock-out or knock-in at the target site, depending on the sequence of the donor DNA polynucleotide. Alternatively, DNA targeting herein can refer to specific association of a Cas/RNA component complex herein to a target DNA sequence, where the Cas protein does or does not cut a DNA strand (depending on the status of the Cas protein's endonucleolytic domains).

[0075] The term "indel" herein refers to an insertion or deletion of a nucleotide base or bases in a target DNA sequence in a chromosome or episome. Such an insertion or deletion may be of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more bases, for example. An indel in certain embodiments can be even larger, at least about 20, 30, 40, 50, 60, 70, 80, 90, or 100 bases. If an indel is introduced within an open reading frame (ORF) of a gene, oftentimes the indel disrupts wild type expression of protein encoded by the ORF by creating a frameshift mutation.

[0076] The terms "knock-out", "gene knock-out" and "genetic knock-out" are used interchangeably herein. A knock-out represents a DNA sequence of a cell herein that has been rendered partially or completely inoperative by targeting with a Cas protein; such a DNA sequence prior to knock-out could have encoded an amino acid sequence, or could have had a regulatory function (e.g., promoter), for example. A knock-out may be produced by an indel (by NHEJ, prompted by Cas-mediated cleavage), or by specific removal of sequence (by HR, prompted by Cas-mediated cleavage or nicking, when a suitable donor DNA polynucleotide is also used), that reduces or completely destroys the function of sequence at, adjoining, or near the targeting site. A knocked out DNA polynucleotide sequence herein can alternatively be characterized as being partially or totally disrupted or downregulated, for example.

[0077] The terms "knock-in", "gene knock-in" and "genetic knock-in" are used interchangeably herein. A knock-in represents the replacement or insertion of a DNA sequence at a specific DNA sequence in a cell by targeting with a Cas protein (by HR, prompted by Cas-mediated cleavage or nicking, when a suitable donor DNA polynucleotide is also used). Examples of knock-ins are a specific insertion of a heterologous amino acid coding sequence in a coding region of a gene, or a specific insertion of a transcriptional regulatory element in a genetic locus.

[0078] The terms "donor polynucleotide", "donor DNA", "targeting polynucleotide" and "targeting DNA" are used interchangeably herein. A donor polynucleotide refers to a DNA sequence that comprises at least one sequence that is homologous to a sequence at or near a DNA target site (e.g., a sequence specifically targeted by a Cas protein herein). A suitable donor polynucleotide is able to undergo HR with a DNA target site if the target site contains a SSB or DSB (such as can be introduced using certain Cas proteins herein associated with an appropriate RNA component). A "homologous sequence" within a donor polynucleotide herein can, for example, comprise or consist of a sequence of at least about 25 nucleotides, for example, having 100% identity with a sequence at or near a target site, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a sequence at or near a target site.

[0079] In certain embodiments, a donor DNA polynucleotide can have two homologous sequences separated by a sequence (or base pair) that is heterologous to sequence at a target site. These two homologous sequences of such a donor polynucleotide can be referred to as "homology arms", which flank the heterologous sequence. HR between a target site and a donor polynucleotide with two homology arms typically results in the replacement of a sequence at the target site with the heterologous sequence of the donor polynucleotide (target site sequence located between DNA sequences homologous to the homology arms of the donor polynucleotide is replaced by the heterologous sequence of the donor polynucleotide). In a donor polynucleotide with two homology arms, the arms can be separated by 1 or more nucleotides (i.e., the heterologous sequence in the donor polynucleotide can be at least 1 nucleotide in length). Various HR procedures that can be performed in a cell herein are disclosed, for example, in DNA Recombination: Methods and Protocols: 1st Edition (H. Tsubouchi, Ed., Springer-Verlag, New York, 2011), which is incorporated herein by reference.

[0080] The terms "cell-penetrating peptide" (CPP) and "protein transduction domain" (PTD) are used interchangeably herein. A CPP refers to a peptide, typically of about 5-60 amino acid residues in length, that can facilitate cellular uptake of molecular cargo, particularly one or more RGEN protein components herein (e.g., Cas9 protein). Such protein cargo can be associated with one or more CPPs through covalent or non-covalent linkage. A CPP can also be characterized in certain embodiments as being able to facilitate the movement or traversal of molecular cargo across/through one or more of a lipid bilayer, micelle, cell membrane, organelle membrane, vesicle membrane, or cell wall. A CPP herein can be cationic, amphipathic, or hydrophobic in certain embodiments. Examples of CPPs useful herein, and further description of CPPs in general, are disclosed in Schmidt et al. (FEBS Lett. 584:1806-1813), Holm et al. (Nature Protocols 1:1001-1005), Yandek et al. (Biophys. J. 92:2434-2444), Morris et al. (Nat. Biotechnol. 19:1173-1176), and U.S. Patent Appl. Publ. No. 2014/0068797, which are all incorporated herein by reference.

[0081] A "cationic", or "polycationic", CPP herein refers to a CPP having a high relative abundance (at least 60%) of positively charged amino acids such as lysine (K), arginine (R), and/or histidine (H).

[0082] An "amphipathic", or "amphiphilic", CPP herein refers to a CPP with an amino acid sequence containing an alternating pattern of polar/charged residues and non-polar, hydrophobic residues. An amphipathic CPP can alternatively be characterized as possessing both hydrophilic and lipophilic properties.

[0083] A "hydrophobic", or "lipophilic", CPP herein contains mostly, or only, non-polar residues with low net charge and/or hydrophobic amino acid groups.

[0084] The terms "covalently linked", "covalently attached", "covalently associated", "covalent linkage", "covalent interaction" and the like are used interchangeably herein. A covalent linkage herein can be via a peptide bond(s) or chemical crosslink(s), for example. A covalent linkage can be direct, for example, where there is a covalent link directly between (directly linking) an RGEN protein component and a CPP (e.g., there is a chemical bond [sharing of electrons] between an atom of an RGEN protein component and an atom of a CPP). A covalent linkage can alternatively be indirect, for example, where an RGEN protein component and a CPP are linked to each other through at least one intermediary factor. Such an intermediary factor, or group of intermediary factors that are themselves covalently linked together, is covalently linked to the RGEN protein component and CPP. Thus, an intermediary factor or group thereof can be characterized as being a bridge between an RGEN protein component and a CPP.

[0085] The terms "fusion protein", "protein fusion", "chimeric protein" and the like are used interchangeably herein. A fusion protein herein contains at least two different (heterologous) amino acid sequences linked together within a single polypeptide. Fusion proteins are typically produced by genetic engineering processes in which DNA sequences encoding different amino acid sequences are joined together to encode a single protein containing the different amino acid sequences. Examples of fusion proteins herein include RGEN protein-CPP fusions (RGEN protein amino acid sequence fused to one or more CPP amino acid sequences).

[0086] The terms "non-covalently linked", "non-covalently attached", "non-covalently associated", "non-covalent linkage", "non-covalent interaction" and the like are used interchangeably herein. A non-covalent linkage herein refers to an interaction between atoms in which electrons are not shared. This type of interaction is weaker than a covalent linkage. Hydrophobic interactions represent an example of a non-covalent linkage that may occur between an RGEN protein component and one or more CPPs. Other examples of non-covalent linkages that may apply herein include electrostatic forces (e.g., ionic, hydrogen bonding) and Van der Waals forces (London Dispersion forces).

[0087] An "RGEN protein-CPP complex" as used herein refers to a complex between a protein component of an RGEN and at least one CPP, where the RGEN and CPP interact via covalent or non-covalent linkage. Both RGEN and CPP components in this complex typically retain all of, or some of (e.g., at least 50%), their respective activity/function as disclosed herein. For example, in embodiment in which the RGEN protein component is Cas9, the Cas9 in a Cas9-CPP complex is capable of associating with a suitable RNA component (e.g., gRNA) and targeting the Cas9-CPP complex to a DNA target site in a cell.

[0088] The terms "traverse", "travel through", "cross through", "goes across" and the like are used interchangeably herein.

[0089] The terms "cell membrane", "plasma membrane", and "cytoplasmic membrane" are used interchangeably herein and refer to a biological membrane that separates the interior of a cell from its exterior. A cell membrane typically comprises a phospholipid bilayer with proteins embedded therein. Among several other functions, a cell membrane can serve as an attachment surface for extracellular structures such as cell wall or glycocalyx structures. Detailed information regarding cell membrane lipid bilayers is provided in Molecular Bioloqy of the Cell. 4th Edition (B. Alberts et al., Eds., Garland Science, New York, 2002), which is incorporated herein by reference.

[0090] The term "cell wall" herein refers to a tough, flexible (but sometimes fairly rigid) layer that surrounds some types of non-mammalian cells (e.g., bacteria, plants, algae, fungi such as yeast). It is located outside the cell membrane and provides structural support and protection to cells. A major function of a cell wall in certain embodiments is to help maintain cell osmotic pressure. Fungal cell (e.g., yeast cell) walls generally comprise chitin, and algal cells walls generally comprise glycoproteins and polysaccharides. Plant cell walls generally comprise mostly polysaccharides with lesser amounts of other components (e.g., phenolic esters, structural proteins). "Primary cell wall" and/or "secondary cell wall" may be used to characterize a plant cell wall, where the secondary wall is located inside the primary wall. Lignin is a major component of secondary walls. Bacterial cell walls generally comprise peptidoglycan as the main constituent. In certain aspects, such as in bacteria, a cell wall can further comprise at its outer layer a glycocalyx, which is generally a coat of polysaccharides.

[0091] The term "leucine zipper domain" herein refers to a dimerization domain characterized by the presence of a leucine residue every seventh residue in a stretch of approximately 35 residues. Leucine zipper domains form dimers held together by an alpha-helical coiled coil. A coiled coil has 3.5 residues per turn, which means that every seventh residue occupies an equivalent position with respect to the helix axis. The regular array of leucines inside the coiled coil stabilizes the structure by hydrophobic and Van der Waals interactions.

[0092] The terms "percent by volume", "volume percent", "vol %" and "v/v %" are used interchangeably herein. The percent by volume of a solute in a solution can be determined using the formula: [(volume of solute)/(volume of solution)].times.100%.

[0093] The terms "percent by weight", "weight percentage (wt %)" and "weight-weight percentage (% w/w)" are used interchangeably herein. Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution.

[0094] The terms "polynucleotide", "polynucleotide sequence", and "nucleic acid sequence" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of DNA or RNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Nucleotides (ribonucleotides or deoxyribonucleotides) can be referred to by a single letter designation as follows: "A" for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for cytidylate or deoxycytidylate (for RNA or DNA, respectively), "G" for guanylate or deoxyguanylate (for RNA or DNA, respectively), "U" for uridylate (for RNA), "T" for deoxythymidylate (for DNA), "R" for purines (A or G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, "W" for A or T, and "N" for any nucleotide (e.g., N can be A, C, T, or G, if referring to a DNA sequence; N can be A, C, U, or G, if referring to an RNA sequence). Any RNA sequence (e.g., crRNA, tracrRNA, gRNA) disclosed herein may be encoded by a suitable DNA sequence.

[0095] The term "isolated" as used herein refers to a polynucleotide or polypeptide molecule that has been completely or partially purified from its native source. In some instances, the isolated polynucleotide or polypeptide molecule is part of a greater composition, buffer system or reagent mix. For example, the isolated polynucleotide or polypeptide molecule can be comprised within a cell or organism in a heterologous manner. Compositions herein comprising a protein component of an RGEN and a cell-penetrating peptide can be considered isolated compositions. These compositions contain heterologous components and do not occur in nature.

[0096] The term "gene" as used herein refers to a DNA polynucleotide sequence that expresses an RNA (RNA is transcribed from the DNA polynucleotide sequence) from a coding region, which RNA can be a messenger RNA (encoding a protein) or a non-protein-coding RNA (e.g., a crRNA, tracrRNA, or gRNA herein). A gene may refer to the coding region alone, or may include regulatory sequences upstream and/or downstream to the coding region (e.g., promoters, 5'-untranslated regions, 3'-transcription terminator regions). A coding region encoding a protein can alternatively be referred to herein as an "open reading frame" (ORF). A gene that is "native" or "endogenous" refers to a gene as found in nature with its own regulatory sequences; such a gene is located in its natural location in the genome of a host cell. A "chimeric" gene refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature (i.e., the regulatory and coding regions are heterologous with each other). Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A "foreign" or "heterologous" gene refers to a gene that is introduced into the host organism by gene transfer. Foreign/heterologous genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes. The polynucleotide sequences in certain embodiments disclosed herein are heterologous. A "transgene" is a gene that has been introduced into the genome by a gene delivery procedure (e.g., transformation). A "codon-optimized" open reading frame has its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.

[0097] A "mutated gene" is a gene that has been altered through human intervention. Such a "mutated gene" has a sequence that differs from the sequence of the corresponding non-mutated gene by at least one nucleotide addition, deletion, or substitution. In certain embodiments of the disclosure, the mutated gene comprises an alteration that is made by using a guide polynucleotide/Cas endonuclease system as disclosed herein. A mutated plant is a plant comprising at least one mutated gene.

[0098] A "non-native" amino acid sequence or polynucleotide sequence comprised in a cell or organism herein does not occur in a native (natural) counterpart of such cell or organism.

[0099] "Regulatory sequences" as used herein refer to nucleotide sequences located upstream of a gene's transcription start site (e.g., promoter), 5' untranslated regions, and 3' non-coding regions, and which may influence the transcription, processing or stability, or translation of an RNA transcribed from the gene. Regulatory sequences herein may include promoters, enhancers, silencers, 5' untranslated leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures, and other elements involved in regulation of gene expression. One or more regulatory elements herein may be heterologous to a coding region herein.

[0100] A "promoter" as used herein refers to a DNA sequence capable of controlling the transcription of RNA from a gene. In general, a promoter sequence is upstream of the transcription start site of a gene. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. Promoters that cause a gene to be expressed in a cell at most times under all circumstances are commonly referred to as "constitutive promoters". One or more promoters herein may be heterologous to a coding region herein.

[0101] A "strong promoter" as used herein refers to a promoter that can direct a relatively large number of productive initiations per unit time, and/or is a promoter driving a higher level of gene transcription than the average transcription level of the genes in a cell.

[0102] A plant promoter is a promoter capable of initiating transcription in a plant cell; for a review of plant promoters, see Potenza et al., (2004) In Vitro Cell Dev Biol 40:1-22. Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al., (1985) Nature 313:810-2); rice actin (McElroy et al., (1990) Plant Cell 2:163-71); ubiquitin (Christensen et al., (1989) Plant Mol Biol 12:619-32; Christensen et al., (1992) Plant Mol Biol 18:675-89); pEMU (Last et al., (1991) Theor Appl Genet 81:581-8); MAS (Velten et al., (1984) EMBO J 3:2723-30); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters are described in, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142 and 6,177,611. In some examples, an inducible promoter may be used. Pathogen-inducible promoters induced following infection by a pathogen include, but are not limited to those regulating expression of PR proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc.

[0103] Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. The promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters include, but are not limited to, the maize In2-2 promoter, activated by benzene sulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-II-27, WO93/01294), activated by hydrophobic electrophilic compounds used as pre-emergent herbicides, and the tobacco PR-la promoter (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7) activated by salicylic acid. Other chemical-regulated promoters include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter (Schena et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-5; McNellis et al., (1998) Plant J 14:247-257); tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156).

[0104] Tissue-preferred promoters can be utilized to target enhanced expression within a particular plant tissue. Tissue-preferred promoters include, for example, Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Hansen et al., (1997) Mol Gen Genet 254:337-43; Russell et al., (1997) Transgenic Res 6:157-68; Rinehart et al., (1996) Plant Physiol 112:1331-41; Van Camp et al., (1996) Plant Physiol 112:525-35; Canevascini et al., (1996) Plant Physiol 112:513-524; Lam, (1994) Results Probl Cell Differ 20:181-96; and Guevara-Garcia et al., (1993) Plant J 4:495-505. Leaf-preferred promoters include, for example, Yamamoto et al., (1997) Plant J 12:255-65; Kwon et al., (1994) Plant Physiol 105:357-67; Yamamoto et al., (1994) Plant Cell Physiol 35:773-8; Gotor et al., (1993) Plant J 3:509-18; Orozco et al., (1993) Plant Mol Biol 23:1129-38; Matsuoka et al., (1993) Proc. Natl. Acad. Sci. USA 90:9586-90; Simpson et al., (1958) EMBO J 4:2723-9; Timko et al., (1988) Nature 318:57-8. Root-preferred promoters include, for example, Hire et al., (1992) Plant Mol Biol 20:207-18 (soybean root-specific glutamine synthase gene); Miao et al., (1991) Plant Cell 3:11-22 (cytosolic glutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol Biol 14:433-43 (root-specific promoter of A. tumefaciens mannopine synthase (MAS)); Bogusz et al., (1990) Plant Cell 2:633-41 (root-specific promoters isolated from Parasponia andersonii and Trema tomentosa); Leach and Aoyagi, (1991) Plant Sci 79:69-76 (A. rhizogenes rolC and rolD root-inducing genes); Teeri et al., (1989) EMBO J 8:343-50 (Agrobacterium wound-induced TR1' and TR2' genes); VfENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol Biol 29:759-72); and rolB promoter (Capana et al., (1994) Plant Mol Biol 25:681-91; phaseolin gene (Murai et al., (1983) Science 23:476-82; Sengopta-Gopalen et al., (1988) Proc. Natl. Acad. Sci. USA 82:3320-4). See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179.

[0105] Seed-preferred promoters include both seed-specific promoters active during seed development, as well as seed-germinating promoters active during seed germination. See, Thompson et al., (1989) BioEssays 10:108. Seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1-phosphate synthase); (WO00/11177; and U.S. Pat. No. 6,225,529). For dicots, seed-preferred promoters include, but are not limited to, bean beta-phaseolin, napin, beta-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-preferred promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin, and nuc1. See also, WO00/12733, where seed-preferred promoters from END1 and END2 genes are disclosed.

[0106] The terms "3' non-coding sequence", "transcription terminator" and "terminator" as used herein refer to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.

[0107] The term "cassette" as used herein refers to a promoter operably linked to a DNA sequence encoding a protein-coding RNA or non-protein-coding RNA. A cassette may optionally be operably linked to a 3' non-coding sequence.

[0108] The terms "upstream" and "downstream" as used herein with respect to polynucleotides refer to "5' of" and "3' of", respectively.

[0109] The term "expression" as used herein refers to (i) transcription of RNA (e.g., mRNA or a non-protein coding RNA such as crRNA, tracrRNA, or gRNA) from a coding region, or (ii) translation of a polypeptide from mRNA.

[0110] When used to describe the expression of a gene or polynucleotide sequence, the terms "down-regulation", "disruption", "inhibition", "inactivation", and "silencing" are used interchangeably herein to refer to instances when the transcription of the polynucleotide sequence is reduced or eliminated. This results in the reduction or elimination of RNA transcripts from the polynucleotide sequence, which results in a reduction or elimination of protein expression derived from the polynucleotide sequence (if the gene comprised an ORF). Alternatively, down-regulation can refer to instances where protein translation from transcripts produced by the polynucleotide sequence is reduced or eliminated. Alternatively still, down-regulation can refer to instances where a protein expressed by the polynucleotide sequence has reduced activity. The reduction in any of the above processes (transcription, translation, protein activity) in a cell can be by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% relative to the transcription, translation, or protein activity of a suitable control cell. Down-regulation can be the result of a targeting event as disclosed herein (e.g., indel, knock-out), for example.

[0111] The terms "control cell" and "suitable control cell" are used interchangeably herein and may be referenced with respect to a cell in which a particular modification (e.g., over-expression of a polynucleotide, down-regulation of a polynucleotide) has been made (i.e., an "experimental cell"). A control cell may be any cell that does not have or does not express the particular modification of the experimental cell. Thus, a control cell may be an untransformed wild type cell or may be genetically transformed but does not express the genetic transformation. For example, a control cell may be a direct parent of the experimental cell, which direct parent cell does not have the particular modification that is in the experimental cell. Alternatively, a control cell may be a parent of the experimental cell that is removed by one or more generations. Alternatively still, a control cell may be a sibling of the experimental cell, which sibling does not comprise the particular modification that is present in the experimental cell.

[0112] The term "increased" as used herein may refer to a quantity or activity that is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% more than the quantity or activity for which the increased quantity or activity is being compared. The terms "increased", "elevated", "enhanced", "greater than", and "improved" are used interchangeably herein. The term "increased" can be used to characterize the expression of a polynucleotide encoding a protein, for example, where "increased expression" can also mean "over-expression".

[0113] The term "operably linked" as used herein refers to the association of two or more nucleic acid sequences such that that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence. That is, the coding sequence is under the transcriptional control of the promoter. Coding sequences can be operably linked to regulatory sequences, for example. Also, for example, a crRNA can be operably linked (fused to) a tracrRNA herein such that the tracrRNA mate sequence of the crRNA anneals with 5' sequence of the tracrRNA. Such operable linkage may comprise a suitable loop-forming sequence such as GAAA (SEQ ID NO:36), CAAA (SEQ ID NO:37), or AAAG (SEQ ID NO:38). Also, for example, an RGEN can be operably linked (fused to) one or more CPPs.

[0114] The term "recombinant" as used herein refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.

[0115] Methods for preparing recombinant constructs/vectors herein (e.g., a DNA polynucleotide encoding an RNA component cassette herein, or a DNA polynucleotide encoding a Cas protein or Cas-CPP fusion protein herein) can follow standard recombinant DNA and molecular cloning techniques as described by J. Sambrook and D. Russell (Molecular Cloninq: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); T. J. Silhavy et al. (Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1984); and F. M. Ausubel et al. (Short Protocols in Molecular Biology, 5th Ed. Current Protocols, John Wiley and Sons, Inc., NY, 2002), for example.

[0116] The term "transformation" as used herein refers to the transfer of a nucleic acid molecule into a host organism or host cell by any method. A nucleic acid molecule that has been transformed into an organism/cell may be one that replicates autonomously in the organism/cell, or that integrates into the genome of the organism/cell, or that exists transiently in the cell without replicating or integrating. Non-limiting examples of nucleic acid molecules suitable for transformation are disclosed herein, such as plasmids and linear DNA molecules.

[0117] A "transgenic plant" herein includes, for example, a plant which comprises within its genome a heterologous polynucleotide introduced by a transformation step. The heterologous polynucleotide can be stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. A transgenic plant can also comprise more than one heterologous polynucleotide within its genome. Each heterologous polynucleotide may confer a different trait to the transgenic plant. Transgenic plant material can include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The alterations of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods, by genome editing procedures that does not result in an insertion of a foreign polynucleotide, or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation are not intended to be regarded as transgenic.

[0118] A "phenotypic marker" is a screenable or selectable marker that includes visual markers and selectable markers whether it is a positive or negative selectable marker. Any phenotypic marker can be used. Specifically, a selectable or screenable marker comprises a DNA segment that allows one to identify, or select for or against a molecule or a cell that contains it, often under particular conditions. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like.

[0119] Examples of selectable markers include, but are not limited to, DNA segments that comprise restriction enzyme sites; DNA segments that encode products which provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT); DNA segments that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA segments that encode products which can be readily identified (e.g., phenotypic markers such as beta-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), and cell surface proteins); the generation of new primer sites for PCR (e.g., the juxtaposition of two DNA sequence not previously juxtaposed), the inclusion of DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; and, the inclusion of a DNA sequences required for a specific modification (e.g., methylation) that allows its identification.

[0120] Additional selectable markers include genes that confer resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See for example, Yarranton, (1992) Curr Opin Biotech 3:506-11; Christopherson et al., (1992) Proc. Natl. Acad. Sci. USA 89:6314-8; Yao et al., (1992) Cell 71:63-72; Reznikoff, (1992) Mol Microbiol 6:2419-22; Hu et al., (1987) Cell 48:555-66; Brown et al., (1987) Cell 49:603-12; Figge et al., (1988) Cell 52:713-22; Deuschle et al., (1989) Proc. Natl. Acad. Sci. USA 86:5400-4; Fuerst et al., (1989) Proc. Natl. Acad. Sci. USA 86:2549-53; Deuschle et al., (1990) Science 248:480-3; Gossen, (1993) Ph.D. Thesis, University of Heidelberg; Reines et al., (1993) Proc. Natl. Acad. Sci. USA 90:1917-21; Labow et al., (1990) Mol Cell Biol 10:3343-56; Zambretti et al., (1992) Proc. Natl. Acad. Sci. USA 89:3952-6; Baim et al., (1991) Proc. Natl. Acad. Sci. USA 88:5072-6; Wyborski et al., (1991) Nucleic Acids Res 19:4647-53; Hillen and Wissman, (1989) Topics Mol Struc Biol 10:143-62; Degenkolb et al., (1991) Antimicrob Agents Chemother 35:1591-5; Kleinschnidt et al., (1988) Biochemistry 27:1094-104; Bonin, (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-51; Oliva et al., (1992) Antimicrob Agents Chemother 36:913-9; Hlavka et al., (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al., (1988) Nature 334:721-4.

[0121] The terms "sequence identity" or "identity" as used herein with respect to polynucleotide or polypeptide sequences refer to the nucleic acid residues or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, "percentage of sequence identity" or "percent identity" refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. It would be understood that, when calculating sequence identity between a DNA sequence and an RNA sequence, T residues of the DNA sequence align with, and can be considered "identical" with, U residues of the RNA sequence. For purposes of determining percent complementarity of first and second polynucleotides, one can obtain this by determining (i) the percent identity between the first polynucleotide and the complement sequence of the second polynucleotide (or vice versa), for example, and/or (ii) the percentage of bases between the first and second polynucleotides that would create canonical Watson and Crick base pairs.

[0122] The Basic Local Alignment Search Tool (BLAST) algorithm, which is available online at the National Center for Biotechnology Information (NCBI) website, may be used, for example, to measure percent identity between or among two or more of the polynucleotide sequences (BLASTN algorithm) or polypeptide sequences (BLASTP algorithm) disclosed herein. Alternatively, percent identity between sequences may be performed using a Clustal algorithm (e.g., ClustalW or ClustalV). For multiple alignments using a Clustal method of alignment, the default values may correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using a Clustal method may be KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids, these parameters may be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. Alternatively still, percent identity between sequences may be performed using an EMBOSS algorithm (e.g., needle) with parameters such as GAP OPEN=10, GAP EXTEND=0.5, END GAP PENALTY=false, END GAP OPEN=10, END GAP EXTEND=0.5 using a BLOSUM matrix (e.g., BLOSUM62).

[0123] Herein, a first sequence that is "complementary" to a second sequence can alternatively be referred to as being in the "antisense" orientation with the second sequence.

[0124] Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments of the disclosed invention. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used. Alternatively, a variant amino acid sequence or polynucleotide sequence can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. The variant amino acid sequence or polynucleotide sequence has the same function/activity of the disclosed sequence, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of the disclosed sequence.

[0125] All the amino acid residues disclosed herein at each amino acid position of Cas9 proteins herein are examples. Given that certain amino acids share similar structural and/or charge features with each other (i.e., conserved), the amino acid at each position in a Cas9 can be as provided in the disclosed sequences or substituted with a conserved amino acid residue ("conservative amino acid substitution") as follows: [0126] 1. The following small aliphatic, nonpolar or slightly polar residues can substitute for each other: Ala (A), Ser (S), Thr (T), Pro (P), Gly (G); [0127] 2. The following polar, negatively charged residues and their amides can substitute for each other: Asp (D), Asn (N), Glu (E), Gin (Q); [0128] 3. The following polar, positively charged residues can substitute for each other: His (H), Arg (R), Lys (K); [0129] 4. The following aliphatic, nonpolar residues can substitute for each other: Ala (A), Leu (L), lie (I), Val (V), Cys (C), Met (M); and [0130] 5. The following large aromatic residues can substitute for each other: Phe (F), Tyr (Y), Trp (W).

[0131] Advances have been made in expressing protein and RNA components in cells for performing RGEN-mediated DNA targeting therein (e.g., U.S. Provisional Appl. Nos. 61/868,706 and 62/036,652). Such strategies typically have entailed recombinant DNA expression in the target cells. Additional means of providing protein and RNA components in a cell to mediate RGEN-mediated DNA targeting are of interest.

[0132] Embodiments of the disclosed invention concern a composition comprising at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein the RGEN protein component and CPP are covalently or non-covalently linked to each other in an RGEN protein-CPP complex. The RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell.

[0133] Significantly, certain embodiments of the disclosed invention can be used to deliver an RGEN already associated (pre-associated) with an RNA component into a cell. Such embodiments may avoid the need to deliver a DNA construct into cells for expressing an RGEN RNA component, thus averting any potentially unwanted effects of introducing exogenous DNA into cells. The disclosed invention is flexible, however, since in certain other embodiments an RNA component can be provided (e.g., expressed) in a cell into which an RGEN protein-CPP complex is being delivered. An RNA component provided in this manner can associate with an RGEN protein component after delivery/entry of the RGEN protein-CPP complex into the cell. Regardless of the mode of RNA component delivery, an RGEN protein-CPP complex herein is able to associate with an RNA component, forming an RGEN-CPP complex that can target a specific DNA sequence in the cell. Thus, the disclosed invention offers substantial flexibility for providing an RGEN in cells to perform RGEN-mediated DNA targeting.

[0134] Compositions disclosed in certain embodiments comprise at least one protein component of an RGEN. An RGEN herein refers to a complex comprising at least one Cas protein and at least one RNA component. Thus, an RGEN protein component can refer to a Cas protein such as Cas9. Examples of suitable Cas proteins include one or more Cas endonucleases of type I, II, or III CRISPR systems (Bhaya et al., Annu. Rev. Genet. 45:273-297, incorporated herein by reference). A type I CRISPR Cas protein can be a Cas3 or Cas4 protein, for example. A type II CRISPR Cas protein can be a Cas9 protein, for example. A type III CRISPR Cas protein can be a Cas10 protein, for example. A Cas9 protein is used in certain preferred embodiments. A Cas protein in certain embodiments may be a bacterial or archaeal protein. Type I-III CRISPR Cas proteins herein are typically prokaryotic in origin; type I and III Cas proteins can be derived from bacterial or archaeal species, whereas type II Cas proteins (i.e., a Cas9) can be derived from bacterial species, for example. In other embodiments, suitable Cas proteins include one or more of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.

[0135] In other aspects of the disclosed invention, a Cas protein herein can be from any of the following genera: Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Haloarcula, Methanobacteriumn, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thernioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx, Porphvromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myrococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Streptococcus, Treponema, Francisella, or Thermotoga. Alternatively, a Cas protein herein can be encoded, for example, by any of SEQ ID NOs:462-465, 467-472, 474-477, 479-487, 489-492, 494-497, 499-503, 505-508, 510-516, or 517-521 as disclosed in U.S. Appl. Publ. No. 2010/0093617, which is incorporated herein by reference.

[0136] An RGEN protein component can comprise a Cas9 amino acid sequence, for example. An RGEN comprising this type of protein component typically can be characterized as having Cas9 as the endonuclease component of the RGEN. The amino acid sequence of a Cas9 protein herein, as well as certain other Cas proteins herein, may be derived from a Streptococcus (e.g., S. pyogenes, S. pneumoniae, S. thermophilus, S. agalactiae, S. parasanguinis, S. oralis, S. salivarius, S. macacae, S. dysgalactiae, S. anginosus, S. constellatus, S. pseudoporcinus, S. mutans), Listeria (e.g., L. innocua), Spiroplasma (e.g., S. apis, S. syrphidicola), Peptostreptococcaceae, Atopobium, Porphyromonas (e.g., P. catoniae), Prevotella (e.g., P. intermedia), Veillonella, Treponema (e.g., T. socranskii, T. denticola), Capnocytophaga, Finegoldia (e.g., F. magna), Coriobacteriaceae (e.g., C. bacterium), Olsenella (e.g., O. profusa), Haemophilus (e.g., H. sputorum, H. pittmaniae), Pasteurella (e.g., P. bettyae), Olivibacter (e.g., O. sitiensis), Epilithonimonas (e.g., E. tenax), Mesonia (e.g., M. mobilis), Lactobacillus, Bacillus (e.g., B. cereus), Aquimarina (e.g., A. muelleri), Chryseobacterium (e.g., C. palustre), Bacteroides (e.g., B. graminisolvens), Neisseria (e.g., N. meningitidis), Francisella (e.g., F. novicida), or Flavobacterium (e.g., F. frigidarium, F. soli) species, for example. An S. pyogenes Cas9 is preferred in certain aspects herein.

[0137] As another example, a Cas9 protein can be any of the Cas9 proteins disclosed in Chylinski et al. (RNA Biology 10:726-737), which is incorporated herein by reference.

[0138] Accordingly, the sequence of a Cas9 protein herein can comprise, for example, any of the Cas9 amino acid sequences disclosed in GenBank Accession Nos. G3ECR1 (S. thermophilus), WP_026709422, WP_027202655, WP_027318179, WP_027347504, WP_027376815, WP_027414302, WP_027821588, WP_027886314, WP_027963583, WP_028123848, WP_028298935, Q03JI6 (S. thermophilus), EGP66723, EGS38969, EGV05092, EH165578 (S. pseudoporcinus), EIC75614 (S. oralis), EID22027 (S. constellatus), ElJ69711, EJP22331 (S. oralis), EJP26004 (S. anginosus), EJP30321, EPZ44001 (S. pyogenes), EPZ46028 (S. pyogenes), EQL78043 (S. pyogenes), EQL78548 (S. pyogenes), ERL10511, ERL12345, ERL19088 (S. pyogenes), ESA57807 (S. pyogenes), ESA59254 (S. pyogenes), ESU85303 (S. pyogenes), ETS96804, UC75522, EGR87316 (S. dysgalactiae), EGS33732, EGV01468 (S. oralis), EHJ52063 (S. macacae), EID26207 (S. oralis), EID33364, EIG27013 (S. parasanguinis), EJF37476, EJO19166 (Streptococcus sp. BS35b), EJU16049, EJU32481, YP_006298249, ERF61304, ERK04546, ETJ95568 (S. agalactiae), TS89875, ETS90967 (Streptococcus sp. SR4), ETS92439, EUB27844 (Streptococcus sp. BS21), AFJ08616, EUC82735 (Streptococcus sp. CM6), EWC92088, EWC94390, EJP25691, YP_008027038, YP_008868573, AGM26527, AHK22391, AHB36273, Q927P4, G3ECR1, or Q99ZW2 (S. pyogenes), which are incorporated by reference. A variant of any of these Cas9 protein sequences may be used, but should have specific binding activity, and optionally cleavage or nicking activity, toward DNA when associated with an RNA component herein. Such a variant may comprise an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the reference Cas9.

[0139] Alternatively, a Cas9 protein herein can be encoded by any of SEQ ID NOs:462 (S. thermophilus), 474 (S. thermophilus), 489 (S. agalactiae), 494 (S. agalactiae), 499 (S. mutans), 505 (S. pyogenes), or 518 (S. pyogenes) as disclosed in U.S. Appl. Publ. No. 2010/0093617 (incorporated herein by reference), for example. Alternatively still, a Cas9 protein herein can comprise the amino acid sequence of SEQ ID NO:3, or residues 1-1368, 2-1368, or 2-1379, of SEQ ID NO:3, for example. Alternatively still, a Cas9 protein may comprise an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing amino acid sequences, for example. Such a variant Cas9 protein should have specific binding activity, and optionally cleavage or nicking activity, toward DNA when associated with an RNA component herein.

[0140] The origin of a Cas protein used herein (e.g., Cas9) may be from the same species from which the RNA component(s) is derived, or it can be from a different species. For example, an RGEN comprising a Cas9 protein derived from a Streptococcus species (e.g., S. pyogenes or S. thermophilus) may be complexed with at least one RNA component having a sequence (e.g., crRNA repeat sequence, tracrRNA sequence) derived from the same Streptococcus species. Alternatively, the origin of a Cas protein used herein (e.g., Cas9) may be from a different species from which the RNA component(s) is derived (the Cas protein and RNA component(s) may be heterologous to each other); such heterologous Cas/RNA component RGENs should have DNA targeting activity.

[0141] Determining binding activity and/or endonucleolytic activity of a Cas protein herein toward a specific target DNA sequence may be assessed by any suitable assay known in the art, such as disclosed in U.S. Pat. No. 8,697,359, which is disclosed herein by reference. A determination can be made, for example, by expressing a Cas protein and suitable RNA component in a cell, and then examining the predicted DNA target site for the presence of an indel (a Cas protein in this particular assay would typically have complete endonucleolytic activity [double-strand cleaving activity]). Examining for the presence of an alteration/modification (e.g., indel) at the predicted target site could be done via a DNA sequencing method or by inferring alteration/modification formation by assaying for loss of function of the target sequence, for example. In another example, Cas protein activity can be determined by expressing a Cas protein and suitable RNA component in a cell that has been provided a donor DNA comprising a sequence homologous to a sequence in at or near the target site. The presence of donor DNA sequence at the target site (such as would be predicted by successful HR between the donor and target sequences) would indicate that targeting occurred. In still another example, Cas protein activity can be determined using an in vitro assay in which a Cas protein and suitable RNA component are mixed together along with a DNA polynucleotide containing a suitable target sequence. This assay can be used to detect binding (e.g., gel-shift) by Cas proteins lacking cleavage activity, or cleavage by Cas proteins that are endonucleolytically competent.

[0142] A Cas protein herein such as a Cas9 can further comprise a heterologous nuclear localization sequence (NLS) in certain aspects. A heterologous NLS amino acid sequence herein may be of sufficient strength to drive accumulation of a Cas protein, or Cas protein-CPP complex, in a detectable amount in the nucleus of a cell herein, for example. An NLS may comprise one (monopartite) or more (e.g., bipartite) short sequences (e.g., 2 to 20 residues) of basic, positively charged residues (e.g., lysine and/or arginine), and can be located anywhere in a Cas amino acid sequence but such that it is exposed on the protein surface. An NLS may be operably linked to the N-terminus or C-terminus of a Cas protein herein, for example. Two or more NLS sequences can be linked to a Cas protein, for example, such as on both the N- and C-termini of a Cas protein. Non-limiting examples of suitable NLS sequences herein include those disclosed in U.S. Pat. Nos. 6,660,830 and 7,309,576 (e.g., Table 1 therein), which are both incorporated herein by reference. Another example of an NLS useful herein includes amino acid residues 1373-1379 of SEQ ID NO:3. A Cas protein as disclosed herein can be fused with a CPP (an example of a Cas protein covalently linked to a CPP), for example. It would be understood that such a Cas-CPP fusion protein can also comprise an NLS as described above. It would also be understood that, in embodiments in which a Cas protein is fused with an amino acid sequence targeting a different organelle (e.g., mitochondria), such a Cas protein typically would not contain an NLS.

[0143] In certain embodiments, a Cas protein and its respective RNA component (e.g., crRNA) that directs DNA-specific targeting by the Cas protein can be heterologous to a cell, in particular a non-prokaryotic cell. The heterologous nature of these RGEN components is due to that Cas proteins and their respective RNA components are only known to exist in prokaryotes (bacteria and archaea).

[0144] In some embodiments, a Cas protein is part of a fusion protein comprising one or more heterologous protein domains (e.g., 1, 2, 3, or more domains in addition to the Cas protein). These embodiments can encompass a Cas protein that is covalently linked to a CPP and one or more additional heterologous amino acid sequences, for example. Other embodiments can encompass a Cas protein that is covalently linked to one or more additional heterologous amino acid sequences not including a CPP, for example (a CPP would be non-covalently linked to a Cas fusion protein in such embodiments). A fusion protein comprising a Cas protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains, such as between Cas and a first heterologous domain. Examples of protein domains that may be fused to a Cas protein herein include, without limitation, epitope tags (e.g., histidine [His, poly-histidine], V5, FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]), reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], beta-galactosidase, beta-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]), and domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity (e.g., VP16 or VP64), transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. A Cas protein in other embodiments may be in fusion with a protein that binds DNA molecules or other molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, and herpes simplex virus (HSV) VP16. Additional domains that may be part of a fusion protein comprising a Cas protein herein are disclosed in U.S. Patent Appl. Publ. No. 2011/0059502, which is incorporated herein by reference. In certain embodiments in which a Cas protein is fused to a heterologous protein (e.g., a transcription factor), the Cas protein has DNA recognition and binding activity (when in complex with a suitable RNA component herein), but no DNA nicking or cleavage activity. A Cas protein as disclosed herein can be fused with a CPP (an example of a Cas protein covalently linked to a CPP), for example. It would be understood that such a Cas-CPP fusion protein can also be fused with one or more heterologous domains as described above, if desired.

[0145] Other examples of heterologous domains that can be linked to a Cas protein herein include amino acid sequences targeting the protein to a particular organelle (i.e., localization signal). Examples of organelles that can be targeted include mitochondria and chloroplasts. Typically, such targeting domains are used instead of an NLS when targeting extra-nuclear DNA sites. A mitochondrial targeting sequence (MTS) can be situated at or near the N-terminus of a Cas protein, for example. MTS examples are disclosed in U.S. Patent Appl. Publ. Nos. 2007/0011759 and 2014/0135275, which are incorporated herein by reference. A chloroplast targeting sequence can be as disclosed in U.S. Patent Appl. Publ. No. 2010/0192262 or 2012/0042412, for example, which are incorporated herein by reference.

[0146] The protein component of an RGEN can be associated with at least one RNA component (thereby constituting a complete RGEN) that comprises a sequence complementary to a target site sequence on a chromosome or episome in a cell, for example. The RGEN in such embodiments can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence. An RGEN can cleave one or both strands of a DNA target sequence, for example. An RGEN can cleave both strands of a DNA target sequence in another example. It would be understood that in all these embodiments, an RGEN protein component can be covalently or non-covalently linked to at least one CPP in an RGEN protein-CPP complex. The association of an RGEN protein-CPP complex with an RNA component herein can be characterized as forming an RGEN-CPP complex. Any disclosure herein regarding an RGEN can likewise apply to the RGEN component of an RGEN-CPP complex, unless otherwise noted.

[0147] An RGEN herein that can cleave both strands of a DNA target sequence typically comprises a Cas protein that has all of its endonuclease domains in a functional state (e.g., wild type endonuclease domains or variants thereof retaining some or all activity in each endonuclease domain). Thus, a wild type Cas protein (e.g., a Cas9 protein disclosed herein), or a variant thereof retaining some or all activity in each endonuclease domain of the Cas protein, is a suitable example of an RGEN that can cleave both strands of a DNA target sequence. A Cas9 protein comprising functional RuvC and HNH nuclease domains is an example of a Cas protein that can cleave both strands of a DNA target sequence. An RGEN herein that can cleave both strands of a DNA target sequence typically cuts both strands at the same position such that blunt-ends (i.e., no nucleotide overhangs) are formed at the cut site.

[0148] An RGEN herein that can cleave one strand of a DNA target sequence can be characterized herein as having nickase activity (e.g., partial cleaving capability). A Cas nickase (e.g., Cas9 nickase) herein typically comprises one functional endonuclease domain that allows the Cas to cleave only one strand (i.e., make a nick) of a DNA target sequence. For example, a Cas9 nickase may comprise (i) a mutant, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., wild type HNH domain). As another example, a Cas9 nickase may comprise (i) a functional RuvC domain (e.g., wild type RuvC domain) and (ii) a mutant, dysfunctional HNH domain.

[0149] Non-limiting examples of Cas9 nickases suitable for use herein are disclosed by Gasiunas et al. (Proc. Natl. Acad. Sci. U.S.A. 109:E2579-E2586), Jinek et al. (Science 337:816-821), Sapranauskas et al. (Nucleic Acids Res. 39:9275-9282) and in U.S. Patent Appl. Publ. No. 2014/0189896, which are incorporated herein by reference. For example, a Cas9 nickase herein can comprise an S. thermophilus Cas9 having an Asp-31 substitution (e.g., Asp-31-Ala) (an example of a mutant RuvC domain), or a His-865 substitution (e.g., His-865-Ala), Asn-882 substitution (e.g., Asn-882-Ala), or Asn-891 substitution (e.g., Asn-891-Ala) (examples of mutant HNH domains). Also for example, a Cas9 nickase herein can comprise an S. pyogenes Cas9 having an Asp-10 substitution (e.g., Asp-10-Ala), Glu-762 substitution (e.g., Glu-762-Ala), or Asp-986 substitution (e.g., Asp-986-Ala) (examples of mutant RuvC domains), or a His-840 substitution (e.g., His-840-Ala), Asn-854 substitution (e.g., Asn-854-Ala), or Asn-863 substitution (e.g., Asn-863-Ala) (examples of mutant HNH domains). Regarding S. pyogenes Cas9, the three RuvC subdomains are generally located at amino acid residues 1-59, 718-769 and 909-1098, respectively, and the HNH domain is located at amino acid residues 775-908 (Nishimasu et al., Cell 156:935-949).

[0150] A Cas9 nickase herein can be used for various purposes in cells, if desired. For example, a Cas9 nickase can be used to stimulate HR at or near a DNA target site sequence with a suitable donor polynucleotide. Since nicked DNA is not a substrate for NHEJ processes, but is recognized by HR processes, nicking DNA at a specific target site should render the site more receptive to HR with a suitable donor polynucleotide.

[0151] As another example, a pair of Cas9 nickases can be used to increase the specificity of DNA targeting. In general, this can be done by providing two Cas9 nickases that, by virtue of being associated with RNA components with different guide sequences, target and nick nearby DNA sequences on opposite strands in the region for desired targeting. Such nearby cleavage of each DNA strand creates a DSB (i.e., a DSB with single-stranded overhangs), which is then recognized as a substrate for NHEJ (leading to indel formation) or HR (leading to recombination with a suitable donor polynucleotide, if provided). Each nick in these embodiments can be at least about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 (or any integer between 5 and 100) bases apart from each other, for example. One or two Cas9 nickase proteins herein can be used in a Cas9 nickase pair as described above. For example, a Cas9 nickase with a mutant RuvC domain, but functioning HNH domain (i.e., Cas9 HNH.sup.+/RuvC.sup.-), could be used (e.g., S. pyogenes Cas9 HNH.sup.+/RuvC.sup.-). Each Cas9 nickase (e.g., Cas9 HNH.sup.+/RuvC.sup.-) would be directed to specific DNA sites nearby each other (up to 100 base pairs apart) by using suitable RNA components herein with guide RNA sequences targeting each nickase to each specific DNA site.

[0152] An RGEN in certain embodiments can bind to a DNA target site sequence, but does not cleave any strand at the target site sequence. Such an RGEN may comprise a Cas protein in which all of its nuclease domains are mutant, dysfunctional. For example, a Cas9 protein herein that can bind to a DNA target site sequence, but does not cleave any strand at the target site sequence, may comprise both a mutant, dysfunctional RuvC domain and a mutant, dysfunctional HNH domain. Non-limiting examples of such a Cas9 protein comprise any of the RuvC and HNH nuclease domain mutations disclosed above (e.g., an S. pyogenes Cas9 with an Asp-10 substitution such as Asp-10-Ala and a His-840 substitution such as His-840-Ala). A Cas protein herein that binds, but does not cleave, a target DNA sequence can be used to modulate gene expression, for example, in which case the Cas protein could be fused with a transcription factor (or portion thereof) (e.g., a repressor or activator, such as any of those disclosed herein). For example, a Cas9 comprising an S. pyogenes Cas9 with an Asp-10 substitution (e.g., Asp-10-Ala) and a His-840 substitution (e.g., His-840-Ala) can be fused to a VP16 or VP64 transcriptional activator domain. The guide sequence used in the RNA component of such an RGEN would be complementary to a DNA sequence in a gene promoter or other regulatory element (e.g., intron), for example.

[0153] An RGEN herein can bind to a target site sequence, and optionally cleave one or both strands of the target site sequence, in a chromosome, episome, or any other DNA molecule in the genome of a cell. This recognition and binding of a target sequence is specific, given that an RNA component of the RGEN comprises a sequence (guide sequence) that is complementary to a strand of the target sequence. A target site in certain embodiments can be unique (i.e., there is a single occurrence of the target site sequence in the subject genome).

[0154] The length of a target sequence herein can be at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides; between 13-30 nucleotides; between 17-25 nucleotides; or between 17-20 nucleotides, for example. This length can include or exclude a PAM sequence. Also, a strand of a target sequence herein has sufficient complementarity with a guide sequence (of a crRNA or gRNA) to hybridize with the guide sequence and direct sequence-specific binding of a Cas protein or Cas protein complex to the target sequence (if a suitable PAM is adjacent to the target sequence, see below). The degree of complementarity between a guide sequence and a strand of its corresponding DNA target sequence is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, for example. A target site herein may be located in a sequence encoding a gene product (e.g., a protein or an RNA) or a non-coding sequence (e.g., a regulatory sequence or a "junk" sequence), for example.

[0155] A PAM (protospacer-adjacent motif) sequence may be adjacent to the target site sequence. A PAM sequence is a short DNA sequence recognized by an RGEN herein. The associated PAM and first 11 nucleotides of a DNA target sequence are likely important to Cas9/gRNA targeting and cleavage (Jiang et al., Nat. Biotech. 31:233-239). The length of a PAM sequence herein can vary depending on the Cas protein or Cas protein complex used, but is typically 2, 3, 4, 5, 6, 7, or 8 nucleotides long, for example. A PAM sequence is immediately downstream from, or within 2, or 3 nucleotides downstream of, a target site sequence that is complementary to the strand in the target site that is in turn complementary to an RNA component guide sequence, for example. In embodiments herein in which an RGEN is an endonucleolytically active Cas9 protein complexed with an RNA component, Cas9 binds to the target sequence as directed by the RNA component and cleaves both strands immediately 5' of the third nucleotide position upstream of the PAM sequence. Consider the following example of a target site:PAM sequence:

TABLE-US-00002 (SEQ ID NO: 43) 5'-NNNNNNNNNNNNNNNNNNNNXGG-3'.

N can be A, C, T, or G, and X can be A, C, T, or G in this example sequence (X can also be referred to as NPAM). The PAM sequence in this example is XGG (underlined). A suitable Cas9/RNA component complex would cleave this target immediately 5' of the double-underlined N. The string of N's in SEQ ID NO:43) represents target sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, for example, with a guide sequence in an RNA component herein (where any T's of the DNA target sequence would align with any U's of the RNA guide sequence). A guide sequence of an RNA component of a Cas9 complex, in recognizing and binding at this target sequence (which is representative of target sites herein), would anneal with the complement sequence of the string of N's; the percent complementarity between a guide sequence and the target site complement is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, for example. If a Cas9 nickase is used to target SEQ ID NO:43) in a genome, the nickase would nick immediately 5' of the double-underlined N or at the same position of the complementary strand, depending on which endonuclease domain in the nickase is dysfunctional. If a Cas9 having no nucleolytic activity (both RuvC and HNH domains dysfuntional) is used to target SEQ ID NO:43 in a genome, it would recognize and bind the target sequence, but not make any cuts to the sequence.

[0156] A PAM herein is typically selected in view of the type of RGEN being employed. A PAM sequence herein may be one recognized by an RGEN comprising a Cas, such as Cas9, derived from any of the species disclosed herein from which a Cas can be derived, for example. In certain embodiments, the PAM sequence may be one recognized by an RGEN comprising a Cas9 derived from S. pyogenes, S. thermophilus, S. agalactiae, N. meningitidis, T. denticola, or F. novicida. For example, a suitable Cas9 derived from S. pyogenes could be used to target genomic sequences having a PAM sequence of NGG (SEQ ID NO:44; N can be A, C, T, or G). As other examples, a suitable Cas9 could be derived from any of the following species when targeting DNA sequences having the following PAM sequences: S. thermophilus (NNAGAA [SEQ ID NO:45]), S. agalactiae (NGG [SEQ ID NO:44]), NNAGAAW [SEQ ID NO:46, W is A or T], NGGNG [SEQ ID NO:47]), N. meningitidis (NNNNGATT [SEQ ID NO:48]), T. denticola (NAAAAC [SEQ ID NO:49]), or F. novicida (NG [SEQ ID NO:50]) (where N's in all these particular PAM sequences are A, C, T, or G). Other examples of Cas9/PAMs useful herein include those disclosed in Shah et al. (RNA Biology 10:891-899) and Esvelt et al. (Nature Methods 10:1116-1121), which are incorporated herein by reference. Examples of target sequences herein follow SEQ ID NO:43, but with the `XGG` PAM replaced by any one of the foregoing PAMs.

[0157] An RNA component herein can comprise a sequence complementary to a target site sequence in a chromosome or episome in a cell. An RGEN can specifically bind to a target site sequence, and optionally cleave one or both strands of the target site sequence, based on this sequence complementary. Thus, the complementary sequence of an RNA component in certain embodiments of the disclosed invention can also be referred to as a guide sequence or variable targeting domain.

[0158] The guide sequence of an RNA component (e.g., crRNA or gRNA) herein can be at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ribonucleotides in length; between 13-30 ribonucleotides in length; between 17-25 ribonucleotides in length; or between 17-20 ribonucleotides in length, for example. In general, a guide sequence herein has sufficient complementarity with a strand of a target DNA sequence to hybridize with the target sequence and direct sequence-specific binding of a Cas protein or Cas protein complex to the target sequence (if a suitable PAM is adjacent to the target sequence). The degree of complementarity between a guide sequence and its corresponding DNA target sequence is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, for example. The guide sequence can be engineered accordingly to target an RGEN to a DNA target sequence in a cell.

[0159] An RNA component herein can comprise a crRNA, for example, which comprises a guide sequence and a repeat (tracrRNA mate) sequence. The guide sequence is typically located at or near (within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bases) the 5' end of the crRNA. Downstream the guide sequence of a crRNA is a "repeat" or "tracrRNA mate" sequence that is complementary to, and can hybridize with, sequence at the 5' end of a tracrRNA. Guide and tracrRNA mate sequences can be immediately adjacent, or separated by 1, 2, 3, 4 or more bases, for example. A tracrRNA mate sequence has, for example, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to the 5' end of a tracrRNA. In general, degree of complementarity can be with reference to the optimal alignment of the tracrRNA mate sequence and 5' end of the tracrRNA sequence, along the length of the shorter of the two sequences. The length of a tracrRNA mate sequence herein can be at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 ribonucleotides in length, for example, and hybridizes with sequence of the same or similar length (e.g., plus or minus 1, 2, 3, 4, or 5 bases) at the 5' end of a tracrRNA. Suitable examples of tracrRNA mate sequences herein comprise SEQ ID NO:51 (guuuuuguacucucaagauuua), SEQ ID NO:52 (guuuuuguacucuca), SEQ ID NO:53 (guuuuagagcua), or SEQ ID NO:54 (guuuuagagcuag), or variants thereof that (i) have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity and (ii) can anneal with the 5'-end sequence of a tracrRNA. The length of a crRNA herein can be at least about 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48 ribonucleotides; or about 18-48 ribonucleotides; or about 25-50 ribonucleotides, for example.

[0160] A tracrRNA can be included along with a crRNA in embodiments in which a Cas9 protein of a type II CRISPR system is comprised in the RGEN. A tracrRNA herein comprises in 5'-to-3' direction (i) a sequence that anneals with the repeat region (tracrRNA mate sequence) of crRNA and (ii) a stem loop-containing portion. The length of a sequence of (i) can be the same as, or similar with (e.g., plus or minus 1, 2, 3, 4, or 5 bases), any of the tracrRNA mate sequence lengths disclosed above, for example. The total length of a tracrRNA herein (i.e., sequence components [i] and [ii]) can be at least about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 (or any integer between 30 and 90) ribonucleotides, for example. A tracrRNA may further include 1, 2, 3, 4, 5, or more uracil residues at the 3'-end, which may be present by virtue of expressing the tracrRNA with a transcription terminator sequence.

[0161] A tracrRNA herein can be derived from any of the bacterial species listed above from which a Cas9 sequence can be derived, for example. Examples of suitable tracrRNA sequences include those disclosed in U.S. Pat. No. 8,697,359 and Chylinski et al. (RNA Biology 10:726-737), which are incorporated herein by reference. A preferred tracrRNA herein can be derived from a Streptococcus species tracrRNA (e.g., S. pyogenes, S. thermophilus). Other suitable examples of tracrRNAs herein may comprise:

TABLE-US-00003 SEQ ID NO: 55: uagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcacc gagucggugc, SEQ ID NO: 56: uagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaagug, or SEQ ID NO: 57: uagcaaguuaaaauaaggcuaguccguuauca,

which are derived from S. pyogenes tracrRNA. Other suitable examples of tracrRNAs herein may comprise:

TABLE-US-00004 SEQ ID NO: 58: uaaaucuugcagaagcuacaaagauaaggcuucaugccgaaaucaacacc cugucauuuuauggcaggguguuuucguuauuuaa, SEQ ID NO: 59: ugcagaagcuacaaagauaaggcuucaugccgaaaucaacacccugucau uuuauggcaggguguuuucguuauuua, or SEQ ID NO: 60: ugcagaagcuacaaagauaaggcuucaugccgaaaucaacacccugucau uuuauggcagggugu,

which are derived from S. thermophilus tracrRNA. Still other examples of tracrRNAs herein are variants of these tracrRNA SEQ ID NOs that (i) have at least about 80%, 85%, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity therewith and (ii) can function as a tracrRNA (e.g., 5'-end sequence can anneal to tracrRNA mate sequence of a crRNA, sequence downstream from the 5'-end sequence can form one or more hairpins, variant tracrRNA can form complex with a Cas9 protein).

[0162] An RNA component of an RGEN disclosed herein (or said another way, an RNA component that may be associated with an RGEN protein component) can comprise, for example, a guide RNA (gRNA) comprising a crRNA operably linked to, or fused to, a tracrRNA. The crRNA component of a gRNA in certain preferred embodiments is upstream of the tracrRNA component (i.e., such a gRNA comprises, in 5'-to-3' direction, a crRNA operably linked to a tracrRNA). Any crRNA and/or tracrRNA (and/or portion thereof, such as a crRNA repeat sequence, tracrRNA mate sequence, or tracrRNA 5'-end sequence) as disclosed herein (e.g., above embodiments) can be comprised in a gRNA, for example.

[0163] The tracrRNA mate sequence of the crRNA component of a gRNA herein should be able to anneal with the 5'-end of the tracrRNA component, thereby forming a hairpin structure. Any of the above disclosures regarding lengths of, and percent complementarity between, tracrRNA mate sequences (of crRNA component) and 5'-end sequences (of tracrRNA component) can characterize the crRNA and tracrRNA components of a gRNA, for example. To facilitate this annealing, the operable linkage or fusion of the crRNA and tracrRNA components preferably comprises a suitable loop-forming ribonucleotide sequence (i.e., a loop-forming sequence may link the crRNA and tracrRNA components together, forming the gRNA). Suitable examples of RNA loop-forming sequences include GAAA (SEQ ID NO:36), CAAA (SEQ ID NO:37) and AAAG (SEQ ID NO:38). However, longer or shorter loop sequences may be used, as may alternative loop sequences. A loop sequence preferably comprises a ribonucleotide triplet (e.g., AAA) and an additional ribonucleotide (e.g., C or G) at either end of the triplet.

[0164] A gRNA herein forms a hairpin ("first hairpin") with annealing of its tracrRNA mate sequence (of the crRNA component) and tracrRNA 5'-end sequence portions. One or more (e.g., 1, 2, 3, or 4) additional hairpin structures can form downstream from this first hairpin, depending on the sequence of the tracrRNA component of the gRNA. A gRNA may therefore have up to five hairpin structures, for example. A gRNA may further include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more residues following the end of the gRNA sequence, which may be present by virtue of expressing the gRNA with a transcription terminator sequence, for example. These additional residues can be all U residues, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% U residues, for example, depending on the choice of terminator sequence.

[0165] Non-limiting examples of suitable gRNAs useful in the disclosed invention may comprise:

TABLE-US-00005 SEQ ID NO: 61: NNNNNNNNNNNNNNNNNNNNguuuuuguacucucaagauuuaGAAAuaaa ucuugcagaagcuacaaagauaaggcuucaugccgaaaucaacacccugu cauuuuauggcaggguguuuucguuauuuaa, SEQ ID NO: 62: NNNNNNNNNNNNNNNNNNNNguuuuuguacucucaGAAAugcagaagcua caaagauaaggcuucaugccgaaaucaacacccugucauuuuauggcagg guguuuucguuauuuaa, SEQ ID NO: 63: NNNNNNNNNNNNNNNNNNNNguuuuuguacucucaGAAAugcagaagcua caaagauaaggcuucaugccgaaaucaacacccugucauuuuauggcagg gugu, SEQ ID NO: 64: NNNNNNNNNNNNNNNNNNNNguuuuuguacucucaGAAAuagcaaguuaa aauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc, SEQ ID NO: 65: NNNNNNNNNNNNNNNNNNNNguuuuagagcuaGAAAuagcaaguuaaaau aaggcuaguccguuaucaacuugaaaaagug, SEQ ID NO: 66: NNNNNNNNNNNNNNNNNNNNguuuuagagcuaGAAAuagcaaguuaaaau aaggcuaguccguuauca, or SEQ ID NO: 67: NNNNNNNNNNNNNNNNNNNNguuuuagagcuaGAAAuagcaaguuaaaau aaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu.

In each of SEQ ID NOs:61-67, the single-underlined sequence represents a crRNA portion of the gRNA. Each "N" represents a ribonucleotide base (A, U, G, or C) of a suitable guide sequence. The first block of lower case letters represents tracrRNA mate sequence. The second block of lower case letters represents a tracrRNA portion of the gRNA. The double-underlined sequence approximates that portion of tracrRNA sequence that anneals with the tracrRNA mate sequence to form a first hairpin. A loop sequence (GAAA, SEQ ID NO:36) is shown in capital letters, which operably links the crRNA and tracrRNA portions of each gRNA. Other examples of gRNAs herein include variants of the foregoing gRNAs that (i) have at least about 80%, 85%, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity (excluding guide sequence in this calculation) with these sequences, and (ii) can function as a gRNA that specifically targets a Cas9 protein to bind with, and optionally nick or cleave, a target DNA sequence.

[0166] A gRNA herein can also be characterized in terms of having a guide sequence (VT domain) followed by a Cas endonuclease recognition (CER) domain. A CER domain comprises a tracrRNA mate sequence followed by a tracrRNA sequence. Examples of CER domains useful herein include those comprised in SEQ ID NOs:61-67 above (the CER domain in each is the sequence following the N's of the VT domain). Another suitable example of a CER domain is SEQ ID NO:24 (see Examples), which comprises in 5'-to-3' direction the tracrRNA mate sequence of SEQ ID NO:53, the loop-forming sequence of SEQ ID NO:36 (GAAA), and the tracrRNA sequence of SEQ ID NO:55.

[0167] An RNA component of an RGEN optionally does not have a 5'-cap (7-methylguanylate [m.sup.7G] cap) (i.e., such an RNA component does not have an m.sup.7G cap at its 5'-terminus). An RNA component herein can have, for example, a 5'-hydroxyl group instead of a 5'-cap. Alternatively, an RNA component herein can have, for example, a 5' phosphate instead of a 5'-cap. It is believed that an RNA component in these embodiments can better accumulate in the nucleus (such as after its transcription in the nucleus, or after its RGEN-mediated import into the nucleus, depending on how the RNA component is provided herein), since 5'-capped RNA (i.e., RNA having 5' m.sup.7G cap) is subject to nuclear export. Preferred examples of uncapped RNA components herein include suitable gRNAs, crRNAs, and/or tracrRNAs. In certain embodiments, an RNA component herein lacks a 5'-cap, and optionally has a 5'-hydroxyl group instead, by virtue of RNA autoprocessing by a ribozyme sequence at the 5'-end of a precursor of the RNA component (i.e., a precursor RNA comprising a ribozyme sequence upstream of an RNA component such as a gRNA undergoes ribozyme-mediated autoprocessing to remove the ribozyme sequence, thereby leaving the downstream RNA component without a 5'-cap). In certain other embodiments, an RNA component herein is not produced by transcription from an RNA polymerase III (Pol III) promoter.

[0168] A cell-penetrating peptide (CPP) herein can be about 5-30, 5-25, 5-20, 10-30, 10-25, or 10-20 amino acid residues in length, for example. As other examples, a CPP can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues in length. Yet in further aspects herein, a CPP can be up to about 35, 40, 45, 50, 55, or 60 amino acid residues in length.

[0169] A CPP disclosed herein can be cationic or amphipathic, for example. A cationic CPP herein typically comprises at least about 60% positively charged amino acids such as lysine (K), arginine (R), and/or histidine (H). Alternatively, a cationic CPP can comprise, for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% positively charged amino acids (e.g., R residues; K residues; K and R residues; K, R and H residues). A cationic CPP can be characterized as being arginine-rich (e.g., comprising at least 70% or 80% R residues) or lysine-rich (e.g., comprising at least 70% or 80% L residues) in certain embodiments. Examples of cationic CPPs useful herein are disclosed in Schmidt et al. (FEBS Lett. 584:1806-1813) and Wender et al. (polylysine; Proc. Natl. Acad. Sci. USA 97:13003-13008), which are incorporated herein by reference. Other examples of cationic CPPs comprise GRKKRRQRRR (SEQ ID NO:68), RKKRRQRRR (SEQ ID NO:69), or RKKRRQRR (SEQ ID NO:70), which were originally derived from HIV Tat protein, and penetratin (RQIKIWFQNRRMKWKK, SEQ ID NO:71), which was originally derived for the Antennapedia homeodomain protein of Drosophila.

[0170] Another example of a cationic CPP comprises a polyarginine sequence having a number of contiguous arginines sufficient to direct entry of the CPP and its cargo (e.g., RGEN protein component or RGEN) into a cell. The number of contiguous arginine residues in such a polyarginine sequence can be at least 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines, for instance. In certain aspects herein, a CPP can have 6 or more contiguous arginine residues (e.g., 6-7, 6-8, 6-9, or 6-10 arginine residues). "PolyR" (GGGGRRRRRRRRRLLLL, SEQ ID NO:15) can be comprised in a polyarginine CPP, if desired. Other polyarginine CPP examples comprise THRLPRRRRRR (SEQ ID NO:72) or GGRRARRRRRR (SEQ ID NO:73).

[0171] In some embodiments, a CPP is an activatable CPP ("ACPP") (Aguilera et al., Integr Biol. (Camb) 1:371-381; incorporated herein by reference). ACPPs typically comprise a polycationic CPP (e.g., nine contiguous arginines) connected via a cleavable linker to a matching polyanion (e.g., nine contiguous glutamates), which reduces the net charge to nearly zero and thereby inhibits CPP adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polycation portion and its inherent adhesiveness, thereby allowing CPP cell entry. Another example herein is a polylysine CPP; any of the above embodiments of polyarginine, but in which R is replaced with K, are examples of polylysine CPPs herein.

[0172] An amphipathic CPP herein comprises an amino acid sequence containing an alternating pattern of polar/charged residues and non-polar, hydrophobic residues. The following CPPs are believed to be amphipathic, and are useful in certain aspects (regardless of whether amphipathic terminology perfectly applies): a CPP comprising transportan-10 (TP10) peptide (e.g., AGYLLGKINLKACAACAKKIL, SEQ ID NO:14); a CPP from a vascular endothelium cadherin protein, such as a CPP comprising a pVEC peptide (e.g., LIILRRRIRKQAHAHSK, SEQ ID NO:74; LLIILRRRIRKQAHAHSK, SEQ ID NO:13); a CPP from an Epstein-Barr virus Zebra trans-activator protein, such as a CPP comprising a Zebra peptide (e.g., ECDSELEIKRYKRVRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMC, SEQ ID NO:12); a CPP comprising a (KFF).sub.3K peptide (e.g., KFFKFFKFFK, SEQ ID NO:75); a CPP comprising a MAP peptide (KLALKLALKALKAALKLA, SEQ ID NO:76); a CPP comprising RRQRRTSKLMKR (SEQ ID NO:77); a CPP comprising KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:78). Other amphipathic CPPs suitable herein include proline-rich CPPs, such as those comprising at least 3, 4, 5, 6, 7, or 8 repeats of VHLPPP (SEQ ID NO:79) or VRLPPP (SEQ ID NO:80).

[0173] As other examples, a CPP herein may comprise an MPG peptide (e.g., GALFLGFLGAAGSTMGAWSQPKSKRKV, SEQ ID NO:81); a Pep-1 peptide (e.g., KETWWETWVTEWSQPKKKRKV, SEQ ID NO:82); or a CPP from a human calcitonin protein, such as an hCT peptide (e.g., LGTYTQDFNKFHTFPQTAIGVGAP, SEQ ID NO:83; CGNLSTCMLGTYTQDFNK, SEQ ID NO:84). Still other examples of CPPs herein include those disclosed in Regberg et al. (Int. J. Pharm. 464:111-116), which is incorporated herein by reference.

[0174] A CPP suitable herein can alternatively comprise an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the CPP amino acid sequences disclosed herein, for example. Such a variant CPP protein should have CPP activity, such as the ability to mediate cellular uptake of molecular cargo (e.g., an amino acid sequence comprising one or more RGEN protein components [e.g., Cas9], or an amino acid sequence comprising one or more RGEN protein components [e.g., Cas9] associated with an RNA component). Testing the activity of a variant CPP can be done any number of ways, such as by covalently linking it with a fluorescent protein (e.g., GFP) and measuring the degree of fluorescence emitted from a cell contacted with a the CPP-fluorescent protein complex.

[0175] A CPP herein can be modified, if desired, to render it even more capable of carrying RGEN protein cargo from outside a cell to inside a cell. For example, a CPP can be modified to have a lipid group at either its N- or C-terminus. Suitable lipid groups herein include acyl groups such as stearyl and myristyl groups. Other examples of lipid groups are acyl groups with 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 carbons. Conditions for modifying peptides with lipid groups useful herein are disclosed in Regberg et al. (Int. J. Pharm. 464:111-116) and Anko et al. (Biochim. Biophys. Acta--Biomembranes 1818:915-924) for example, which are incorporated herein by reference.

[0176] An RGEN protein component and at least one CPP herein can be covalently linked to each other in an RGEN protein-CPP complex in certain aspects herein. For example, an RGEN protein component and at least one CPP can be fused together in a single amino acid sequence (i.e., an RGEN protein component and at least one CPP can be comprised within a fusion protein). Thus, an example of covalent linkage herein can be via a peptide bond in which the amino acid sequence of an RGEN protein component is fused with the amino acid sequence of a CPP, such that both these amino acid sequences are contained in a single amino acid sequence. Such a fusion protein (or "chimeric protein"), can be characterized as an RGEN protein-CPP fusion herein. In those embodiments in which an RNA component is associated with an RGEN protein component, such a fusion protein can be characterized as an RGEN-CPP fusion.

[0177] One or more CPPs can be located at the N-terminus or C-terminus of an RGEN protein-CPP fusion, for example. Alternatively, one or more CPPs can be located at both the N- and C-termini of an RGEN protein-CPP fusion. Alternatively still, one or more CPPs can be located within the amino acid sequence of an RGEN protein-CPP fusion. Embodiments herein comprising more than one CPP can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 CPPs, or 5-10, 5-20, or 10-20 CPPs. The CPPs fused to the RGEN protein component can be the same or different (e.g., 2, 3, 4, or more different types of CPPs). One or more CPPs can be fused directly to the amino acid sequence of an RGEN protein, and/or can be fused to a heterologous domain(s) (e.g., NLS or other organelle-targeting sequence such as an MTS) that is fused with an RGEN protein.

[0178] A fusion between a CPP and an RGEN protein component herein can be direct (i.e., CPP amino acid sequence is directly linked to RGEN amino acid sequence by a peptide bond). Alternatively, a fusion between a CPP and an RGEN protein component can be via an intermediary amino acid sequence (this is an example of a CPP and RGEN protein component being indirectly linked). Examples of an intermediary amino acid sequence include suitable linker sequences comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues such as glycine, serine, alanine and/or proline. Suitable amino acid linkers are disclosed in U.S. Pat. Nos. 8,828,690, 8,580,922 and 5,990,275, for example, which are incorporated herein by reference. Other examples of intermediary amino acid sequences can comprise one or more other types of proteins and/or domains. For example, a marker protein (e.g., a fluorescent protein such as any of those disclosed herein) can be comprised in an intermediary amino acid sequence.

[0179] A composition comprising a covalent complex of an RGEN protein component and at least one CPP, such as in a fusion protein, can be used with any cell type disclosed herein. Optionally, however, this composition can be used with non-mammalian cells such as yeast, fungi, and plants, but excludes use on mammalian cells.

[0180] Examples of RGEN protein-CPP fusion proteins herein can comprise SEQ ID NO:39 (Zebra CPP-Cas9-NLS fusion protein), 40 (PolyR CPP-Cas9-NLS fusion protein), 41 (TP10 CPP-Cas9-NLS fusion protein), or 42 (pVEC CPP-Cas9-NLS fusion protein). SEQ ID NOs:39-42 are examples of Cas9-CPP fusion proteins. Other examples of RGEN protein-CPP fusion proteins comprise an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs:39-42. Such a variant fusion protein should have (i) a CPP domain that can mediate cellular uptake of the fusion protein, and (ii) a Cas9 protein with specific binding activity, and optionally cleavage or nicking activity, toward DNA when associated with an RNA component. SEQ ID NO:39, 40, 41 and 42 comprise Zebra CPP (SEQ ID NO:12), PolyR CPP (SEQ ID NO:15), TP10 CPP (SEQ ID NO:14) and pVEC CPP (SEQ ID NO:13), respectively, operably linked to Cas9 (S. pyogenes)-NLS protein (residues 2-1379 of SEQ ID NO:3).

[0181] In certain embodiments, the protein component of a guide polynucleotide/Cas endonuclease system can be fused to a CPP, wherein the CPP comprises:

[0182] (i) a CPP from an Epstein-Barr virus Zebra trans-activator protein,

[0183] (ii) a CPP having 6 or more contiguous arginine residues,

[0184] (iii) a transportan-10 (TP10) CPP,

[0185] (iv) a CPP from a vascular endothelium cadherin protein, or

[0186] (vi) a CPP selected from the group consisting of a synthetic non-arginine CPP, a histidine-rich nona-arginine CPP and a Pas nona-arginine CPP. Examples of synthetic nona-arginine, histidine-rich nona-arginine, and Pas nona-arginine CPPs are disclosed in, for example, Liu et al. (Advanced Studies in Biology 5(2):71-88, HIKARI Ltd), which is incorporated herein by reference.

[0187] Another example of how an RGEN protein component and at least one CPP can be covalently linked is via crosslinking (chemical crosslinking). Thus, an example of an RGEN protein-CPP complex herein can comprise an RGEN protein crosslinked to at least one CPP. Crosslinking herein refers to a process of chemically joining two or more molecules (an RGEN protein component and at least one CPP, in this case) by a covalent bond(s). Crosslinking can be performed using any number of processes known in the art, such as those disclosed in U.S. Patent Appl. Publ. No. 2011/0190813, U.S. Pat. No. 8,642,744, and Bioconjugate Techniques, 2nd Edition (G. T. Hermanson, Academic Press, 2008), which are all incorporated herein by reference.

[0188] Typically, a CPP can be modified and/or synthesized to contain a suitable protein linking group at its N-terminus, C-terminus, and/or an amino acid side group, for the purpose of crosslinking the CPP to an RGEN protein component. A "protein linking group" refers to a group that is capable of reacting directly, either spontaneously or after activation (e.g., light), with an accessible side chain functional group of an RGEN protein component under suitable conditions (e.g., aqueous conditions) to produce a covalently link the CPP to the RGEN protein. A protein linking group may react with the side chain functional groups of a Lys, Cys, Ser, Thr, Tyr, His, or Arg amino acid residue in an RGEN protein, for example, to produce a covalent linkage to the protein. Either a homobifunctional (e.g., capable of linking amine to amine) or heterobifunctional (e.g., capable of linking amine to thiol) protein linking group can be used, for example. A protein linking group on a CPP can also react with a terminal group (e.g., N-terminus) of an RGEN protein in certain embodiments. Suitable protein linking groups herein include amino-reactive (e.g., NHS ester or imidoester), thiol (sulfhydryl)-reactive (e.g., a maleimide such as BMOE, BMB, or BMH), hydroxyl-reactive, imidazolyl-reactive, or guanidinyl-reactive groups. Exemplary protein linking groups include active esters (e.g., an amino-reactive NHS ester), and thiol-reactive maleimide or iodoacetamide groups. Further exemplary protein linking groups useful herein and methods of using them are described in Bioconiuqate Techniques, 2nd Edition (G. T. Hermanson, Academic Press, 2008), for example.

[0189] A protein linking group herein typically can produce a link between a CPP and an RGEN protein with a backbone of 20 atoms or less in length. For example, such a link can be between 1 and 20 atoms in length, or about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 or 20 carbon atoms in length. A link may be linear, branched, cyclic or a single atom in certain embodiments. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be substituted with a sulfur, nitrogen or oxygen heteroatom. The bonds between backbone atoms may be saturated or unsaturated (usually not more than one, two, or three unsaturated bonds in the linker backbone). A linker may include, without limitation, an oligo (ethylene glycol); ether; thioether; tertiary amine; or alkyl group, which may be straight or branched (e.g., methyl, ethyl, n-propyl, iso-propyl, n-butyl, n-pentyl, t-butyl). As other examples, a linker backbone may include a cyclic group such as an aryl, a heterocycle, or a cycloalkyl group, where 2 or more atoms (e.g., 2, 3 or 4 atoms) of the cyclic group are included in the backbone.

[0190] More than one type of CPP (e.g., 2, 3, 4, or more different types of CPPs) can be crosslinked to an RGEN protein component in certain embodiments. The ratio (molar ratio) of CPP(s) to RGEN protein that can be used when crosslinking can be at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 15:1, 20:1, 30:1, 40:1, or 50:1, for example. In other aspects, the average number of CPPs crosslinked to an RGEN protein may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or at least 5-10, 5-15, 5-20, or 5-25.

[0191] An RGEN protein component and at least one CPP can be crosslinked into a complex further comprising one or more other proteins/peptides/domains, if desired. Such other elements can optionally be used to bridge an RGEN protein component with a CPP, and may include any of the intermediary amino acid sequences described above.

[0192] An RGEN protein component and at least one CPP herein can be non-covalently linked to each other in an RGEN protein-CPP complex in certain aspects herein. Though not intending to be held to any particular theory or mechanism, it is contemplated that a non-covalent linkage between an RGEN protein component and at least one CPP can be due to electrostatic, Van der Waals, and/or hydrophobic forces. In those embodiments in which an RNA component is associated with an RGEN protein component, such embodiments can be characterized as comprising an RGEN that is non-covalently linked to at least one CPP in an RGEN-CPP complex. A composition comprising an RGEN protein component and CPP that are non-covalently linked can optionally be characterized as a mixture of these components.

[0193] In certain embodiments, an RGEN protein component is non-covalently linked to at least one CPP with an amino acid sequence consisting of the CPP amino acid sequence only. Such a CPP, while not having any "non-CPP" amino acid sequence, can optionally comprise a modification such as a lipid group as disclosed herein.

[0194] Alternatively, a CPP that is non-covalently linked to an RGEN protein component may be comprised in a fusion protein having both CPP amino acid sequence and one or more heterologous amino acid sequences (non-RGEN protein sequences). A heterologous sequence in such embodiments can be that of a domain or a protein (e.g., a fluorescent protein such as any of those disclosed herein, or any domain/protein listed in the above disclosure regarding Cas fusions). Another example is fusing a dimerization domain to a CPP, which dimerization domain is able to bind to a dimerization domain linked or fused to an RGEN protein component.

[0195] Leucine zipper domains are examples of dimerization domains herein. Leucine zipper domains can represent those from natural proteins known to contain such domains (e.g., transcription factors), or can be synthetically designed. A leucine zipper domain linked to a CPP can associate ("zip together") with a leucine zipper domain of an RGEN protein component, thereby linking the CPP and RGEN protein component in a non-covalent complex. A pair of leucine zipper domains for non-covalently linking a CPP and an RGEN protein component can be the same (such a domain pair forms a homodimeric leucine zipper) or different (such a domain pair forms a heterodimeric leucine zipper). Examples of leucine zipper domains include those disclosed in U.S. Patent Appl. Publ. Nos. 2003/0108869 and 2004/0147721. In certain aspects, a homodimeric leucine zipper can be formed using a leucine zipper domain from a GCN4 transcription factor, while in other aspects a heterodimeric leucine zipper can be formed using leucine zipper domains from fos and jun transcription factors, respectively.

[0196] A non-covalent complex of an RGEN protein component and at least one CPP can further comprise one or more other proteins/peptides/domains, if desired. Such other elements can optionally be used to bridge an RGEN protein component with a CPP, and may include any of the intermediary amino acid sequences described above.

[0197] More than one type of CPP (e.g., 2, 3, 4, or more different types of CPPs) can be non-covalently linked to an RGEN protein component in certain embodiments. The ratio (molar ratio) of CPP(s) to RGEN protein that can be used to prepare such a complex can be at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 15:1, 20:1, 30:1, 40:1, or 50:1, for example. In other aspects, the average number of CPPs non-covalently linked to an RGEN protein may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or at least 5-10, 5-15, 5-20, or 5-25.

[0198] In certain embodiments, a non-covalent complex of an RGEN protein component and at least one CPP can be prepared by mixing an appropriate amount of each component (e.g., such as to obtain a ratio of CPP to RGEN protein disclosed above) in an aqueous medium. A suitable aqueous medium can comprise a buffer solution such as PBS or a serum-free medium such as DMEM, for example. The mixture can be incubated for about 30, 60, 90, or 120 minutes at a temperature of about 4 to 45.degree. C., for example, to allow formation of a non-covalent RGEN protein-CPP complex. A suitable volume (e.g., a minimum volume that adequately covers/immerses cells being treated) of this solution comprising the complex can be applied to a cell in a cell type-appropriate manner. In embodiments in which an RNA component is associated with an RGEN protein component, such formation of an RGEN can comprise adding an RNA component before, at the same time of, or after incubating a CPP with the RGEN protein component.

[0199] A composition comprising a non-covalent complex of an RGEN protein component and at least one CPP can be used with any cell type disclosed herein. Optionally, however, this composition can be used with non-mammalian cells such as yeast, fungi, and plants, but excludes use on mammalian cells.

[0200] An RGEN protein-CPP complex, as it may exist in a composition before application to cells can be at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% pure, for example. Such purity can be on a protein basis in certain embodiments. As an example, if the purity of a complex is at least 80%, this would mean that at least 80% of all the protein in a composition is constituted by the complex. Complex purity alternatively can take into account not only purity on a protein basis, but also in account of other biomolecules (e.g., lipids, saccharides, and/or nucleic acids). As an example, if the purity of a complex is at least 80%, this could mean that at least 80% of all the biomolecules in the composition herein is constituted by the complex. In certain embodiments, compounds such as carbohydrates, salts, and/or lipids and the like do not affect the determination of percent purity herein. All these disclosures regarding purity can also apply to an RGEN-CPP complex (i.e., RGEN protein component of complex is associated with an RNA component).

[0201] A composition herein is preferably aqueous, wherein the solvent in which an RGEN protein-CPP complex or RGEN-CPP complex is dissolved is at least about 70, 75, 80, 85, 90, 95, 98, or 99 wt % water. The concentration of a complex in a composition can be at least about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 .mu.M, or about 0.5 to 5.0 .mu.M, 0.5 to 2.5 .mu.M, 1.0 to 5.0 .mu.M, 1.0 to 2.5 .mu.M, or 2.5 to 5.0 .mu.M, for example. It would be understood that such compositions can be in a liquid state.

[0202] The pH of a composition in certain embodiments can be between about 4.0 to about 10.0. Alternatively, the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10.0. pH can be adjusted or controlled by the addition or incorporation of a suitable buffer, including but not limited to: HEPES, phosphate (e.g., PBS), Tris, Tris-HCl, citrate, or a combination thereof. Buffer concentration in a composition herein can be from 0 mM to about 100 mM, or about 10, 20, or 50 mM, for example. A HEPES buffer (e.g., .about.25 mM HEPES, such as 25 mM HEPES/KOH pH 7.5, 200 mM KCl, 20% glycerol, 1 mM DTT) can be used in certain aspects.

[0203] A composition herein can optionally comprise other components in addition to an RGEN protein-CPP complex or RGEN-CPP complex. For example, the composition can comprise one or more salts such as a sodium salt (e.g., NaCl, Na.sub.2SO.sub.4). Other non-limiting examples of salts include those having (i) an aluminum, ammonium, barium, calcium, chromium (II or III), copper (I or II), iron (II or III), hydrogen, lead (II), lithium, magnesium, manganese (II or III), mercury (I or II), potassium, silver, sodium strontium, tin (II or IV), or zinc cation, and (ii) an acetate, borate, bromate, bromide, carbonate, chlorate, chloride, chlorite, chromate, dichromate, dihydrogen phosphate, ferricyanide, ferrocyanide, fluoride, hydrogen carbonate, hydrogen phosphate, hydrogen sulfate, hydrogen sulfide, hydrogen sulfite, hydride, hydroxide, hypochlorite, iodate, iodide, nitrate, nitride, nitrite, oxalate, oxide, perchlorate, permanganate, peroxide, phosphate, phosphide, phosphite, silicate, stannate, stannite, sulfate, sulfide, sulfite, tartrate, or thiocyanate anion. Thus, any salt having a cation from (i) above and an anion from (ii) above can be in a composition herein, for example. A salt can be present at a wt % of about 0.01 to about 10.00 (or any hundredth increment between 0.01 and 10.00), for example.

[0204] An RGEN protein-CPP complex herein can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell. In those embodiments in which an RGEN protein component is associated with an RNA component (thereby constituting a complete RGEN), an RGEN-CPP complex similarly has this cell membrane/cell wall-traversing ability. Either an RGEN protein-CPP complex or an RGEN-CPP complex can traverse a cell wall and cell membrane in certain aspects herein.

[0205] An RGEN protein-CPP or RGEN-CPP complex herein can optionally traverse a cell wall that comprises a glycocalyx (capsule). These embodiments typically are with regard to prokaryotic cells (e.g., bacteria), some of which may have a glycocalyx depending on species type and growth conditions.

[0206] Though not intending to be held to any particular theory or mechanism, it is believed that a CPP herein may deliver an RGEN protein component into a cell via an endocytic process. Examples of such a process might include macropinocytosis, clathrin-mediated endocytosis, caveolae/lipid raft-mediated endocytosis, and/or receptor mediated endocytosis mechanisms (e.g., scavenger receptor-mediated uptake, proteoglycan-mediated uptake).

[0207] Once an RGEN protein-CPP or RGEN-CPP complex is inside a cell, it can traverse an organelle membrane such as a nuclear membrane or mitochondrial membrane, for example. This ability depends on, in certain embodiments, the presence of at least one organelle-targeting sequence (e.g., NLS, MTS) being included with the RGEN protein. Still, in other embodiments, the ability to traverse an organelle membrane such as a nuclear membrane or mitochondrial membrane does not depend on the presence of an organelle-targeting sequence (i.e., a CPP[s] in such embodiments may be responsible for allowing RGEN traversal into an organelle such as the nucleus or mitochondria).

[0208] A cell herein can be a mammalian cell or a non-mammalian cell, the latter of which is used in certain preferred embodiments. In certain other aspects, a cell herein can be as it exists (i) in an organism/tissue in vivo, (ii) in a tissue or group of cells ex vivo, or (iii) in an in vitro state.

[0209] A microbial cell herein can be as it exists in an isolated state (e.g., in vitro cells, cultured cells) or a non-isolated state.

[0210] A microbial cell in certain embodiments is a fungal cell such as a yeast cell. A yeast in certain aspects herein can be one that reproduces asexually (anamorphic) or sexually (teleomorphic). While yeast herein typically exist in unicellular form, certain types of these yeast may optionally be able to form pseudohyphae (strings of connected budding cells). In still further aspects, a yeast may be haploid or diploid, and/or may have the ability to exist in either of these ploidy forms.

[0211] Examples of yeast herein include conventional yeast and non-conventional yeast. Conventional yeast in certain embodiments are yeast that favor homologous recombination (HR) DNA repair processes over repair processes mediated by non-homologous end-joining (NHEJ). Examples of conventional yeast herein include species of the genera Saccharomyces (e.g., S. cerevisiae, which is also known as budding yeast, baker's yeast, and/or brewer's yeast; S. bayanus; S. boulardii; S. bulderi; S. cariocanus; S. cariocus; S. chevalieri; S. dairenensis; S. ellipsoideus; S. eubayanus; S. exiguus; S. florentinus; S. kluyveri; S. martiniae; S. monacensis; S. norbensis; S. paradoxus; S. pastorianus; S. spencerorum; S. turicensis; S. unisporus; S. uvarum; S. zonatus) and Schizosaccharomyces (e.g., S. pombe, which is also known as fission yeast; S. cryophilus; S. japonicus; S. octosporus).

[0212] A non-conventional yeast herein is not a conventional yeast such as a Saccharomyces (e.g., S. cerevisiae) or Schizosaccharomyces (e.g., S. pombe) species. Non-conventional yeast in certain embodiments can be yeast that favor NHEJ DNA repair processes over repair processes mediated by HR. Conventional yeasts such as S. cerevisiae and S. pombe typically exhibit specific integration of donor DNA with short flanking homology arms (30-50 bp) with efficiencies routinely over 70%, whereas non-conventional yeasts such as Pichia pastoris, Pichia stipitis, Hansenula polymorpha, Yarrowia lipolytica and Kluyveromyces lactis usually show specific integration with similarly structured donor DNA at efficiencies of less than 1% (Chen et al., PLoS ONE 8:e57952). Thus, a preference for HR processes can be gauged, for example, by transforming yeast with a suitable donor DNA and determining the degree to which it is specifically recombined with a genomic site predicted to be targeted by the donor DNA. A preference for NHEJ (or low preference for HR), for example, would be manifest if such an assay yielded a high degree of random integration of the donor DNA in the yeast genome. Assays for determining the rate of specific (HR-mediated) and/or random (NHEJ-mediated) integration of DNA in yeast are known in the art (e.g., Ferreira and Cooper, Genes Dev. 18:2249-2254; Corrigan et al., PLoS ONE 8:e69628; Weaver et al., Proc. Natl. Acad. Sci. U.S.A. 78:6354-6358; Keeney and Boeke, Genetics 136:849-856).

[0213] Given their low level of HR activity, non-conventional yeast herein can (i) exhibit a rate of specific targeting by a suitable donor DNA having 30-50 bp flanking homology arms of less than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 8%, for example, and/or (ii) exhibit a rate of random integration of the foregoing donor DNA of more than about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, or 75%, for example. These rates of (i) specific targeting and/or (ii) random integration of a suitable donor DNA can characterize a non-conventional yeast as it exists before being provided an RGEN as disclosed herein. An aim for providing an RGEN to a non-conventional yeast in certain embodiments is to create site-specific DNA single-strand breaks (SSB) or double-strand breaks (DSB) for biasing the yeast toward HR at the specific site. Thus, providing a suitable RGEN in a non-conventional yeast typically should allow the yeast to exhibit an increased rate of HR with a particular donor DNA. Such an increased rate can be at least about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold higher than the rate of HR in a suitable control (e.g., same non-conventional yeast transformed with the same donor DNA, but lacking a suitable RGEN).

[0214] A non-conventional yeast herein can be cultivated following any means known in the art, such as described in Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology: Practical Protocols (K. Wolf, K. D. Breunig, G. Barth, Eds., Springer-Verlag, Berlin, Germany, 2003), Yeasts in Natural and Artificial Habitats (J. F. T. Spencer, D. M. Spencer, Eds., Springer-Verlag, Berlin, Germany, 1997), and/or Yeast Biotechnoloqy: Diversity and Applications (T. Satyanarayana, G. Kunze, Eds., Springer, 2009), all of which are incorporated herein by reference.

[0215] Non-limiting examples of non-conventional yeast herein include yeasts of the following genera: Yarrowia, Pichia, Schwanniomyces, Kluyveromyces, Arxula, Trichosporon, Candida, Ustilago, Torulopsis, Zygosaccharomyces, Trigonopsis, Cryptococcus, Rhodotorula, Phaffia, Sporobolomyces, Pachysolen, and Moniliella. A suitable example of a Yarrowia species is Y. lipolytica. Suitable examples of Pichia species include P. pastoris, P. methanolica, P. stipitis, P. anomala and P. angusta. Suitable examples of Schwanniomyces species include S. castellii, S. alluvius, S. hominis, S. occidentalis, S. capriottii, S. etchellsii, S. polymorphus, S. pseudopolymorphus, S. vanrijiae and S. yamadae. Suitable examples of Kluyveromyces species include K. lactis, K. marxianus, K. fragilis, K. drosophilarum, K. thermotolerans, K. phaseolosporus, K. vanudenii, K. waltii, K. africanus and K. polysporus. Suitable examples of Arxula species include A. adeninivorans and A. terrestre. Suitable examples of Trichosporon species include T. cutaneum, T. capitatum, T. inkin and T. beemeri. Suitable examples of Candida species include C. albicans, C. ascalaphidarum, C. amphixiae, C. antarctica, C. apicola, C. argentea, C. atlantica, C. atmosphaerica, C. blattae, C. bromeliacearum, C. carpophila, C. carvajalis, C. cerambycidarum, C. chauliodes, C. corydali, C. dosseyi, C. dubliniensis, C. ergatensis, C. fructus, C. glabrata, C. fermentati, C. guilliermondii, C. haemulonii, C. insectamens, C. insectorum, C. intermedia, C. jeffresii, C. kefyr, C. keroseneae, C. krusei, C. lusitaniae, C. lyxosophila, C. maltosa, C. marina, C. membranifaciens, C. milleri, C. mogii, C. oleophila, C. oregonensis, C. parapsilosis, C. quercitrusa, C. rugosa, C. sake, C. shehatea, C. temnochilae, C. tenuis, C. theae, C. tolerans, C. tropicalis, C. tsuchiyae, C. sinolaborantium, C. sojae, C. subhashii, C. viswanathii, C. utilis, C. ubatubensis and C. zemplinina. Suitable examples of Ustilago species include U. avenae, U. esculenta, U. hordei, U. maydis, U. nuda and U. tritici. Suitable examples of Torulopsis species include T. geochares, T. azyma, T. glabrata and T. candida. Suitable examples of Zygosaccharomyces species include Z. bailii, Z. bisporus, Z. cidri, Z. fermentati, Z. florentinus, Z. kombuchaensis, Z. lentus, Z. mellis, Z. microellipsoides, Z. mrakii, Z. pseudorouxii and Z. rouxii. Suitable examples of Trigonopsis species include T. variabilis. Suitable examples of Cryptococcus species include C. laurentii, C. albidus, C. neoformans, C. gattii, C. uniguttulatus, C. adeliensis, C. aerius, C. albidosimilis, C. antarcticus, C. aquaticus, C. ater, C. bhutanensis, C. consortionis, C. curvatus, C. phenolicus, C. skinneri, C. terreus and C. vishniacci. Suitable examples of Rhodotorula species include R. acheniorum, R. tula, R. acuta, R. americana, R. araucariae, R. arctica, R. armeniaca, R. aurantiaca, R. auriculariae, R. bacarum, R. benthica, R. biourgei, R. bogoriensis, R. bronchialis, R. buffonii, R. calyptogenae, R. chungnamensis, R. cladiensis, R. corallina, R. cresolica, R. crocea, R. cycloclastica, R. dairenensis, R. diffluens, R. evergladiensis, R. ferulica, R. foliorum, R. fragaria, R. fujisanensis, R. futronensis, R. gelatinosa, R. glacialis, R. glutinis, R. gracilis, R. graminis, R. grinbergsii, R. himalayensis, R. hinnulea, R. histolytica, R. hylophila, R. incarnata, R. ingeniosa, R. javanica, R. koishikawensis, R. lactosa, R. lamellibrachiae, R. laryngis, R. lignophila, R. lini, R. longissima, R. ludwigii, R. lysinophila, R. marina, R. martyniae-fragantis, R. matritensis, R. meli, R. minuta, R. mucilaginosa, R. nitens, R. nothofagi, R. oryzae, R. pacifica, R. pallida, R. peneaus, R. philyla, R. phylloplana, R. pilatii, R. pilimanae, R. pinicola, R. plicata, R. polymorpha, R. psychrophenolica, R. psychrophila, R. pustula, R. retinophila, R. rosacea, R. rosulata, R. rubefaciens, R. rubella, R. rubescens, R. rubra, R. rubrorugosa, R. rufula, R. rutila, R. sanguinea, R. sanniei, R. sartoryi, R. silvestris, R. simplex, R. sinensis, R. slooffiae, R. sonckii, R. straminea, R. subericola, R. suganii, R. taiwanensis, R. taiwaniana, R. terpenoidalis, R. terrea, R. texensis, R. tokyoensis, R. ulzamae, R. vanillica, R. vuilleminii, R. yarrowii, R. yunnanensis and R. zsoltii. Suitable examples of Phaffia species include P. rhodozyma. Suitable examples of Sporobolomyces species include S. alborubescens, S. bannaensis, S. beijingensis, S. bischofiae, S. clavatus, S. coprosmae, S. coprosmicola, S. corallinus, S. dimmenae, S. dracophylli, S. elongatus, S. gracilis, S. inositophilus, S. johnsonii, S. koalae, S. magnisporus, S. novozealandicus, S. odorus, S. patagonicus, S. productus, S. roseus, S. sasicola, S. shibatanus, S. singularis, S. subbrunneus, S. symmetricus, S. syzygii, S. taupoensis, S. tsugae, S. xanthus and S. yunnanensis. Suitable examples of Pachysolen and Moniliella species include P. tannophilus and M. pollinis, respectively. Still other examples of non-conventional yeasts herein include Pseudozyma species (e.g., S. antarctica), Thodotorula species (e.g., T. bogoriensis), Wickerhamiella species (e.g., W. domercqiae), and Starmerella species (e.g., S. bombicola).

[0216] Yarrowia lipolytica is preferred in certain embodiments disclosed herein. Examples of suitable Y. lipolytica include the following isolates available from the American Type Culture Collection (ATCC, Manassas, Va.): strain designations ATCC #20362, #8862, #8661, #8662, #9773, #15586, #16617, #16618, #18942, #18943, #18944, #18945, #20114, #20177, #20182, #20225, #20226, #20228, #20327, #20255, #20287, #20297, #20315, #20320, #20324, #20336, #20341, #20346, #20348, #20363, #20364, #20372, #20373, #20383, #20390, #20400, #20460, #20461, #20462, #20496, #20510, #20628, #20688, #20774, #20775, #20776, #20777, #20778, #20779, #20780, #20781, #20794, #20795, #20875, #20241, #20422, #20423, #32338, #32339, #32340, #32341, #34342, #32343, #32935, #34017, #34018, #34088, #34922, #34922, #38295, #42281, #44601, #46025, #46026, #46027, #46028, #46067, #46068, #46069, #46070, #46330, #46482, #46483, #46484, #46436, #60594, #62385, #64042, #74234, #76598, #76861, #76862, #76982, #90716, #90811, #90812, #90813, #90814, #90903, #90904, #90905, #96028, #201241, #201242, #201243, #201244, #201245, #201246, #201247, #201249, and/or #201847.

[0217] A fungal cell herein can be a yeast (e.g., as described above) or of any other fungal type such as a filamentous fungus. For instance, a fungus herein can be a Basidiomycetes, Zygomycetes, Chytridiomycetes, or Ascomycetes fungus.

[0218] Examples of filamentous fungi herein include those of the genera Trichoderma, Chrysosporium, Thielavia, Neurospora (e.g., N. crassa, N. sitophila), Cryphonectria (e.g., C. parasitica), Aureobasidium (e.g., A. pullulans), Filibasidium, Piromyces, Cryplococcus, Acremonium, Tolypocladium, Scytalidium, Schizophyllum, Sporotrichum, Penicillium (e.g., P. bilaiae, P. camemberti, P. candidum, P. chrysogenum, P. expansum, P. funiculosum, P. glaucum, P. marneffei, P. roqueforti, P. verrucosum, P. viridicatum), Gibberella (e.g., G. acuminata, G. avenacea, G. baccata, G. circinata, G. cyanogena, G. fujikuroi, G. intricans, G. pulicaris, G. stilboides, G. tricincta, G. zeae), Myceliophthora, Mucor (e.g., M. rouxii, M. circinelloides), Aspergillus (e.g., A. niger, A. oryzae, A. nidulans, A. flavus, A. lentulus, A. terreus, A. clavatus, A. fumigatus), Fusarium (e.g., F. graminearum, F. oxysporum, F. bubigenum, F. solani, F. oxysporum, F. verticillioides, F. proliferatum, F. venenatum), and Humicola, and anamorphs and teleomorphs thereof. The genus and species of fungi herein can be defined, if desired, by morphology as disclosed in Barnett and Hunter (Illustrated Genera of Imperfect Funqi, 3rd Edition, Burgess Publishing Company, 1972). A fungus can optionally be characterized as a pest/pathogen of a plant or animal (e.g., human) in certain embodiments.

[0219] Trichoderma species in certain aspects herein include T. aggressivum, T. amazonicum, T. asperellum, T. atroviride, T. aureoviride, T. austrokoningii, T. brevicompactum, T. candidum, T. caribbaeum, T. catoptron, T. cremeum, T. ceramicum, T. cerinum, T. chlorosporum, T. chromospermum, T. cinnamomeum, T. citrinoviride, T. crassum, T. cremeum, T. dingleyeae, T. dorotheae, T. effusum, T. erinaceum, T. estonicum, T. fertile, T. gelatinosus, T. ghanense, T. hamatum, T. harzianum, T. helicum, T. intricatum, T. konilangbra, T. koningii, T. koningiopsis, T. longibrachiatum, T. longipile, T. minutisporum, T. oblongisporum, T. ovalisporum, T. petersenii, T. phyllostahydis, T. piluliferum, T. pleuroticola, T. pleurotum, T. polysporum, T. pseudokoningii, T. pubescens, T. reesei, T. rogersonii, T. rossicum, T. saturnisporum, T. sinensis, T. sinuosum, T. spirale, T. stramineum, T. strigosum, T. stromaticum, T. surrotundum, T. taiwanense, T. thailandicum, T. thelephoricolum, T. theobromicola, T. tomentosum, T. velutinum, T. virens, T. viride and T. viridescens. A Trichoderma species herein can be cultivated and/or manipulated as described in Trichoderma: Bioloqy and Applications (P. K. Mukherjee et al., Eds., CABI, Oxfordshire, U K, 2013), for example, which is incorporated herein by reference.

[0220] A microbial cell in certain embodiments is an algal cell. For example, an algal cell can be from any of the following: Chlorophyta (green algae), Rhodophyta (red algae), Phaeophyceae (brown algae), Bacillariophycaeae (diatoms), and Dinoflagellata (dinoflagellates). An algal cell can be of a microalgae (e.g., phytoplankton, microphytes, or planktonic algae) or macroalgae (kelp, seaweed) in other aspects. As further examples, an algal cell herein can be a Porphyra (purple laver), Palmaria species such as P. palmata (dulse), Arthrospira species such as A. platensis (spirulina), Chlorella (e.g., C. protothecoides), a Chondrus species such as C. crispus (Irish moss), Aphanizomenon, Sargassum, Cochayuyo, Botryococcus (e.g., B. braunii), Dunaliella (e.g., D. tertiolecta), Gracilaria, Pleurochrysis (e.g., P. carterae), Ankistrodesmus, Cyclotella, Hantzschia, Nannochloris, Nannochloropsis, Nitzschia, Phaeodactylum (e.g., P. tricornutum), Scenedesmus, Stichococcus, Tetraselmis (e.g., T. suecica), Thalassiosira (e.g., T. pseudonana), Crypthecodinium (e.g., C. cohnii), Neochloris (e.g., N. oleoabundans), or Schiochytrium. An algal species herein can be cultivated and/or manipulated as described in Thompson (Alqal Cell Culture. Encyclopedia of Life Support System (EOLSS), Biotechnoloqy Vol 1, available at eolss.net/sample-chapters internet site), for example, which is incorporated herein by reference.

[0221] In one embodiment, the method comprises a method of delivering a protein component of an RNA-guided endonuclease (RGEN) into a microbial cell, said method comprising: contacting the microbial cell with a composition comprising the protein component of the RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein said protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, wherein said RGEN protein-CPP complex traverses (i) a cell membrane, or (ii) a cell wall and cell membrane, of the cell, thereby entering the microbial cell. Microbial cells useful for the methods and composition described herein include cells selected from Phytophtora species such as Phytophtora capsici (Lamour et al. 2012. The oomycete broad-host-range pathogen Phytophthora capsici. Mol. Plant Pathol. May 13(4): 329-337), Zymoseptoria species such as Septoria tritici (Testa et al. 2015. Overview of genomic and bioinformatics resources for Zymoseptoria tritici. Fungal Genet. Biol. Jun. 79:13-16) and Botrytis species such as Botrytis cinerea (Hahn M. 2014. The rising threat of fungicide resistance in plant pathogenic fungi: Botrytis as a case study. J. Chem. Biol 7:133-141).

[0222] A protist cell herein can be selected from the class Ciliata (e.g., the genera Tetrahymena, Paramecium, Coipidium, Colpoda, Glaucoma, Platyphrya, Vorticella, Potomacus, Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonichia), the subphylum Mastigophora (flagellates), the class Phytomastigophorea (e.g., the genera Euglena, Astasia, Haematococcus, and Crypthecodinium), the class Zoomastigophorea, the superclass Rhizopoda, the class Lobosea (e.g., the genus Amoeba), and the class Eumycetozoea (e.g., the genera Dictyostelium and Physarum), for example. Certain protist species herein can be cultivated and/or manipulated as described in ATCC.RTM. Protistology Culture Guide: tips and techniques for propaqatinq protozoa and algae (2013, available at American Type Culture Collection internet site), for example, which is incorporated herein by reference. A protist can optionally be characterized as a pest/pathogen of a plant or animal (e.g., human) in certain embodiments.

[0223] A bacterial cell in certain embodiments can be those in the form of cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Other non-limiting examples of bacteria include those that are Gram-negative and Gram-positive. Still other non-limiting examples of bacteria include those of the genera Salmonella (e.g., S. typhi, S. enteritidis), Shigella (e.g., S. dysenteriae), Escherichia (e.g., E. coli), Enterobacter, Serratia, Proteus, Yersinia, Citrobacter, Edwardsiella, Providencia, Klebsiella, Hafnia, Ewingella, Kluyvera, Morganella, Planococcus, Stomatococcus, Micrococcus, Staphylococcus (e.g., S. aureus, S. epidermidis), Vibrio (e.g., V. cholerae), Aeromonas, Plessiomonas, Haemophilus (e.g., H. influenzae), Actinobacillus, Pasteurella, Mycoplasma (e.g., M. pneumonia), Ureaplasma, Rickettsia, Coxiella, Rochalimaea, Ehrlichia, Streptococcus (e.g., S. pyogenes, S. mutans, S. pneumoniae), Enterococcus (e.g., E. faecalis), Aerococcus, Gemella, Lactococcus (e.g., L. lactis), Leuconostoc (e.g., L. mesenteroides), Pedicoccus, Bacillus (e.g., B. cereus, B. subtilis, B. thuringiensis), Corynebacterium (e.g., C. diphtheriae), Arcanobacterium, Actinomyces, Rhodococcus, Listeria (e.g., L. monocytogenes), Erysipelothrix, Gardnerella, Neisseria (e.g., N. meningitidis, N. gonorrhoeae), Campylobacter, Arcobacter, Wolinella, Helicobacter (e.g., H. pylori), Achromobacter, Acinetobacter, Agrobacterium (e.g., A. tumefaciens), Alcaligenes, Chryseomonas, Comamonas, Eikenella, Flavimonas, Flavobacterium, Moraxella, Oligella, Pseudomonas (e.g., P. aeruginosa), Shewanella, Weeksella, Xanthomonas, Bordetella, Franciesella, Brucella, Legionella, Afipia, Bartonella, Calymmatobacterium, Cardiobacterium, Streptobacillus, Spirillum, Peptostreptococcus, Peptococcus, Sarcinia, Coprococcus, Ruminococcus, Propionibacterium, Mobiluncus, Bifidobacterium, Eubacterium, Lactobacillus (e.g., L. lactis, L. acidophilus), Rothia, Clostridium (e.g., C. botulinum, C. perfringens), Bacteroides, Porphyromonas, Prevotella, Fusobacterium, Bilophila, Leptotrichia, Wolinella, Acidaminococcus, Megasphaera, Veilonella, Norcardia, Actinomadura, Norcardiopsis, Streptomyces, Micropolysporas, Thermoactinomycetes, Mycobacterium (e.g., M. tuberculosis, M. bovis, M. leprae), Treponema, Borrelia (e.g., B. burgdorferi), Leptospira, and Chlamydiae. A bacteria can optionally be characterized as a pest/pathogen of a plant or animal (e.g., human) in certain embodiments. Bacteria can be comprised in a mixed microbial population (e.g., containing other bacteria, or containing yeast and/or other bacteria) in certain embodiments.

[0224] An archaeal cell in certain embodiments can be from any Archaeal phylum, such as Euryarchaeota, Crenarchaeota, Nanoarchaeota, Korarchaeota, Aigarchaeota, or Thaumarchaeota. Archaeal cells herein can be extremophilic (e.g., able to grow and/or thrive in physically or geochemically extreme conditions that are detrimental to most life), for example. Some examples of extremophilic archaea include those that are thermophilic (e.g., can grow at temperatures between 45-122.degree. C.), hyperthermophilic (e.g., can grow at temperatures between 80-122.degree. C.), acidophilic (e.g., can grow at pH levels of 3 or below), alkaliphilic (e.g., can grow at pH levels of 9 or above), and/or halophilic (e.g., can grow in high salt concentrations [e.g., 20-30% NaCl]). Examples of archaeal species include those of the genera Halobacterium (e.g., H. volcanii), Sulfolobus (e.g., S. solfataricus, S. acidocaldarius), Thermococcus (e.g., T. alcaliphilus, T. celer, T. chitonophagus, T. gammatolerans, T. hydrothermalis, T. kodakarensis, T. litoralis, T. peptonophilus, T. profundus, T. stetteri), Methanocaldococcus (e.g., M. thermolithotrophicus, M. jannaschii), Methanococcus (e.g., M. maripaludis), Methanothermobacter (e.g., M. marburgensis, M. thermautotrophicus), Archaeoglobus (e.g., A. fulgidus), Nitrosopumilus (e.g., N. maritimus), Metallosphaera (e.g., M. sedula), Ferroplasma, Thermoplasma, Methanobrevibacter (e.g., M. smithii), and Methanosphaera (e.g., M. stadtmanae).

[0225] Examples of insect cells herein include Spodoptera frugiperda cells, Trichoplusia ni cells, Bombyx mori cells and the like. S. frugiperda cells include Sf9 and Sf21, for instance. T. ni ovary cells include HIGH FIVE cells (alias BTI-TN-5B1-4, manufactured by Invitrogen), for example. B. mori cells include N4, for example. Certain insect cells herein can be cultivated and/or manipulated as described in Growth and Maintenance of Insect cell lines (2010, Invitrogen, Manual part no. 25-0127, MAN0000030), for example, which is incorporated herein by reference. In other aspects, an insect cell can be a cell of a plant pest/pathogen such as an armyworm, black cutworm, corn earworm, corn flea beetle, corn leaf aphid, corn root aphid, European corn borer, fall armyworm, granulate cutworm, Japanese beetle, lesser cornstalk borer, maize billbug, melanotus communis, seedcorn maggot, sod webworms, sorghum midge, sorghum webworm, southern corn billbug, southern corn rootworm, southern cornstalk borer, southern potato wireworm, spider mite, stalk borer, sugarcane beetle, tobacco wireworm, white grub, aphid, boll weevil, bollworm complex, cabbage looper, tarnished plant bug, thrip, two spotted spider mite, yellow striped armyworm, alfalfa weevil, clover leaf weevil, clover root curculio, fall armyworm, grasshopper, meadow spittlebug, pea aphid, potato leafhopper, sod webworm, variegated cutworm, lesser cornstalk borer, tobacco thrip, wireworm, cereal leaf beetle, chinch bug, English grain aphid, greenbug, hessian fly, bean leaf beetle, beet armyworm, blister beetle, grape colaspis, green cloverworm, Mexican bean beetle, soybean looper, soybean stem borer, stink bug, three-cornered alfalfa hopper, velvetbean caterpillar, budworm, cabbage looper, cutworm, green june beetle, green peach aphid, hornworm, potato tuberworm, southern mole cricket, suckfly, tobacco flea beetle, vegetable weevil, or whitefringed beetle. Alternatively, an insect cell can be a cell of a pest/pathogen of an animal (e.g., human).

[0226] A nematode cell, for example, can be of a nematode from any of the following genera: Meloidogyne (root-knot nematode), Pratylenchus (lesion nematode), Heterodera (cyst nematode), Globodera (cyst nematode), Ditylenchus (stem and bulb nematode), Tylenchulus (citrus nematode), Xiphinema (dagger nematode), Radopholus (burrowing nematode), Rotylenchulus (reniform nematode), Helicotylenchus (spiral nematode), or Belonolaimus (sting nematode). A nematode can optionally be characterized as a pest/pathogen of a plant or animal (e.g., human) in certain embodiments. A nematode can be C. elegans in other aspects.

[0227] A fish cell herein can be any of those as disclosed in U.S. Pat. Nos. 7,408,095 and 7,217,564, and Tissue Culture of Fish Cell Lines (T. Ott, NWFHS Laboratory Procedures Manual--Second Edition, Chapter 10, 2004), for example, which are incorporated herein by reference. These references also disclose information regarding cultivating and/or manipulating fish cells. Non-limiting examples of fish cells can be from a teleost such as zebrafish, medaka, Giant rerio, or puffer fish.

[0228] A plant cell herein can be, for example, a monocot plant cell or dicot plant cell. Examples of monocot plants herein include corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet, Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), wheat (Triticum aestivum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), banana (Musa spp.), palm, ornamentals, and turfgrasses. Examples of dicot plants herein include soybean (Glycine max), canola (Brassica napus and B. campestris), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (A. thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum), peanut (Arachis hypogaea), tomato (Solanum lycopersicum), and potato (Solanum tuberosum). A plant cell may be from any part of a plant and/or from any stage of plant development.

[0229] Plant cells herein may be grown or regenerated into plants using conventional conditions, see for example, McCormick et al., (1986) Plant Cell Rep 5:81-4. Regenerated plants may then be grown, and either pollinated with the same strain or with a different strain, and resulting progeny having the desired characteristic (e.g., alteration) and/or comprising an introduced polynucleotide or polypeptide identified. Two or more generations may be grown to ensure that an alteration is stably maintained and inherited, and seeds harvested.

[0230] Mammalian cells in certain embodiments can be human, non-human primate (e.g., monkey, ape), rodent (e.g., mouse, rat, hamster, guinea pig), rabbit, dog, cat, cow, pig, horse, goat, or sheep cells. Other examples of mammalian cells herein include primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells, retinal epithelial cells); established cell lines (e.g., 293 embryonic kidney cells, HeLa cervical epithelial cells, PER-C6 retinal cells, MDBK, CRFK, MDCK, CHO, BeWo, Chang cells, Detroit 562, Hep-2, KB, LS 180, LS 174T, NCI-H-548, RPMI 2650, SW-13, T24, WI-28 VA13, 2RA, WISH, BS--C-I, LLC-MK2, Clone M-3, RAG, TCMK-1, LLC-PK1, PK-15, GH1, GH3, L2, LLC-RC 256, MH1C1, XC, MDOK, VSW, TH-I, B1 cells); any epithelial, mesenchymal (e.g., fibroblast), neural, or muscular cell from any tissue or organ (e.g., skin, heart; liver; kidney; colon; intestine; esophagus; stomach; neural tissue such as brain or spinal cord; lung; vascular tissue; lymphoid tissue such as lymph gland, adenoid, tonsil, bone marrow, or blood; spleen); and fibroblast or fibroblast-like cell lines (e.g., TRG-2, IMR-33, Don cells, GHK-21, citrullinemia cells, Dempsey cells, Detroit 551, Detroit 510, Detroit 525, Detroit 529, Detroit 532, Detroit 539, Detroit 548, Detroit 573, HEL 299, IMR-90, MRC-5, WI-38, WI-26, MiCI1, CV-1, COS-1, COS-3, COS-7, Vero, DBS-FrhL-2, BALB/3T3, F9, SV-T2, M-MSV-BALB/3T3, K-BALB, BLO-11, NOR-10, C3H/IOTI/2, HSDM1C3, KLN205, McCoy cells, Mouse L cells, SCC-PSA1, Swiss/3T3 cells, Indian muntjac cells, SIRC, Jensen cells). Methods of culturing and manipulating mammalian cells lines are known in the art.

[0231] In certain embodiments, a microbial cell can be of any pathogen and/or pest of an animal or plant. Examples of such pathogens/pests include various types of bacteria, fungi, yeast, protists, nematodes, and insects. Those skilled in the art would recognize examples of such pathogens/pests disclosed above.

[0232] As described herein (see Example 10), cell-penetrating peptides were able to deliver cargo to different eukaryotic species including Phytophthora capsici, Septoria tritici, and Botrytis cinerea.

[0233] In one embodiment, the method described herein is a method of delivering a protein component of an RNA-guided endonuclease (RGEN) into a microbial cell selected from the group consisting of Phytophthora capsici, Septoria tritici, and Botrytis cinerea, said method comprising: contacting the microbial cell with a composition comprising the protein component of the RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein said protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, wherein said RGEN protein-CPP complex traverses (i) a cell membrane, or (ii) a cell wall and cell membrane, of the cell, thereby entering the microbial cell.

[0234] A composition in certain embodiments herein can comprise at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one cell-penetrating peptide (CPP), wherein the protein component and CPP are covalently, or non-covalently, linked to each other in a polynucleotide/endonuclease protein-CPP complex, and wherein the polynucleotide/endonuclease protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell (such as a microbial cell).

[0235] The guide polynucleotide and Cas endonuclease are capable of forming a complex, referred to as a "guide polynucleotide/Cas endonuclease complex", that enables the Cas endonuclease to introduce a double-strand break at a DNA target site.

[0236] The disclosed invention also concerns a method of delivering a protein component of an RNA-guided endonuclease (RGEN) into a cell (such as a microbial cell). This method comprises contacting a cell with a composition comprising the RGEN protein component and at least one cell-penetrating peptide (CPP), wherein the RGEN protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex. As a result of this contacting step, the RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of the cell, and thereby gain entry to the cell. In certain embodiments in which an RGEN protein component is associated with an RNA component (thereby forming an RGEN), the disclosed method is directed to delivering an RGEN-CPP complex into a cell. Additionally, since an RGEN can be used in RGEN-mediated DNA targeting in certain embodiments, this method can optionally be characterized as a method of targeting DNA in a cell.

[0237] This method can be practiced using any of the above-disclosed embodiments or below Examples regarding each of the method features (e.g., cell type, RGEN protein component, CPP, organelle-targeting sequence, etc.), for example. Thus, any of the features disclosed above or in the Examples, or any combination of these features, can be used appropriately to characterize embodiments of a delivery method herein. The following delivery method features are examples.

[0238] Embodiments of a delivery method herein comprise contacting a cell (such as a microbial cell) with a composition comprising an RGEN protein-CPP complex. It is believed that such contacting results in interaction of the complex with the outer surface of the cell (e.g., cell membrane, cell wall), thereby allowing the CPP component of the complex to initiate traversal of the complex across (i) a cell membrane, or (ii) a cell wall and cell membrane.

[0239] Contacting a composition comprising an RGEN protein-CPP complex with a cell (such as a microbial cell) can be done at a temperature that allows the complex to enter the cell. Such contacting can be done at any temperature between about 4 and 45.degree. C., for example. The contacting temperature can be about 4, 15, 20, 30, 37, or 42.degree. C. in non-limiting embodiments. The same temperature or temperature range can be maintained during the contacting step, or modified appropriately (e.g., two or more different temperatures).

[0240] Contacting a composition comprising an RGEN protein-CPP complex with a cell can be done for an amount of time that is adequate for allowing the complex to enter the cell. For example, cells can be incubated with an RGEN protein-CPP complex for at least about 15, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 240, 300, 360, 420, 480, 540, 600, 660, or 720 minutes.

[0241] The milieu (e.g., buffer, water and salt concentrations, pH, purity of RGEN protein-CPP complex) in which the contacting is performed may be any of those conditions disclosed above regarding a composition comprising an RGEN protein-CPP complex. For example, cells can be incubated with a complex in a HEPES buffer (e.g., .about.25 mM HEPES, such as 25 mM HEPES/KOH pH 7.5, 200 mM KCl, 20% glycerol, 1 mM DTT) or PBS (e.g., 1.times.PBS, pH 7).

[0242] One or more cells (such as microbial cells) may be contacted with a composition comprising an RGEN protein-CPP complex. A cell herein may be as it exists (i) in an organism/tissue in vivo, (ii) in a tissue or group of cells ex vivo, or (iii) in an in vitro state (e.g., cultured cells).

[0243] Entry of an RGEN protein-CPP complex into a cell herein typically refers to when a complex has completely traversed (i) a cell membrane, or (ii) a cell wall and cell membrane, and is comprised within at least the cell cytoplasm. Though not intending to be held to any particular theory or mechanism, it is believed that an RGEN protein-CPP complex held together by non-covalent linkage either remains in a complete or partial complex, or the RGEN protein component separates from the CPP component(s) of the complex, after the RGEN protein-CPP complex gains cell entry. In either case, the RGEN protein component is able to associate with a suitable RNA component herein; such association can occur in the cytoplasm, nucleus, or mitochondria, for example. This capability likewise applies to an RGEN protein-CPP complex held together by covalent linkage.

[0244] In certain embodiments of an RGEN protein delivery method, a composition herein further comprises at least one RNA component that is associated with the RGEN protein component of the RGEN protein-CPP complex (i.e., the composition comprises an RGEN-CPP complex). The RNA component in this embodiment can be as disclosed herein, comprising a sequence complementary to a target site sequence on a chromosome or episome in the microbial cell. The RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence. Such an embodiment can also be characterized as a method of delivering an RGEN-CPP complex into a microbial cell, or alternatively as a method of delivering an RNA into a microbial cell.

[0245] An RNA component (e.g., gRNA) for use in this embodiment can be prepared using any number of means known in the art. For example, an in vitro transcription process can be used to prepare an RNA component herein. Bacterial RNA polymerases (e.g., T7, T3, SP6) can be used to transcribe an RNA component from a suitable DNA construct encoding the RNA component in certain non-limiting embodiments. An RNA component may be processed to at least about 70%, 80%, 90%, or 95% purity with respect to other biomolecules (e.g., protein, saccharides, lipids), if desired.

[0246] To prepare a composition comprising an RNA component and an RGEN protein-CPP complex, the RNA component can be dissolved in a composition in which an RGEN protein-CPP complex is already dissolved, or vice versa (or these components can be dissolved at the same time). A molar ratio of RNA component to RGEN protein-CPP complex of at least about 0.5:1, 1.0:1, 1.5:1, 2.0:1, 2.5:1, 3.0:1, 3.5:1, or 4.0:1, for example, can be used when mixing these elements together. In certain aspects, the molar ratio of RNA component to RGEN protein-CPP complex can be about 3.0:1, or can range from about 2.5:1 to 3.5:1, 2.75:1 to 3.25:1, or 2.9:1 to 3.1:1. In these and other aspects, the concentration of an RGEN protein-CPP complex with which an RNA component is mixed can be at least about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0 .mu.M, or about 0.5 to 5.0 .mu.M, 0.5 to 2.5 .mu.M, 1.0 to 5.0 .mu.M, 1.0 to 2.5 .mu.M, or 2.5 to 5.0 .mu.M. The amount of time allowed for RNA association with an RGEN protein-CPP complex to form an RGEN-CPP complex can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, or 60 minutes, for example. Other conditions (e.g., temperature, buffer, water and salt concentrations, pH, purity of RGEN protein-CPP complex) in which an RNA component can be associated with an RGEN protein-CPP complex may be any of those conditions disclosed above regarding (i) a composition comprising an RGEN protein-CPP complex, or (ii) contacting an RGEN protein-CPP complex with a cell. For example, an RNA component such as a gRNA can be contacted with an RGEN protein-CPP complex in a HEPES buffer (e.g., .about.25 mM HEPES, such as 25 mM HEPES/KOH pH 7.5, 200 mM KCl, 20% glycerol, 1 mM DTT), or PBS (e.g., 1.times.PBS, pH 7), at room temperature (e.g., about 20-25.degree. C.) for about 15 minutes. In those embodiments in which an RGEN protein-CPP complex is held together by non-covalent linkage, association of an RNA component to an RGEN protein can comprise adding an RNA component before, at the same time of, or after incubating a CPP with the RGEN protein component.

[0247] After associating an RNA component with an RGEN protein-CPP complex the resulting composition comprising an RGEN-CPP complex (e.g., CPP-Cas9/gRNA) can be immediately contacted with cells, for example. Contact can be made in the milieu in which the RNA component and RGEN protein-CPP complex were associated (e.g., see above), for example. A composition comprising an RGEN-CPP complex can be stored at about room temperature, 4.degree. C., or frozen (e.g., -20 or -80.degree. C.) for later use, if desired. RGEN-CPP complex stability, and/or ability to enter cells and effect DNA targeting, can remain unchanged, or can have at least about 50%, 60%, 70%, 80%, 90%, or 95% of either respective activity, even if the complex is in a composition that has been through one, two, or more freeze-thaw cycles.

[0248] A composition comprising an RGEN protein-CPP complex or RGEN-CPP complex, for contacting with a cell, may optionally comprise one or more volume exclusion agents, which are contemplated to enhance contact points between the cell and complexes. Examples of suitable volume exclusion agents herein include glycerol and polyethylene glycol (PEG). Other examples include anionic polymer such as polyacrylate, polymethylacrylate, or anionic polysaccharidic polymers (e.g., dextran sulfate). Still other examples of volume exclusion agents are disclosed in U.S. Pat. No. 4,886,741, which is incorporated herein by reference.

[0249] In certain embodiments of an RGEN protein delivery method, a cell (such as a microbial cell) comprises an RNA component that associates with an RGEN protein component of an RGEN protein-CPP complex after the RGEN protein-CPP complex enters the cell (i.e., thereby forming an RGEN-CPP complex in the cell). The RNA component in this embodiment can be as disclosed herein, comprising a sequence complementary to a target site sequence on a chromosome or episome in the cell. The RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence.

[0250] One or more RNA components herein can be stably or transiently expressed in a cell (such as a microbial cell) to which an RGEN protein-CPP complex is introduced, for example. As examples of transient expression, an RGEN protein-CPP complex can be (i) delivered into a cell that has previously been modified to transiently express an RNA component, (ii) co-delivered into a cell with an RNA component, or (iii) delivered into a cell after which the cell is modified for transient RNA component expression.

[0251] A DNA polynucleotide sequence comprising (i) a promoter operably linked to (ii) a nucleotide sequence encoding an RNA component can typically be used for stable and/or transient RNA component expression herein. Such a polynucleotide sequence can be comprised within a plasmid, yeast artificial chromosome (YAC), cosmid, phagemid, bacterial artificial chromosome (BAC), virus, or linear DNA (e.g., linear PCR product), for example, or any other type of vector or construct useful for transferring a polynucleotide sequence into a cell. This polynucleotide sequence can be capable of existing transiently (i.e., not integrated into the genome) or stably (i.e., integrated into the genome) in a cell. Also, this polynucleotide sequence can comprise, or lack, one or more suitable marker sequences (e.g., selection or phenotype marker).

[0252] A suitable promoter comprised in a polynucleotide sequence for expressing an RNA component herein can be constitutive or inducible, for example. A promoter in certain aspects can comprise a strong promoter, which is a promoter that can direct a relatively large number of productive initiations per unit time, and/or is a promoter driving a higher transcription level than the average transcription level of the genes in a cell comprising the strong promoter.

[0253] Examples of strong promoters useful in certain aspects herein (e.g., fungal and/or yeast cells) herein include those disclosed in U.S. Patent Appl. Publ. Nos. 2012/0252079 (DGAT2), 2012/0252093 (EL1), 2013/0089910 (ALK2), 2013/0089911 (SPS19), 2006/0019297 (GPD and GPM), 2011/0059496 (GPD and GPM), 2005/0130280 (FBA, FBAIN, FBAINm), 2006/0057690 (GPAT) and 2010/0068789 (YAT1), which are incorporated herein by reference. Other examples of strong promoters include those listed in Table 2, which also may be useful in fungal and/or yeast cells, for example.

TABLE-US-00006 TABLE 2 Strong Promoters Promoter Name Native Gene Reference.sup.a XPR2 alkaline extracellular protease U.S. Pat. No. 4,937,189; EP220864 TEF translation elongation factor U.S. Pat. No. 6,265,185 EF1-.alpha. (tef) GPD, GPM glyceraldehyde-3-phosphate- U.S. Pat. Nos. 7,259,255 dehydrogenase (gpd), and 7,459,546 phosphoglycerate mutase (gpm) GPDIN glyceraldehyde-3-phosphate- U.S. Pat. No. 7,459,546 dehydrogenase (gpd) GPM/FBAIN chimeric phosphoglycerate U.S. Pat. No. 7,202,356 mutase (gpm)/fructose- bisphosphate aldolase (fba1) FBA, FBAIN, fructose-bisphosphate aldolase U.S. Pat. No. 7,202,356 FBAINm (fba1) GPAT glycerol-3-phosphate U.S. Pat. No. 7,264,949 O-acyltransferase (gpat) YAT1 ammonium transporter U.S. patent application enzyme (yat1) Pub. No. 2006/0094102 EXP1 export protein U.S. Pat. No. 7,932,077 .sup.aEach reference in this table is incorporated herein by reference.

[0254] Other examples of strong promoters useful in certain embodiments herein include PGK1, ADH1, TDH3, TEF1, PHO5, LEU2, and GAL1 promoters, as well as strong yeast promoters disclosed in Velculescu et al. (Cell 88:243-251), which is incorporated herein by reference.

[0255] A promoter for stable and/or transient expression of an RNA component herein can be an RNA polymerase II (Pol II) promoter, for example. It is believed that all the above-listed strong promoters are examples of suitable Pol II promoters. Transcription from a Pol II promoter may involve formation of an RNA polymerase II complex of at least about 12 proteins (e.g., RPB1-RPN12 proteins), for example. RNA transcribed from a Pol II promoter herein typically is 5'-capped (e.g., contains an m.sup.7G group at the 5'-end) and/or has a polyadenylate (polyA) tail, for example. Means for removing a 5'-cap and/or polyA tail from an RNA component can be employed, if desired, when expressing an RNA component from a Pol II promoter. Suitable means for effectively removing a 5'-cap and/or polyA tail from a Pol II-transcribed RNA component herein include appropriate use of one or more ribozymes (see below), group 1 self-splicing introns, and group 2 self-splicing introns, for example.

[0256] Alternatively, a promoter for stable and/or transient expression of an RNA component herein can be an RNA polymerase III (Pol III) promoter, for example. Such a promoter typically allows for expressing an RNA component with defined 5'- and 3'-ends, since initiation and termination of transcription with an RNA polymerase III can be controlled. Examples of Pol III promoters useful herein include U6 and H1 promoters. Other suitable Pol III promoters are disclosed in U.S. Appl. Publ. No. 2010/0160416, for example, which is incorporated herein by reference.

[0257] One or more ribozyme sequences may be used to create defined 5' and/or 3' transcript ends, such as in those embodiments in which a Pol II promoter is used for expressing an RNA component in a cell. For example, a nucleotide sequence herein encoding an RNA component may further encode a ribozyme that is upstream of the sequence encoding the RNA component. Thus, a cell in certain embodiments further comprises a DNA polynucleotide sequence comprising (i) a promoter operably linked to (ii) a nucleotide sequence encoding, in 5'-to-3' direction, a ribozyme and an RNA component. Transcripts expressed from such a polynucleotide sequence autocatalytically remove the ribozyme sequence to yield an RNA with a defined 5'-end (without a 5'-cap) but which comprises the RNA component sequence. This "autoprocessed" RNA can comprise a crRNA or gRNA, for example, and can complex with an RGEN protein component such as a Cas9, thereby forming an RGEN.

[0258] A ribozyme herein can be a hammerhead (HH) ribozyme, hepatitis delta virus (HDV) ribozyme, group I intron ribozyme, RnaseP ribozyme, or hairpin ribozyme, for example. Other non-limiting examples of ribozymes herein include Varkud satellite (VS) ribozymes, glucosamine-6-phosphate activated ribozymes (glmS), and CPEB3 ribozymes. Lilley (Biochem. Soc. Trans. 39:641-646) discloses information pertaining to ribozyme structure and activity. Examples of ribozymes that should be suitable for use herein include ribozymes disclosed in EP0707638 and U.S. Pat. Nos. 6,063,566, 5,580,967, 5,616,459, and 5,688,670, which are incorporated herein by reference. Further information regarding using ribozymes to express RNA components with defined 5' and/or 3' ends is disclosed in U.S. Patent Appl. No. 62/036,652 (filed Aug. 13, 2014).

[0259] In certain embodiments, a DNA polynucleotide comprising a cassette for expressing an RNA component comprises a suitable transcription termination sequence downstream of the RNA component sequence. Examples of transcription termination sequences useful herein are disclosed in U.S. Pat. Appl. Publ. No. 2014/0186906, which is herein incorporated by reference. Such embodiments typically comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more residues following the end of the RNA component sequence, depending on the choice of terminator sequence. These additional residues can be all U residues, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% U residues, for example, depending on the choice of terminator sequence. Alternatively, a ribozyme sequence (e.g., hammerhead or HDV ribozyme) can be 3' of (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides downstream) the RNA component sequence, for example. A 3' ribozyme sequence can be positioned accordingly such that it cleaves itself from the RNA component sequence; such cleavage would render a transcript ending exactly at the end of the RNA component sequence, or with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more residues following the end of the RNA component sequence, for example.

[0260] An RNA component in other examples can be provided in the nucleus and/or cytoplasm of a cell into which an RGEN protein-CPP complex is delivered. For example, an RNA component expressed from a Pol II promoter without use of a 5'-located ribozyme sequence can be expected to exist in both the nucleus and cytoplasm. An RNA component expressed from any type of promoter (e.g. Pol II or III promoter) and using a 5'-located ribozyme sequence can be expected to exist mostly in the nucleus in other embodiments. An RNA component expressed from a Pol III promoter in certain aspects can be expected to exist mostly in the nucleus. In certain aspects, an RNA component is uncapped (e.g., by virtue of being expressed from a Pol III promoter, and/or by ribozyme autoprocessing) and typically is located in the nucleus, while in other aspects is capped and located in nuclear and cytoplasmic locations. In general, the RGEN protein component of an RGEN protein-CPP complex, once delivered into a cell, can associate with an RNA component (thereby forming an RGEN) in the cytoplasm and/or nucleus (depending on RNA component location). Such association in the nucleus is generally due to the ability of an RGEN protein component herein to localize to the nucleus as directed by an NLS.

[0261] An RGEN herein is useful for RGEN-mediated DNA targeting. Any of the above embodiments regarding delivering an RGEN protein component into a cell can be applied to a DNA targeting method. For example, an RGEN protein-CPP complex can be contacted with at least one RNA component outside of a microbial cell to form an RGEN-CPP complex for delivery into a cell for DNA targeting therein. As another example, an RGEN protein-CPP complex, after its delivery into a microbial cell, can be contacted with at least one RNA component inside a microbial cell to form an RGEN-CPP complex therein that can then mediate DNA targeting. The following disclosure regarding targeting methods refers to an "RGEN", as opposed to referring to an "RGEN-CPP complex". It would be understood that, depending on whether a covalent or non-covalent RGEN-CPP complex is used in an RGEN delivery method herein (and depending on how strong a non-covalent linkage is in embodiments employing a non-covalent RGEN-CPP complex), reference to an RGEN below refers to such an RGEN-CPP complex, accordingly.

[0262] An RGEN herein that can cleave one or both DNA strands of a DNA target sequence can be used in a DNA targeting method, for example. Such DNA targeting methods can involve HR-mediated DNA targeting if a suitable donor DNA is provided in the method. Thus, in certain embodiments, a microbial cell in a targeting method herein can comprise a donor polynucleotide comprising at least one sequence homologous to a sequence at or near a target site sequence (a sequence specifically targeted by an RGEN herein). Such embodiments can optionally be characterized in that the targeting method further comprises a step of providing a suitable donor polynucleotide to the microbial cell.

[0263] A donor polynucleotide herein can undergo HR with a sequence at or near a DNA target site if the target site contains a SSB or DSB (such as can be introduced using an RGEN herein). A "homologous sequence" within a donor polynucleotide herein can, for example, comprise or consist of a sequence of at least about 25, 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 nucleotides, or about 50-500, 50-550, 50-600, 50-650, or 50-700 nucleotides, that have 100% identity with a sequence at or near the target site sequence, or at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a sequence at or near the target site sequence, for example.

[0264] A donor polynucleotide herein can have two homologous sequences (homology arms), for example, separated by a sequence that is heterologous to sequence at or near a target site sequence. HR between such a donor polynucleotide and a target site sequence typically results in the replacement of a sequence at the target site with the heterologous sequence of the donor polynucleotide (i.e., a target site sequence located between target site sequences homologous to the homology arms of the donor polynucleotide is replaced by the heterologous sequence of the donor polynucleotide). In a donor polynucleotide with two homology arms, the arms can be separated by at least about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, 1000, 2500, 5000, 10000, 15000, 20000, 25000, or 30000 nucleotides (i.e., the heterologous sequence in the donor polynucleotide can be at least about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, 100, 250, 500, 1000, 2500, 5000, 10000, 15000, 20000, 25000, or 30000 nucleotides in length), for example. The length (e.g., any of the lengths disclosed above for a homologous sequence) of each homology arm may be the same or different. The percent identity (e.g., any of the % identities disclosed above for a homologous sequence) of each arm with respective homologous sequences at or near the target site can be the same or different.

[0265] A DNA sequence at or near (alternatively, in the locality or proximity of) the target site sequence that is homologous to a corresponding homologous sequence in a donor polynucleotide can be within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 450, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, or 60000 (or any integer between 1 and 60000) nucleotides (e.g., about 1-1000, 100-1000, 500-1000, 1-500, or 100-500 nucleotides), for example, from the predicted RGEN cut site (DSB or nick) in the target sequence. These nucleotide distances can be marked from the cut site to the first nucleotide of the homologous sequence, going either in the upstream or downstream direction from the cut site.

[0266] For example, a sequence near a target sequence that is homologous to a corresponding sequence in a donor polynucleotide can start at 500 nucleotide base pairs downstream the predicted RGEN cut site in a target sequence. In embodiments herein employing a donor polynucleotide with two homology arms (e.g., first and second homology arms separated by a heterologous sequence), a homologous sequence (corresponding in homology with the first homology arm of a donor) can be upstream the predicted RGEN cut site, and a homologous sequence (corresponding in homology with the second homology arm of a donor) can be downstream the predicted RGEN cut site, for example. The nucleotide distances of each of these upstream and downstream homologous sequences from the predicted cut site can be the same or different, and can be any of the nucleotide distances disclosed above, for example. For instance, the 3' end of a homologous sequence (corresponding in homology with the first homology arm of a donor) may be located 600 nucleotide base pairs upstream a predicted RGEN cut site, and the 5' end of a homologous sequence (corresponding in homology with the second homology arm of a donor) may be located 400 nucleotide base pairs downstream the predicted RGEN cut site.

[0267] A donor polynucleotide in various aspects can be delivered into a cell (such as a microbial cell) at or near (e.g., within 1, 2, 3 or more hours) the time when an RGEN protein-CPP complex is delivered into the cell. Such delivery can be via by any means known in the art suitable for the particular type of cell being used. These techniques include transformation (e.g., lithium acetate transformation [Methods in Enzymology, 194:186-187]), transfection, biolistic impact, electroporation, and microinjection, for example. As examples, U.S. Pat. Nos. 4,880,741 and 5,071,764, and Chen et al. (Appl. Microbiol. Biotechnol. 48:232-235), which are incorporated herein by reference, describe DNA transfer techniques for Y. lipolytica. Examples of delivery modes useful in plants include Agrobacterium-mediated transformation and biolistic particle bombardment.

[0268] An RGEN that cleaves one or both DNA strands of a DNA target sequence can be used to create an indel in other non-limiting embodiments of DNA targeting herein. A method of forming an indel in a cell can be performed as disclosed above for HR-mediated targeting, but without further providing a donor DNA polynucleotide that could undergo HR at or near the target DNA site (i.e., NHEJ is induced in this method). Examples of indels that can be created are disclosed herein. The size of an indel may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more bases, for example. An indel in certain embodiments can be even larger such as at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 bases. In still other embodiments, insertions or deletions can be at least about 500, 750, 1000, or 1500 bases. When attempting to create an indel in certain embodiments, a single base substitution may instead be formed in a target site sequence. Thus, a targeting method herein can be performed for the purpose of creating single base substitution, for example.

[0269] In certain embodiments of a targeting method herein aimed at indel formation, the frequency of indel formation in a non-conventional yeast (e.g., Y. lipolytica) is significantly higher than what would be observed using the same or similar targeting strategy in a conventional yeast such as S. cerevisiae. For example, while the frequency of indel formation in a conventional yeast may be about 0.0001 to 0.001 (DiCarlo et al., Nucleic Acids Res. 41:4336-4343), the frequency in a non-conventional yeast herein may be at least about 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, or 0.80. Thus, the frequency of indel formation in a non-conventional yeast herein may be at least about 50, 100, 250, 500, 750, 1000, 2000, 4000, or 8000 times higher, for example, than what would be observed using the same or similar RGEN-mediated targeting strategy in a conventional yeast.

[0270] A targeting method in certain embodiments can be performed to disrupt one or more DNA polynucleotide sequences encoding a protein or a non-coding RNA. An example of such a sequence that can be targeted for disruption is one encoding a marker (i.e., a marker gene). Non-limiting examples of markers herein include screenable markers and selectable markers. A screenable marker herein can be one that renders a cell visually different under appropriate conditions. Examples of screenable markers include polynucleotides encoding beta-glucuronidase (GUS), beta-galactosidase (lacZ), and fluorescent proteins (e.g., GFP, RFP, YFP, BFP). A selectable marker herein can be one that renders a cell resistant to a selective agent or selective environment. Examples of selectable markers are auxotrophic markers such as HIS3, LEU2, TRP1, MET15, or URA3, which allow cells such as yeast cells to survive in the absence of exogenously provided histidine, leucine, tryptophan, methionine, or uracil, respectively. Other examples of selectable markers are antibiotic- or antifungal-resistance markers such as those rendering a cell resistant to ampicillin, chloramphenicol, hygromycin B, nourseothricin, phleomycin, puromycin, or neomycin (e.g., G418). Examples of these methods can optionally be characterized as marker recycling methods.

[0271] At least one purpose for disrupting a marker in certain embodiments can be for marker recycling. Marker recycling is a process, for example, comprising (i) transforming a cell with a marker and heterologous DNA sequence, (ii) selecting a transformed cell comprising the marker and the heterologous DNA sequence (where a marker-selectable cell typically has a higher chance of containing the heterologous DNA sequence), (iii) disrupting the marker, and then repeating steps (i)-(iii) as many times as necessary (using the same [or different] marker, but each cycle using a different heterologous DNA sequence) to transform cells with multiple heterologous DNA sequences. One or more heterologous sequences in this process may comprise the marker itself in the form of a donor polynucleotide (e.g., marker flanked by homology arms for targeting a particular locus). Examples of marker recycling processes herein include those using URA3 as a marker, such as in certain methods employing a yeast (e.g., a non-conventional yeast such as Y. lipolytica).

[0272] An RGEN herein that can bind to a DNA target site sequence, but does not cleave any strand at the target site sequence, can be used in a DNA targeting method in other embodiments. Any RGEN disclosed herein that has only dysfunctional nuclease domains, but retains specific DNA-binding activity, can be used in this type of targeting method.

[0273] In certain embodiments of DNA targeting with an RGEN having no functional nuclease domains, an RGEN can bind to a target site and modulate transcription of a polynucleotide sequence (i.e., gene transcription). Typically, an RGEN is targeted to a regulatory sequence such as a promoter (e.g., within 1-1000, 1-500, 1-250, 1-125, or 1-50 bases upstream a transcription start site), a sequence encoding a 5'-untranslated RNA sequence, or an intron (e.g., first intron) to effect transcriptional modulation of a polynucleotide sequence.

[0274] As a non-limiting example, an RGEN linked or fused to a repressor transcription factor or repressor domain thereof can be used to repress, or silence, expression of one or more polynucleotide sequences. An RGEN in certain alternative embodiments can, by itself (without a repressor or domain thereof), inhibit gene expression; such an RGEN can be targeted such that it inhibits binding and/or movement of RNA transcriptional machinery necessary for transcription. A method incorporating any repressing RGEN can optionally be characterized as a gene silencing or transcriptional silencing method. The level of transcriptional down-regulation in a silencing method can be about 100% (gene completely silenced), or at least about 30% (gene moderately silenced), 40%, 50%, 60%, 70%, 80%, 90%, or 95% (gene substantially silenced), for example, compared to the transcription level before application of a repressing RGEN.

[0275] An RGEN linked or fused to an activator transcription factor or activator domain thereof can be used to upregulate expression of one or more polynucleotide sequences. A method incorporating such an activating RGEN can optionally be characterized as a transcriptional up-regulation or activation method. The level of transcriptional up-regulation in such a method can be at least about 25%, 50%, 75%, 100%, 250%, 500%, or 1000%, for example, compared to the transcription level before application of an activating RGEN.

[0276] In certain embodiment, an RGEN that can bind to a DNA target site sequence, but preferably does not cleave any strand at the target site sequence, can be used as a diagnostic tool (e.g., probe for detecting a DNA sequence). An RGEN protein component in DNA probe can be linked to a reporter agent such as a reporter protein (e.g., fluorescent protein such as GFP), for example. Specific DNA binding of the RGEN-reporter protein, as specified by the RNA component of the RGEN, can be incorporated in a detection system accordingly, taking advantage of the activity of the reporter agent. Flow cytometry (e.g., flow-activated cell sorting [FACS]) and fluorescence in situ hybridization (FISH) are examples of suitable detection systems herein that use a fluorescent reporter.

[0277] A targeting method herein can be performed in such a way that two or more DNA target sites are targeted in the method, for example. Such a method can optionally be characterized as a multiplex method. Two, three, four, five, six, seven, eight, nine, ten, or more target sites can be targeted at the same time in certain embodiments. A multiplex method is typically performed by a targeting method herein in which multiple different RNA components are provided, each designed to guide an RGEN to a unique DNA target site. For example, two or more different RNA components can be used to prepare a mix of RGEN-CPP complexes in vitro (e.g., following a procedure disclosed herein for associating an RNA component with an RGEN protein-CPP complex), which mix is then contacted with a cell.

[0278] Another aspect of multiplex targeting herein can comprise providing two or more different RNA components in a cell which associate with the RGEN protein components of RGEN protein-CPP complexes that have traversed into the cell. Such a method can comprise, for example, providing to the cell (i) individual DNA polynucleotides, each of which express a particular RNA component that, and/or (ii) at least one DNA polynucleotide encoding two or more RNA components (e.g., see below disclosure regarding tandem ribozyme-RNA component cassettes).

[0279] A multiplex method can optionally target DNA sites very close to the same sequence (e.g., a promoter or open reading frame, and/or sites that are distant from each other (e.g., in different genes and/or chromosomes). A multiplex method in other embodiments can be performed with (for HR) or without (for NHEJ leading to indel and/or base substitution) suitable donor DNA polynucleotides, depending on the desired outcome of the targeting (if an endonuclease- or nickase-competent RGEN is used). In still other embodiments, a multiplex method can be performed with a repressing or activating RGEN as disclosed herein. For example, multiple repressing RGENs can be provided that down-regulate a set of genes, such as genes involved in a particular metabolic pathway.

[0280] A multiplex method in certain embodiments can comprise providing to a cell a DNA polynucleotide comprising (i) a promoter operably linked to (ii) a sequence comprising more than one ribozyme-RNA component cassettes (i.e., tandem cassettes). A transcript expressed from such a DNA polynucleotide can have, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cassettes. A 3' ribozyme sequence can optionally be included following all or some RNA component sequences to allow cleavage and separation of the RNA component from downstream transcript sequence (i.e., tandem cassettes may comprise one or more ribozyme-RNA component-ribozyme cassettes). A DNA polynucleotide herein for expressing tandem ribozyme-RNA component-ribozyme cassettes can be designed such that there are about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more nucleotides between each cassette (e.g., non-coding spacer sequence). The distances between each cassette may be the same or different.

[0281] Any construct or vector comprising a DNA polynucleotide encoding an RNA component described herein can be introduced into a cell by any means known in the art suitable for the particular type of cell being used. For example, any of the means disclosed above for delivering a donor DNA into a cell can be employed.

[0282] Certain embodiments herein concern a method of modifying or altering a target site in the genome of a microbial cell, wherein the method comprises contacting the microbial cell with a guide polynucleotide and Cas endonuclease covalently or non-covalently linked to a CPP, wherein the guide polynucleotide and CPP-Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double-strand break at the target site in the genome of the microbial cell. The modification or alteration of the target site can include (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0283] Certain embodiments herein concern a polynucleotide sequence comprising a nucleotide sequence encoding a fusion protein that comprises a protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP). Any fusion protein as disclosed herein, for example, can be encoded by the nucleotide sequence. The nucleotide sequence may optionally be in operable linkage with a promoter sequence. Certain embodiments include, for example, a polynucleotide (e.g., vector or construct) comprising at least one open reading frame encoding any RGEN protein-CPP fusion disclosed herein. Such a coding region can optionally be operably linked to a promoter sequence suitable for expressing an RGEN protein-CPP fusion in a cell (e.g., bacteria cell; eukaryotic cell such as a yeast, insect, or mammalian cell) or in an in vitro protein expression system, for example. Examples of a vector or construct include circular (e.g., plasmid) and non-circular (e.g., linear DNA such as an amplified DNA sequence) polynucleotide molecules.

[0284] Certain embodiments herein concern a method of producing an RGEN protein-CPP fusion protein comprising the steps of: providing a polynucleotide sequence having a nucleotide sequence encoding the RGEN protein-CPP fusion protein, and expressing the RGEN protein-CPP fusion protein from the polynucleotide sequence, thereby producing the RGEN protein-CPP fusion protein.

[0285] The expression step in such a method can optionally be performed in a cell (e.g., bacteria cell such as E. coli; eukaryotic cell such as a yeast [e.g., S. cerevisiae], insect, or mammalian cell). Alternatively, expression of an RGEN protein-CPP fusion protein can be performed in an in vitro protein expression system (e.g., cell-free protein expression systems such as those employing rabbit reticulocyte lysate or wheat germ extract). Also, the RGEN protein-CPP fusion protein produced in the expression step can optionally be isolated. Such isolation can be performed in a manner that produces a composition having any of the above-disclosed features (e.g., purity, pH, buffer, and/or salt level), for example.

[0286] Non-limiting examples of compositions and methods disclosed herein include: [0287] 1. A composition comprising at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein the protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, and wherein the RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell. [0288] 2. The composition of embodiment 1, wherein the protein component of the RGEN is associated with at least one RNA component that comprises a sequence complementary to a target site sequence on a chromosome or episome in the cell, wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence. [0289] 3. The composition of embodiment 2, wherein the RNA component comprises a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) operably linked to a trans-activating CRISPR RNA (tracrRNA). [0290] 4. The composition of embodiment 2, wherein the RGEN can cleave one or both DNA strands at the target site sequence. [0291] 5. The composition of embodiment 1, wherein the RGEN comprises a CRISPR-associated (Cas) protein-9 (Cas9) amino acid sequence. [0292] 6. The composition of embodiment 1, wherein the RGEN protein component and CPP are covalently linked. [0293] 7. The composition of embodiment 1, wherein the RGEN protein component and CPP are non-covalently linked. [0294] 8. The composition of embodiment 1, wherein the CPP is cationic or amphipathic. [0295] 9. The composition of embodiment 1, wherein the CPP comprises: [0296] (i) a CPP from an Epstein-Barr virus Zebra trans-activator protein, [0297] (ii) a CPP having 6 or more contiguous arginine residues, [0298] (iii) a transportan-10 (TP10) CPP, or [0299] (iv) a CPP from a vascular endothelium cadherin protein. [0300] 10. The composition of embodiment 1, wherein the RGEN protein-CPP complex can traverse a cell wall and cell membrane of a cell. [0301] 11. A cell comprising the composition according to embodiment 1. [0302] 12. A method of delivering a protein component of an RNA-guided endonuclease (RGEN) into a cell, the method comprising: [0303] contacting the cell with a composition comprising the protein component of the RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), [0304] wherein the protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, [0305] wherein the RGEN protein-CPP complex traverses (i) a cell membrane, or (ii) a cell wall and cell membrane, of the cell, thereby entering the cell. [0306] 13. The method of embodiment 12, wherein: [0307] (i) the composition further comprises at least one RNA component that is associated with the protein component of the RGEN; or [0308] (ii) the cell comprises the RNA component, wherein the RNA component associates with the protein component of the RGEN after the RGEN protein-CPP complex enters the cell; [0309] wherein the RNA component comprises a sequence complementary to a target site sequence on a chromosome or episome in the cell, wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence. [0310] 14. The method of embodiment 13, wherein the RGEN can cleave one or both DNA strands at the target site sequence. [0311] 15. The method of embodiment 14, wherein the cell further comprises a donor polynucleotide comprising at least one sequence homologous to a sequence at or near the target site sequence. [0312] 16. The method of embodiment 12, wherein the cell is a non-mammalian cell. [0313] 17. A composition comprising at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one cell-penetrating peptide (CPP), wherein the protein component and CPP are covalently, or non-covalently, linked to each other in a guide polynucleotide/Cas endonuclease-CPP complex, and wherein the guide polynucleotide/Cas endonuclease-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell, wherein the cell is optionally a plant cell. [0314] 18. The composition of embodiment 17, wherein the Cas endonuclease is a plant-optimized Cas9 endonuclease. [0315] 19. The composition of embodiment 17, wherein the guide polynucleotide comprises [0316] (i) a first nucleotide sequence domain that is complementary to a nucleotide sequence in a target DNA, and [0317] (ii) a second nucleotide sequence domain that interacts with a Cas endonuclease, [0318] wherein the first nucleotide sequence domain and the second nucleotide sequence domain are composed of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or a combination thereof. [0319] 20. The composition of embodiment 17, wherein the guide polynucleotide/Cas endonuclease-CPP complex can traverse the cell wall of a plant cell. [0320] 21. The composition of embodiment 17, wherein the CPP comprises: [0321] (i) a CPP from an Epstein-Barr virus Zebra trans-activator protein, [0322] (ii) a CPP having 6 or more contiguous arginine residues, [0323] (iii) a transportan-10 (TP10) CPP, [0324] (iv) a CPP from a vascular endothelium cadherin protein, or [0325] (vi) a CPP selected from the group consisting of a synthetic nona-arginine CPP, a histidine-rich nona-arginine CPP, and a Pas nona-arginine CPP. [0326] 22. The composition of embodiment 20, wherein the plant cell is a monocot or a dicot cell. [0327] 23. The composition of embodiment 22, wherein the monocot is selected from the group consisting of maize, rice, sorghum, rye, barley, wheat, millet, oats, sugarcane, turfgrass, and switchgrass. [0328] 24. The composition of embodiment 22, wherein the dicot is selected from the group consisting of soybean, canola, alfalfa, sunflower, cotton, tobacco, peanut, potato, tobacco, Arabidopsis, and safflower. [0329] 25. A method for modifying a target site in the genome of a cell, the method comprising providing a guide polynucleotide, a cell-penetrating peptide (CPP) and a Cas endonuclease to the cell, wherein the guide polynucleotide, Cas endonuclease and CPP are covalently, or non-covalently, linked to each other in a guide polynucleotide/Cas endonuclease-CPP complex, and wherein the guide polynucleotide/Cas endonuclease-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a cell, wherein the cell is optionally a plant cell. [0330] 26. The method of embodiment 25, further comprising identifying at least one plant cell that has a modification at the target site, wherein the modification at the target site is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii). [0331] 27. The method of embodiment 25, wherein the plant cell is a monocot or dicot cell. [0332] 28. A composition comprising at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein the protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, and wherein the RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a microbial cell. [0333] 29. The composition of embodiment 28, wherein the protein component of the RGEN is associated with at least one RNA component that comprises a sequence complementary to a target site sequence on a chromosome or episome in the microbial cell, wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence. [0334] 30. The composition of embodiment 28, wherein the RGEN protein-CPP complex can traverse a cell wall and cell membrane of a microbial cell. [0335] 31. A microbial cell comprising the composition according to embodiment 28. [0336] 32. A method of delivering a protein component of an RNA-guided endonuclease (RGEN) into a microbial cell, the method comprising: [0337] contacting the microbial cell with a composition comprising the protein component of the RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), [0338] wherein the protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, [0339] wherein the RGEN protein-CPP complex traverses (i) a cell membrane, or (ii) a cell wall and cell membrane, of the microbial cell, thereby entering the microbial cell. [0340] 33. The method of embodiment 32, wherein: [0341] (i) the composition further comprises at least one RNA component that is associated with the protein component of the RGEN; or [0342] (ii) the microbial cell comprises the RNA component, wherein the RNA component associates with the protein component of the RGEN after the RGEN protein-CPP complex enters the microbial cell; [0343] wherein the RNA component comprises a sequence complementary to a target site sequence on a chromosome or episome in the microbial cell, wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence. [0344] 34. The method of embodiment 33, wherein the RGEN can cleave one or both DNA strands at the target site sequence. [0345] 34. The method of embodiment 34, wherein the microbial cell further comprises a donor polynucleotide comprising at least one sequence homologous to a sequence at or near the target site sequence. [0346] 36. The method of embodiment 32, wherein the microbial cell is a yeast cell. [0347] 37. A composition comprising at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one cell-penetrating peptide (CPP), wherein the protein component and CPP are covalently, or non-covalently, linked to each other in a guide polynucleotide/Cas endonuclease-CPP complex, and wherein the guide polynucleotide/Cas endonuclease-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a microbial cell. [0348] 38. The composition of embodiment 37, wherein the guide polynucleotide/Cas endonuclease-CPP complex can traverse the cell wall of the microbial cell. [0349] 39. A method for modifying a target site in the genome of a microbial cell, the method comprising providing a guide polynucleotide, a cell-penetrating peptide (CPP) and a Cas endonuclease to the microbial cell, wherein the guide polynucleotide, Cas endonuclease and CPP are covalently, or non-covalently, linked to each other in a guide polynucleotide/Cas endonuclease-CPP complex, and wherein the guide polynucleotide/Cas endonuclease-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a microbial cell. [0350] 23. The method of embodiment 39, further comprising identifying at least one microbial cell that has a modification at the target site, wherein the modification at the target site is selected from the group consisting of (i) a replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii).

EXAMPLES

[0351] The disclosed invention is further defined in the following Examples. It should be understood that these Examples, while indicating certain preferred aspects of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

Example 1

Vectors for Expressing a Cas9-CPP (Cell-Penetrating Peptide) Fusion Protein in E. coli

[0352] In this example, vectors designed for inducible expression of translational fusion proteins comprising Cas9 protein and a cell-penetrating peptide (CPP) were produced and tested for expression in E. coli. Cas9-CPP fusion proteins were shown to express in E. coli as expected, and subsequently purified.

[0353] The open reading frame of the Cas9 gene from Streptococcus pyogenes M1 GAS (SF370) was codon-optimized for expression in Yarrowia per standard techniques, yielding SEQ ID NO:1. DNA sequence encoding a simian virus 40 (SV40) monopartite nuclear localization signal (NLS) plus a short linker (4 amino acids) was incorporated after the last sense codon of SEQ ID NO:1 to render SEQ ID NO:2. SEQ ID NO:2 encodes the amino acid sequence shown in SEQ ID NO:3. The last seven amino acids of SEQ ID NO:3 encode the added NLS, whereas residues at positions 1369-1372 of SEQ ID NO:3 encode the added linker. The Yarrowia codon-optimized Cas9-NLS sequence (SEQ ID NO:2) was linked to a Yarrowia constitutive promoter, FBA1 (SEQ ID NO:4), by standard molecular biology techniques. A Yarrowia codon-optimized Cas9 expression cassette containing the constitutive FBA1 promoter, Yarrowia codon-optimized Cas9, and the SV40 NLS is set forth in SEQ ID NO:5. This Cas9 expression cassette (SEQ ID NO:5) was cloned into the plasmid pZUF rendering construct pZUFCas9 (FIG. 1, SEQ ID NO:6).

[0354] The Yarrowia codon-optimized Cas9-NLS sequence was PCR-amplified from pZUFCas9 (SEQ ID NO:6) using standard molecular biology techniques. Primers for the PCR reaction were SEQ ID NO:7 (Forward) and SEQ ID NO:8 (Reverse), which added a 5' EcoRI site and 3' HindIII site, respectively, to the amplified DNA product. The added 5' EcoRI site replaced the ATG start codon of the Cas9-NLS open reading frame (ORF) in the amplified product. The amplified product (SEQ ID NO:9) was digested with EcoRI and HindIII, and then purified using Zymoclean.TM. and concentrator columns (Zymo Research, Irvine, Calif.). The purified DNA fragment was cloned into the EcoRI and HindIII sites of plasmid pBAD/HisB from Life Technologies (Carlsbad, Calif.) (FIG. 2A, SEQ ID NO:10) to create plasmid construct pRF48 (FIG. 2B, SEQ ID NO:11). Plasmid pRF48 is capable of expressing, in E. coli, a Cas9-NLS comprising a hexahistidine (6.times.His) tag at its N-terminus.

[0355] To fuse a cell-penetrating peptide (CPP) sequence to Cas9-NLS, individual DNA polynucleotide sequences were prepared, each codon-optimized for expression in E. coli and comprising sequence encoding a 6.times.His tag linked to a particular CPP amino acid sequence: Zebra peptide (ECDSELEIKRYKRVRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMC, SEQ ID NO:12), from the Epstein-Barr virus Zebra trans-activator protein; pVEC peptide (LLIILRRRIRKQAHAHSK, SEQ ID NO:13), from a murine endothelial cadherin protein; TP10 peptide (AGYLLGKINLKACAACAKKIL, SEQ ID NO:14), from a neuropeptide galanin protein; and synthetic arginine-rich "PolyR" peptide (GGGGRRRRRRRRRLLLL, SEQ ID NO:15). Each DNA polynucleotide sequence included a 5'-end NcoI restriction site and a 3'-end EcoRI site to create cloning sequences structured as follows: NcoI-6.times.His-CPP-EcoRI (SEQ ID NO:16-19). Each of SEQ ID NOs:16-19 was individually cloned into the NcoI and EcoRI sites of pRF48, thereby creating plasmid constructs capable of expressing certain 6.times.His-CPP-Cas9-NLS fusion proteins in E. coli. In particular, plasmid construct pRF144 (FIG. 3A, SEQ ID NO:20) was prepared for expressing a 6.times.His-Zebra CPP-Cas9-NLS fusion; plasmid construct pRF145 (FIG. 3B, SEQ ID NO:21) was prepared for expressing a 6.times.His-PolyR CPP-Cas9-NLS fusion; plasmid construct pRF146 (FIG. 3C, SEQ ID NO:22) was prepared for expressing a 6.times.His-TP10 CPP-Cas9-NLS fusion, and plasmid construct pRF162 (FIG. 3D, SEQ ID NO:23) was prepared for expressing a 6.times.His-pVEC CPP-Cas9-NLS fusion.

[0356] Each of plasmids pRF48, pRF144, pRF145, pRF146 and pRF162 was individually transformed into TOP10 competent cells (Life Technologies). Cells were grown overnight at 37.degree. C. with shaking (220 rpm) in L broth (Miller) containing 0.4% (w/v) glucose and 100 .mu.g/mL ampicillin. Each pre-culture was diluted 1:100 in 2.times.YT medium containing 100 .mu.g/mL ampicillin and further grown at 37.degree. C. with shaking (220 rpm). When cultures reached an OD.sub.600 of about 0.5, protein expression from each plasmid was induced by adding L-arabinose to a final concentration of 0.2% (w/v). The cultures were grown for an additional 18 hours at 18.degree. C. with shaking (200 rpm). Cells were pelleted at 5000.times.g for 15 minutes at 4.degree. C. Medium was disposed of and cell pellets were frozen at -80.degree. C. for at least 4 hours. Cell pellets were thawed for 15 minutes on ice and resuspended in 15 mL of lysis buffer (20 mM tris pH 7.5, 500 mM NaCl, 1 mM MgCl.sub.2, 10 mM imidazole, 120 units/mL DNaseI, 1 mM PMSF, 1 mM DTT) per liter of original culture. Cells were lysed by passage twice through a large French pressure cell at 16000 psi. Cell debris was pelleted at 20000.times.g for 30 minutes at 4.degree. C. Supernatants were transferred to a 50-mL conical tubes, to which 2 ml of a 50% slurry of Ni-NTA resin (Qiagen) was added for binding the 6.times.His Tag of each expressed fusion protein. Each tube was slowly rotated at 4.degree. C. for 1 hour and then applied to an empty gravity column through which the supernatant was allowed to flow. Flow-through sample (75 .mu.L) was taken, added to 25 .mu.L of 4.times.-reduced Laemmeli buffer, and stored on ice. The resin was washed four times in each column with 5 ml of wash buffer (20 mM tris pH 7.5, 500 mM NaCl, 10 mM imidazole, 1 mM PMSF, 1 mM DTT). A sample (75 .mu.L) was taken from each wash, added to 25 .mu.L of 4.times.-reduced Laemmeli buffer, and stored on ice. 1-ml aliquots of elution buffer (20 mM Tris pH 7.5, 500 mM NaCl, 1 mM MgCl.sub.2, 500 mM imidazole, 1 mM PMSF, 1 mM DTT) were applied to the resin in each column and allowed to incubate for 10 minutes. Protein elution was monitored by absorbance at 280 nm. A sample (75 .mu.L) was taken from each elution, added to 25 .mu.L of 4.times.-reduced Laemmeli buffer, and stored on ice. For each plasmid expression experiment, fractions containing eluted protein from the column were combined, loaded into 10000 MWCO dialysis membrane, and dialyzed against dialysis buffer (25 mM HEPES/KOH pH 7.5, 200 mM KCl, 20% glycerol, 1 mM DTT) at 4.degree. C. for at least 14 hours. The protein concentration of each dialysate was determined using the Bradford assay and absorbance at 565 nm. Purified protein was split into two aliquots, one of which was frozen at -80.degree. C. and the other stored on ice at 4.degree. C. Samples taken during the column purification process for each plasmid expression experiment were heated at 95.degree. C. for 5 minutes and loaded onto an 8% (w/v) tris-glycine polyacrylamide resolving gel with a 4% (w/v) stacking gel. Proteins were electrophoretically separated at 200 volts for 30 minutes and stained with Coomassie blue. The gel for the 6.times.His-Zebra-Cas9-NLS purification process is shown in FIG. 4 as an example.

[0357] Thus, four different CPP-Cas9 fusion proteins were expressed and isolated. These fusion proteins represent examples of RGEN protein-CPP complexes herein.

Example 2

Expressing Short Guide RNA (sqRNA) by In Vitro Transcription

[0358] In this example, a DNA sequence was designed that encodes an sgRNA fused to ribozymes at its 5'- and 3'-ends (referred to as "RGR"), respectively. The RGR sequence allowed for in vitro transcription by T7 RNA polymerase of an sgRNA with precisely defined ends.

[0359] FIG. 5 illustrates an sgRNA molecule, which is a single RNA molecule containing two regions, a variable targeting domain (VT) (guide sequence) and Cas endonuclease recognition (CER) domain (SEQ ID NO:24 represents an example of a CER). The VT region can be a 20mer of RNA polynucleotide that has identity to a targeted nucleic acid molecule, for example. The VT domain specifies a target site for cleavage in the target site that lies 5' of a PAM motif. The CER domain interacts with Cas9 protein and allows the VT domain to interact and direct the Cas9 protein cleavage (Jinek et al., Science 337:816-821). Both VT and CER domains are required for the function of an sgRNA.

[0360] The addition of 5' HammerHead (HH) and 3' Hepatitis Delta Virus (HDV) ribozymes to an sgRNA sequence allows expression of the sgRNA from any promoter without consideration for certain transcriptional requirements of some RNA polymerases (e.g., T7 RNA polymerase requires one transcribed G residue directly after initiation of transcription, but works best with three transcribed G residues). When such sgRNA is expressed, the ribozymes present in the pre-sgRNA transcript autocleave, thereby separating from the transcript leaving an unmodified sgRNA.

[0361] A DNA sequence encoding an sgRNA that targets the Can1-1 locus (SEQ ID NO:25) in Yarrowia lipolytica was prepared; this sgRNA comprises SEQ ID NO:24 as its CER domain. The sgRNA-encoding sequence was linked at its 5'-end to sequence encoding an HH ribozyme (SEQ ID NO:26) and at its 3'-end to a sequence encoding an HDV ribozyme (SEQ ID NO:27), such that the first 6 bases of the HH ribozyme were a reverse compliment to the first 6 bases of the VT region of the sgRNA. This particular RGR sgRNA is encoded by SEQ ID NO:28. The RGR sgRNA of SEQ ID NO:28 was then linked to a T7 RNA polymerase promoter (SEQ ID NO:29) via standard molecular biology techniques to create plasmid pRF46 (SEQ ID NO:30).

[0362] T7-RGR sgRNA-encoding sequence was PCR-amplified from plasmid pRF46 (SEQ ID NO:30) using standard techniques. Primers for the PCR reaction were SEQ ID NO:31 (T7 forward primer) and SEQ ID NO:32 (gRNArev1 reverse primer).

[0363] The PCR product was purified by ethanol precipitation and resuspended in ddH.sub.2O; this DNA was used as template in an in vitro transcription reaction. Template DNA was added to a final concentration of 150 nM in 20-.mu.L in vitro transcription reactions (MEGAshortscript.TM. T7 Kit, Life Technologies). Reactions were allowed to proceed for various times (2 hours, 4 hours, 6 hours, and overnight) to determine suitable conditions for in vitro transcription (FIG. 6). The reactions were then treated with 10 units of DNaseI for 15 minutes at 37.degree. C. to remove template DNA. RNA was precipitated using ethanol and standard protocols. Each 20-.mu.l in vitro transcription reaction produced between 60 and 100 .mu.g of RNA.

[0364] Thus, sgRNA with defined 5'- and 3'-ends was synthesized in vitro. As demonstrated in Example 3 below, in vitro transcribed sgRNA can be associated with a Cas9-CPP fusion protein to form an RGEN-CPP complex.

Example 3

Specific In Vitro Cleavage of Target DNA Sequence Using Cas9-CPP Fusion Protein Complexed with sqRNA

[0365] In this example, the targeting endonuclease function of Zebra CPP-Cas9 fusion protein (comprising SEQ ID NO:39) in complex with an sgRNA was tested to confirm that fusion with a CPP does not hinder Cas9 endonuclease activity.

[0366] An in vitro Can1 cleavage assay DNA polynucleotide (SEQ ID NO:35) containing the Can1-1 target sequence of SEQ ID NO:25 was PCR-amplified from Y. lipolytica cells (ATCC 20362) and purified using standard techniques. Primers for the PCR reaction were SEQ ID NO:33 (IV-up forward primer) and SEQ ID NO:34 (IV-down reverse primer).

[0367] Purified Zebra CPP-Cas9 fusion protein (600 ng, prepared in Example 1), sgRNA targeting the Can1-1 target site (250 ng, prepared in Example 2), NEBuffer 3.1 (New England BioLabs, Ipswich, Mass.), and Can1 cleavage assay DNA (150 ng, SEQ ID NO:35) were mixed in a 10-.mu.L reaction (volume brought up to final volume with ddH.sub.2O). As negative controls, reactions lacking either Zebra CPP-Cas9 fusion protein or sgRNA were also prepared. As a positive control, wild type Cas9 protein (PNA Bio, Thousand Oaks, Calif.) was used in a reaction instead of Zebra CPP-Cas9. The reactions were incubated at 37.degree. C. for 60 minutes. RNaseI (4 .mu.g) was then added to each reaction and incubated at 37.degree. C. for 15 minutes to degrade the sgRNA. Stop solution (1 .mu.L; 30% [w/v] glycerol, 1.2% [w/v] SDS, 250 mM EDTA, pH 8.0) was added to terminate the reactions, which were then further incubated for 15 minutes at 37.degree. C. Each reaction was loaded onto a 1.2% FlashGel.TM. (Lonza, Basel, Switzerland) and electrophoresed for 10 minutes at 200 volts (FIG. 7). The target DNA cleavage pattern rendered by Zebra CPP-Cas9 was consistent with the cleavage pattern rendered by wild type Cas9 (FIG. 7), thereby indicating that Zebra CPP-Cas9 functions normally in vitro. Furthermore, this activity was not inhibited using Zebra CPP-Cas9/sgRNA that had been subjected to two freeze-thaw cycles.

[0368] Thus, a CPP-Cas9 fusion protein complexed with a suitable sgRNA (i.e., an example of an RGEN-CPP complex) had specific DNA cleavage activity. This activity was shown to be similar with the activity of a wild type Cas9-sgRNA complex, thereby indicating that CPP fusion does not inhibit Cas9-sgRNA endonucleolytic function. While the CPP-Cas9 fusion protein in this example comprised SEQ ID NO:39 (Zebra CPP-Cas9), it is contemplated that a CPP-Cas9 fusion protein comprising SEQ ID NO:40, 41, or 42, for example, also has cleavage activity when associated with a suitable sgRNA as an RNA component.

Example 4

Delivery of a CPP-Cas9/sqRNA Complex into Yeast Cells and Cleavage of Target DNA Therein

[0369] In this example, Zebra CPP-Cas9 fusion protein (comprising SEQ ID NO:39) in complex with an sgRNA (Zebra CPP-Cas9/sgRNA) was tested for the ability to enter yeast cells after simple contact with the cells. Zebra CPP-Cas9/sgRNA specific for Can1-1 was able to enter cells and cleave the Can1 gene, thereby rendering cells to be canavanine-resistant.

[0370] Y. lipolytica yeast cells (ATCC 20362) were grown in YPD (2% glucose, 2% peptone, 1% yeast extract) liquid medium at 30.degree. C. with shaking (220 rpm) to OD.sub.600=0.5 (approximately 5.times.10.sup.6 cells per mL of culture). Purified Zebra CPP-Cas9 fusion protein (prepared in Example 1) and sgRNA targeting the Can1-1 target site (prepared in Example 2) were mixed in a 1:3 molar ratio, respectively, in the dialysis buffer used in Example 1 and pre-incubated at room temperature for 15 minutes to allow the sgRNA to associate with the Zebra CPP-Cas9. 5.times.10.sup.5 Y. lipolytica cells were mixed into the Zebra CPP-Cas9/sgRNA preparation such that the final concentration of Zebra CPP-Cas9 was 1 .mu.M, 2.5 .mu.M, or 5 .mu.M. Cells were also mixed with 5 .mu.M final concentration Zebra CPP-Cas9 alone (no sgRNA as RNA component) as a negative control. All the cell-Cas9 preparations were incubated at 30.degree. C. with shaking (220 rpm) for 2 hours. The cells were then serially diluted 1000- and 10000-fold. Each serial dilution (100 .mu.L) was plated onto complete medium lacking arginine (CM-Arg) and allowed to recover for 48 hours at 30.degree. C.

[0371] Colonies of the 10.sup.-3-dilution plates were counted to determine the total number of cells plated. Colonies were transferred to CM-Arg plates with canavanine (60 .mu.g/mL) via replica-plating technique. Colonies were allowed to grow at 30.degree. C. for 48 hours. The number of canavanine-resistant colonies were scored and divided by the total number of colonies (from plates without canavanine) to determine a mutation frequency for each case. Contacting cells with Zebra CPP-Cas9/sgRNA complexes yielded colonies that were resistant to canavanine at frequencies of about 2% to 10% of the total colonies (FIG. 8). This canavanine-resistance is expected to be due to loss of Can1 gene function by indel formation at/near the predicted Cas9 cleavage site in the Can1 gene coding sequence. However, contacting cells with Zebra CPP-Cas9 alone (no sgRNA) did not yield canavanine-resistant colonies (FIG. 8), indicating that canavanine-resistance in the experimental cells was dependent on sgRNA-based specificity given to CPP-Cas9 protein. Given the nature of yeast cells, the CPP-Cas9/sgRNA complexes likely had to traverse both cell wall and cell membrane structures to mediate specific DNA targeting.

[0372] Thus, a CPP-Cas9 fusion protein complexed with a suitable sgRNA (i.e., an example of an RGEN-CPP complex) is able to enter yeast cells (traverse cell wall and cell membrane) and target a specific DNA sequence therein. While the CPP-Cas9 fusion protein in this example comprised SEQ ID NO:39 (Zebra CPP-Cas9), it is contemplated that a CPP-Cas9 fusion protein comprising SEQ ID NO: 40, 41, or 42, for example, also has cell-entry activity, and specific DNA targeting activity in cells, when associated with a suitable sgRNA as an RNA component.

Example 5

CPP-Facilitated Cas9/sqRNA Complex Delivery into Plant Cells and Cleavage of Target DNA Therein

[0373] CPP-facilitated protein delivery into soybean cells can be tested by incubating soybean callus cells with DS-RED fluorescent proteins fused to CPPs. Fluorescent signals are expected in CPP-DS-RED treatments, but not in controls incubated with DS-RED proteins only. Various CPPs can be tested in this manner to help identify the most effective CPPs for plant cell penetration and delivery of protein cargo. Some examples of CPPs that can be tested include: [0374] (i) a CPP from an Epstein-Barr virus Zebra trans-activator protein, [0375] (ii) a CPP having 6 or more contiguous arginine residues, [0376] (iii) a transportan-10 (TP10) CPP, [0377] (iv) a CPP from a vascular endothelium cadherin protein, or [0378] (vi) a CPP selected from the group consisting of a synthetic nona-arginine CPP, a histidine-rich nona-arginine CPP and a Pas nona-arginine CPP. Examples of a synthetic nona-arginine CPP, a histidine-rich nona-arginine CPP and a Pas nona-arginine CPP are disclosed in, for example, Liu et al. (Advanced Studies in Biology 5(2):71-88, HIKARI Ltd).

[0379] In vitro translated Cas9 proteins and synthetic sgRNA can be mixed with CPPs, by themselves or in a fusion (e.g., CPP-DS-RED above), and incubated with soybean callus to test if Cas9/sgRNA can be transported into the cells. Once in the cells, the Cas9/sgRNA complex can recognize a genomic target specified by the sgRNA targeting sequence to make DNA double strand breaks (DSBs). Spontaneous repair of the DSBs by cell machinery can result in mutations through non-homologous end joining (NHEJ), or gene integration through homologous recombination if appropriate donor DNA is present. CPPs can also be covalently linked to Cas9 proteins for potentially better efficiency. The success of CPP-Cas9/sgRNA delivery into soybean cells, and thus the transfer of the CPP-Cas endonuclease complex across a plant cell wall and plant cell membrane, can be verified by the detection of mutations or gene integrations at the specific target site by PCR analysis, for example.

Example 6

Expression and Purification of CPP-dsREDexpress Proteins from E. coli Cells

[0380] To rapidly assess the ability of a given cell-penetrating peptide to enter a specific cell type CPP fusions to the dsREDexpress protein (SEQ ID NO: 85) were created, expressed in E. coli cells, and purified. The CPP-dsREDexpress protein fusions are a tool that allows rapid assessment of cargo delivery into a given cell type by a given CPP. This allows selection of a species, cell type, or strain specific CPP molecule to maximize delivery of cargo in a rapid and high-throughput manner by assessing cellular fluorescence by microscopic or flow cytometric analysis.

[0381] An E. coli codon optimized dsREDexpress gene (SEQ ID NO: 86) was synthesized (IDT DNA) and cloned into the NcoI/HinDIII sites of pBAD/HisB (SEQ ID NO: 87) creating pRF161 (SEQ ID NO: 88). The E. coli codon optimized dsREDexpress contained an internal EcoRI site such that digestion of the plasmid with NcoI/EcoRI would allow replacement of the his tag with various his tag-CPP sequences to create his tag-CPP-dsREDexpress fusion expression plasmids. Various his-tag-CPP fusions; TAT (SEQ ID NO: 89), TLM (SEQ ID NO: 90), MPG1 (SEQ ID NO: 91), pep1 (SEQ ID NO: 92), and CFFKDEL (SEQ ID NO: 93); were codon optimized for E. coli and flanked with in frame 5' NcoI and 3' EcoRI sites (SEQ ID NO: 94-98 respectively) and cloned using standard techniques into the NcoI/EcoRI sites of pRF161 (SEQ ID NO: 88) replacing the his tag sequence with the corresponding his tag-CPP fusion and generating plasmids pRF224 (his-TAT-dsREDexpress SEQ ID NO: 99), pRF214 (his-TLM-dsREDexpress SEQ ID NO: 100), pRF213 (his-MPG1-dsREDexpress SEQ ID NO: 101), pRF217 (his-pepl-dsREDexpress SEQ ID NO: 102), pRF216 (his-CFFKDEL-dsREDexpress SEQ ID NO: 103). Sequences of the inserted fragments were verified using standard sequencing techniques and oligo 36 (SEQ ID NO: 104).

[0382] E. coli codon optimized His-Zebra (SEQ ID NO: 105), His-tp10 (SEQ ID NO: 106), and His-pVEC (SEQ ID NO: 107) were PCR amplified from pRF144 (SEQ ID NO 108), pRF162 (SEQ ID NO 109), and pRF146 (SEQ ID NO: 110) respectively using oligo 36 (SEQ ID NO: 104) and oligo 153 (SEQ ID NO: 111) with standard PCR techniques. PCR fragments were cloned into the NcoI/EcoRI sites of pRF161 (SEQ ID NO: 88) creating plasmids pRF186 (his-Zebra-dsREDexpress SEQ ID NO:112), pRF192 (his-tp10-dsREDexpress SEQ ID NO: 113), and pRF190 (his-pVEC-dsREDexpress SEQ ID NO: 114). Sequences were verified using oligo 36 (SEQ ID NO: 104).

[0383] His tagged CPP-dsREDexpress fusion proteins were expressed using standard techniques. In brief, cells were precultured in either 10 ml ZYM-505 (1% N-Z amine, 0.5% yeast extract, 5% glycerol, 1.0% dextrose, 25 mM Na.sub.2HPO.sub.4, 25 mM KH.sub.2PO.sub.4, 50 mM NH.sub.4Cl, 5 mM Na.sub.2SO.sub.4, 1.times. trace metals (Teknova), 5.times.10.sup.-5% Thiamine, 2 mM MgCl.sub.2, 100 .mu.g/ml Ampicillin) or lysogeny broth (1% Tryptone, 0.5% yeast extract, 1% sodium chloride, 100 .mu.g/ml Ampicillin, 0.4% dextrose) in 125 ml flasks for 12-16 hours at 37.degree. C. and 220 RPM. Precultures were diluted 1:1000 (ZYM-505) in 500 ml ZYM-5052 (1% N-Z amine, 0.5% yeast extract, 5% glycerol, 0.5% dextrose, 2% L-arabinose, 25 mM Na.sub.2HPO.sub.4, 25 mM KH.sub.2PO.sub.4, 50 mM NH.sub.4Cl, 5 mM Na.sub.2SO.sub.4, 1.times. trace metals (Teknova), 5.times.10.sup.-5% Thiamine, 2 mM MgCl.sub.2, 100 .mu.g/ml Ampicillin) or 1:100 (Lysis broth) in 500 ml 2.times.YT (1.6% Tryptone, 1% Yeast extract, 0.5% NaCl, 100 .mu.g/ml ampicillin) and grown at 37.degree. C. 220RPM in 2.9 L Fernbach flasks to OD.sub.600.about.0.5. L-arabinose was added to a final concentration of 0.1% to 2.times.YT cultures and all cultures were shifted to 18.degree. C. 220RPM for 20-30 hours for protein expression. Cells were harvested at 5000 RPM for 10 minutes, spent medium was discarded and cell pellets frozen at -80.degree. C.

[0384] Cell pellets were thawed and resuspended in Denaturing lysis buffer (50 mM Tris pH8.0, 150 mM NaCl, 8M Urea, 20 mM Imidazole) and lysed via passage through a French pressure cell at 16,000 PSI twice. Solid precipitates were removed from the supernatant by centrifugation at 10,000 g 4.degree. C. for 15 minutes. 20 .mu.l of clarified extract was mixed with 20 .mu.l of 2.times. Laemmli buffer (4% SDS, 20% Glycerol, 100 mM DTT, 0.004% bromophenol blue, 125 mM Tris pH 6.8), heated to 95.degree. C. for 5 minutes and frozen at -20.degree. C. to save for analysis. Clarified extract was mixed with 6 ml of 50% (v/v) Nickel-NTA-agarose slurry for 1 hour at room temperature. Beads were pelleted from mixture at 2000 RPM for 5 minutes. Supernatant was removed and a 20 .mu.l sample was taken as for the clarified extract. The pelleted beads were resuspended in 10 ml of denaturing lysis buffer and applied to a gravity flow chromatography column. The liquid was allowed to flow out leaving a bed of packed beads. The bed was washed with a series washes using different ratios of wash buffer 1 (50 mM Tris pH8.0, 150 mM NaCl, 8M Urea, 20 mM Imidazole) and wash buffer 2 (50 mM Tris pH 8.0, 500 mM NaCl, 20 mM Imidazole) to step down the concentration of the denaturant (urea) and step up the concentration of NaCl and allow the protein to refold on the column. In brief the column was washed with (Buffer 1: Buffer 2): 10 ml of 1:0 (8M urea 150 mM NaCl), 10 ml of 7:1 (7M Urea, 194 mM NaCl), 10 ml of 3:1 (6M Urea, 238 mM NaCl) 10 ml of 5:3 (5M Urea, 281 mM NaCl), 10 ml of 1:1 (4M Urea, 325 mM NaCl), 20 ml of 3:5 (3M Urea, 369 mM NaCl), 20 ml of 1:3 (2M Urea, 413 mM NaCl), 20 ml of 3:13 (1.5M Urea, 434 mM NaCl), 20 ml of 1:5 (1M urea, 456 mM NaCl), 20 ml of 1:15 (0.5M Urea, 478 mM NaCl), and 30 ml of 0:1 (0M Urea, 500 mM NaCl). Protein was eluted in native elution buffer (50 mM Tris pH8.0, 500 mM NaCl, 10% Glycerol, 500 mM Imidazole) in 10.times.1 ml fractions.

[0385] Fractions containing the eluted dsREDexpress or CPP-dsREDexpress protein were red in color. Red fractions were combined and dialyzed in 10,000 MWCO regenerated cellulose dialysis membrane against 1000 volumes of dialysis buffer (50 mM Tris pH 8.0, 10% glycerol) overnight at room temperature. Protein solution was removed from dialysis membrane and filter sterilized using a 0.22 .mu.M Tuffryn.RTM. membrane. 20 .mu.l of protein solution was processed as for the clarified cell extract.

[0386] Samples taken during the purification in Laemmli buffer were heated to 95.degree. C. for 5 minutes and loaded onto a 12.5% PAGE gel. The gel was run at 200 volts constant for 1 hour and stained using simply blue stain. An example of a representative PAGE gel for the purification of CPP-dsREDexpress tagged proteins is shown in FIG. 9. Total protein concentration for each purified protein was determined using Pierce.TM. Coomassie Plus assay with bovine serum albumin as a standard. The concentration of each purified CPP-dsREDexpress fusion is given in Table 3.

TABLE-US-00007 TABLE 3 Concentration of purified dsREDexpress protein fusions. Protein mg/ml .mu.M dsREDexpress (SEQ ID NO: 700) 3.8 137 MPG1-dsREDexpress (SEQ ID NO: 751) 0.5 17 pVEC-dsREDexpress (SEQ ID NO: 752) 2.0 68 CFFKDEL-dsREDexpress (SEQ ID NO: 753) 1.5 54 TLM-dsREDexpress (SEQ ID NO: 754) 2.5 86 Zebra-dsREDexpress (SEQ ID NO: 755) 0.5 18 pep1-dsREDexpress (SEQ ID NO: 756) 0.3 10 tp10-dsREDexpress (SEQ ID NO: 757) 0.9 33

Example 7

Expression and Purification of Additional CPP-Cas9 Proteins from E. coli Cells

[0387] The delivery of Cas9 into different cell types may require Cas9 tagged with different CPP molecules. In order to isolate various CPP-Cas9 fusion proteins different CPPs were fused to Cas9 in an E. coli expression vector. These proteins were expressed and purified from E. coli cells for use in CPP mediated delivery of Cas9/sgRNA ribonucleoprotein complex to cells.

[0388] In order to make His-CFFKDEL-Cas9 (SEQ ID NO: 115) and His-MPG1-Cas9 (SEQ ID NO: 116) fusion expression cassettes the NcoI/EcoRI fragments of pRF216 (CFFKDEL SEQ ID NO: 103) or pRF213 (MPG1 SEQ ID NO: 101) were cloned into the same sites of the Cas9 protein expression plasmid pRF48 (SEQ ID NO: 117) using standard techniques generating plasmids pRF243 (his-CFFKDEL-Cas9 SEQ ID NO: 118) and pRF238 (his-MPG1-Cas9, SEQ ID NO: 119) respectively. Correct construction of the MPG1-Cas9 or CFFKDEL-Cas9 fusion cassettes was confirmed via Sanger sequencing with oligo 36 (SEQ ID NO: 104).

[0389] His tagged CPP-Cas9 fusion proteins were expressed using standard techniques. In brief, cells were precultured in either 10 ml ZYM-505 (1% N-Z amine, 0.5% yeast extract, 5% glycerol, 1.0% dextrose, 25 mM Na.sub.2HPO.sub.4, 25 mM KH.sub.2PO.sub.4, 50 mM NH.sub.4Cl, 5 mM Na.sub.2SO.sub.4, 1.times. trace metals (Teknova), 5.times.10.sup.-5% Thiamine, 2 mM MgCl.sub.2, 100 .mu.g/ml Ampicillin) or lysogeny broth (1% Tryptone, 0.5% yeast extract, 1% sodium chloride, 100 .mu.g/ml Ampicillin, 0.4% dextrose) in 125 ml flasks for 12-16 hours at 37.degree. C. and 220 RPM. Precultures were diluted 1:1000 (ZYM-505) in 500 ml ZYM-5052 (1% N-Z amine, 0.5% yeast extract, 5% glycerol, 0.5% dextrose, 2% L-arabinose, 25 mM Na.sub.2HPO.sub.4, 25 mM KH.sub.2PO.sub.4, 50 mM NH.sub.4Cl, 5 mM Na.sub.2SO.sub.4, 1.times. trace metals (Teknova), 5.times.10.sup.-5% Thiamine, 2 mM MgCl.sub.2, 100 .mu.g/ml Ampicillin) or 1:100 (Lysis broth) in 500 ml 2.times.YT (1.6% Tryptone, 1% Yeast extract, 0.5% NaCl, 100 .mu.g/ml ampicillin) and grown at 37.degree. C. 220RPM in 2.9 L Fernbach flasks to OD.sub.600.about.0.5. L-arabinose was added to a final concentration of 0.1% to 2.times. YT cultures and all cultures were shifted to 18.degree. C. 220RPM for 20-30 hours for protein expression. Cells were harvested at 5000 RPM for 10 minutes, spent medium was discarded and cell pellets frozen at -80.degree. C. Proteins were purified as described in Example 1. The final concentrations of the purified CPP-Cas9 proteins as determined by Coomasie Plus assay (Pierce.TM.) are listed in Table 4.

TABLE-US-00008 TABLE 4 Concentration of purified CPP-Cas9 proteins. Protein mg/ml .mu.M Zebra-Cas9 (SEQ ID NO: 758) 1.5 9 CFFKDEL-Cas9 (SEQ ID NO: 730) 4.6 28 MPG1-Cas9 (SEQ ID NO: 731) 3.8 23 pVEC-Cas9 (SEQ ID NO: 759) 2.5 15

Example 8

CPP-Cas9/qRNA Mediated Gene Targeting in E. coli Cells

[0390] This example demonstrates the treatment of Escherichia coli cells with CPP-Cas9/sgRNA ribonucleoprotein complexes with sgRNAs targeting the galK gene of E. coli. The entry of the CPP-Cas9/sgRNA into the cell allows targeting and cleavage to occur within the galK gene leading to gene inactivation by error-prone DNA repair mechanisms which can be phenotypically monitored as resistance to galactose. This method depends on delivery of Cas9/sgRNA cargo to the cells via CPP-mediated delivery.

[0391] The galK gene of E. coli (SEQ ID NO: 120) is responsible for a galactose sensitive phenotype seen in gal/E mutants in the presence of the sugar galactose. As galactose enters the cell it is phosphorylated by galactokinase, the product of the galK gene (SEQ ID NO: 120). Galactose phosphate is toxic to the cell. In wild-type cells the galactose phosphate is further metabolized by the products of the gale (SEQ ID NO: 121) and galT (SEQ ID NO: 122) genes and used as a carbon source. In gal/E or galT loss-of-function mutants galactose phosphate accumulates leading to cell death. Therefore, loss of function mutations in the galK gene can be selected in the background of a gal/E mutant as allowing colony formation in the presence of galactose.

[0392] In order to produce sgRNA (SEQ ID NO: 135) targeting the galK gene (SEQ ID NO: 120) at the galK2-1 target site (SEQ ID NO: 134) an in vitro transcription template (SEQ ID NO: 131) was produced. First a PCR product of the DNA encoding the CER domain (SEQ ID NO: 123) was amplified from pRF291 (SEQ ID NO: 125) using CER forward (SEQ ID NO: 126) and universal reverse primers (SEQ ID NO: 127) in a standard PCR reaction (SEQ ID NO: 124). The CER encoding PCR product (SEQ ID NO: 124) was purified using Zymo.TM. clean and concentrate 25 columns and eluted in 35 .mu.l of ddH.sub.2O. Amplification of the sgRNA in vitro transcription template used a multiplex PCR containing 4 primers, a universal forward primer containing the T7 promoter (SEQ ID NO: 128), a target specific forward primer containing some of the T7 promoter and some of the target site (SEQ ID NO: 129), a target reverse primer containing some of the target site and overlap with the CER domain (SEQ ID NO: 130), and the universal reverse primer (SEQ ID NO: 127). A PCR reaction was run using Phusion flash master mix containing 15 nM CER domain PCR product (SEQ ID NO: 124), 1 .mu.M each the universal forward (SEQ ID NO: 128) and reverse primers (SEQ ID NO: 127) and 300 nM each target forward (SEQ ID NO: 129) and target reverse (SEQ ID NO: 130) primers. The PCR reaction was cycled as for a standard reaction. sgRNA in vitro transcription template (SEQ ID NO: 131) was purified using Zymo clean and concentrate 25 columns and eluted in 35 .mu.l of ddH.sub.2O. The sgRNA in vitro transcription template (SEQ ID NO: 131) contained the T7 promoter (SEQ ID NO: 132), the DNA encoding the galK2-1 variable targeting domain (SEQ ID NO: 133), and the DNA encoding the CER domain (SEQ ID NO: 125) The in vitro transcription reaction to create the galK2-1 sgRNA (SEQ ID NO: 135) was performed as described in Example 2.

[0393] CPP delivery of Cas9/sgRNA nucleoprotein complexes was performed by growing a strain of E. coli deleted for galE in lysogeny broth (1% Tryptone, 0.5% Yeast Extract, 1% NACl) overnight at 37.degree. C., 220RPM. The culture was diluted 1:100 in fresh lysogeny broth and grown at 37.degree. C., 220RPM for 2 hours to obtain cells in exponential growth phase. CPP-Cas9 (pvEC-Cas9 (SEQ ID NO: 144), Zebra-Cas9 (SEQ ID NO: 143), MPG1-Cas9 (SEQ ID NO: 116), CFFKDEL-Cas9 (SEQ ID NO: 115)) were incubated at 10 .mu.M final concentration either in the presence or absence of 10 .mu.M galK2-1 sgRNA (SEQ ID NO: 135) in a 50 .mu.l volume for 30 minutes at room temperature. For the treatment 1.2 ml of cells were pelleted at 3000 RPM for 3 minutes, supernatant was discarded and cells were resuspended in 600 .mu.l of LB containing 2.times. nuclease buffer (200 mM NaCl, 100 mM Tris-HCl, 20 mM MgCl.sub.2, 200 .mu.g/ml BSA pH 7.9). 50 .mu.l of the cell suspension was mixed with each reaction as well as gRNA only control and no treatment. Samples were incubated at 37.degree. C., 220 RPM for 4 hours. 100 .mu.l of 10.sup.-3, 10.sup.-4, and 10.sup.-5 dilutions of the samples were plated on lysogeny broth plates to obtain a viable cell count at the end of the treatment, the remainder of the reaction was plated onto lysogeny broth plates and incubated overnight at 37.degree. C. Viable cells were counted from the 10.sup.-5 dilution to determine the number of viable colony forming units (CFU) plated on the sample lysogeny broth plate. The sample plates were replica plated via standard techniques to minimal A medium (1 g/L (NH.sub.4).sub.2SO.sub.4, 4.5 g/L KH.sub.2PO.sub.4, 10.5 g/L K.sub.2HPO.sub.4, 0.5 g/L sodium Citrate. 2H.sub.2O, 1 mM MgSO.sub.4.7H.sub.2O, 5.times.10.sup.-5% Thiamine) solidified with 1.5% (w/v) Bacto agar containing 0.2% (w/v) glycerol and 0.2% (w/v) galactose as carbon sources. The plates were incubated at 37.degree. C. for 24 hours and then scored for formation of colonies. Each CFU from a galE strain on a plate containing galactose represents a gene inactivation event of the galK gene. The results of the replica plating are shown in Table 5.

TABLE-US-00009 TABLE 5 Frequency of galK gene inactivation in galE mutant E. coli cells treated with CPP-Cas9/sgRNA. Fold Frequency CFU Gal.sup.R/ plated Frequency untreated Cas9 CFU on on of Gal.sup.R protein sgRNA galactose galactose Gal.sup.R CFU frequency None None 21 1.65 .times. 10.sup.8 1.27 .times. 10.sup.-7 1.00 pVEC-Cas9 None 21 1.18 .times. 10.sup.8 1.78 .times. 10.sup.-7 1.39 pVEC-Cas9 galK2-1 15 1.23 .times. 10.sup.8 1.22 .times. 10.sup.-7 0.96 MPG1-Cas9 None 22 1.34 .times. 10.sup.8 1.65 .times. 10.sup.-7 1.29 MPG1-Cas9 galK2-1 16 1.11 .times. 10.sup.8 1.44 .times. 10.sup.-7 1.13 Zebra-Cas9 None 29 1.89 .times. 10.sup.8 1.53 .times. 10.sup.-7 1.20 Zebra-Cas9 galK2-1 25 8.88 .times. 10.sup.7 2.82 .times. 10.sup.-7 2.21 CFFKDEL- None 29 1.24 .times. 10.sup.8 2.34 .times. 10.sup.-7 1.84 Cas9 CFFKDEL- galK2-1 63 1.24 .times. 10.sup.8 5.10 .times. 10.sup.-7 4.00 Cas9 None galK2-1 31 1.42 .times. 10.sup.8 2.19 .times. 10.sup.-7 1.72

[0394] The treatment of E. coli cells with CPP-Cas9/sgRNA ribonucleoprotein complexes in some cases enhanced the frequency of galK inactivation around 4 fold over the background of untreated cells. This enhancement was not seen in cells treated with only CPP-Cas9 or sgRNA only suggesting that the increased inactivation of the galK gene was due to the CPP-Cas9/sgRNA ribonucleoprotein entering the cell and making DNA double-stranded breaks at the galK2-1 target site within the galK gene.

Example 9

Delivery of CPP-dsREDexpress Protein to Archeal Cells

[0395] In order to test the delivery of cargo using cell-penetrating peptides to Archeal cells and determine candidate CPPs that cross the archeal cell wall which includes elements that are similar to bacterial and eukaryotic cell walls (eg. phospholipids) and membranes and elements that are distinctly archeal (eg. S-layer) archeal cells were treated with CPP-dsREDexpress protein fusions. The CPPs identified in this screen could be used to deliver other cargo (eg. Cas9/sgRNA ribonucleoprotein complex) to Archeal cells.

[0396] The archeon Halobacterium salinarum ATCC19700 was grown on medium 213 (250 g/L NaCl, 10 g/L MgSO.sub.4.7H.sub.2O, 5 g/L KCl, 0.2 g/L CaCl.sub.2.6H.sub.2O, 10 g/L Yeast extract, 2.5 g/L Tryptone) solidified with 1.5% Bacto agar at 37.degree. C. until colonies formed (4 days). A single colony was used to inoculate 50 ml of medium 213 in a 250 ml flask. The culture was grown at 37.degree. C. 220 RPM until the OD.sub.600 reached approximately 0.5 indicating exponential growth phase. 100 .mu.l of cells were mixed with either No protein, 5 .mu.M dsREDexpress (SEQ ID NO: 85), 5 .mu.M MPG1-dsREDexpress (SEQ ID NO: 136), 5 .mu.M pVEC-dsREDexpress (SEQ ID NO: 137), 5 .mu.M CFFKDEL-dsREDexpress (SEQ ID NO: 138), 5 .mu.M TLM-dsREDexpress (SEQ ID NO: 139), 5 .mu.M pep1-dsREDexpress (SEQ ID NO: 141), or 5 .mu.M tp10 dsRED-express (SEQ ID NO: 142) in a 24 well block. Mixtures were incubated for 4 hours at 37.degree. C. 220 RPM. Cells were washed twice with medium 213 lacking tryptone and yeast extract and resusepended in 100 .mu.l of medium 213 lacking tryptone and yeast extract. Cells were analyzed for flourecense in the red channel of an Accuri C5 flow cytometer to determine which CPP tags had delivered the dsREDexpress cargo to H. salinarum cells. The untreated cells were used to create an analysis gate for the flow cytometry data between non-red and red cells such that the gate created a false positive frequency of 0.2% of the untreated cells falling in the red gate (Table 6).

TABLE-US-00010 TABLE 6 CPP delivery of dsREDexpress to H. salinarum. Percent of population Fold increase in in red cell gate .+-. red population over Treatment standard deviation.sup.1 dsREDexpress alone No dsREDexpress 0.21 .+-. 0.06 0.73 dsREDexpress 0.29 .+-. 0.21 1.00 MPG1-dsREDexpress 0.37 .+-. 0.08 1.27 pVEC-dsREDexpress 16.87 .+-. 9.90 57.50 CFFKDEL-dsREDexpress 0.33 .+-. 0.14 1.14 TLM-dsREDexpress 2.03 .+-. 1.02 6.93 pep1-dsREDexpress 0.36 .+-. 0.18 1.23 tp10-dsREDexpress 0.91 .+-. 0.27 3.09 .sup.1Data represents three replicates .+-. standard deviation.

[0397] The delivery of the dsREDexpress cargo into archeal cells demonstrates that at least three of the cell-penetrating peptides (pVEC, TLM, tp10) are capable of delivering a protein cargo to the archeal cells with an efficiency as high as more than 50 fold that of the delivery of the dsREDexpress protein alone suggesting that these three CPP motifs can be used to deliver other cargo to archeal cells (eg. Cas9 ribonucleoprotein complex). Additionally the CPP motifs deliver cargo to as much 16% of the entire cell population suggesting that deliver of cargo by CPP to archeal cells is an efficient process.

Example 10

Delivery of CPP-dsREDexpress Protein to Eukaryotic Cells

[0398] To test the ability of cell-penetrating peptides to deliver cargo to different eukaryotic species a panel of three species, Phytophthora capsici (Oomycete), Septori tritici (True Fungus), and Botrytis cinerea (True Fungus) was treated with various CPP-dsREDexpress fusions. The delivery of dsREDexpress cargo was monitored for various CPP moieties by FACS analysis to determine the percentage of cells to which the cargo was delivered. CPPs that are capable of delivering the dsREDexpress cargo to these cells which suggests that the CPPs would be capable of delivering other cargos to these classes of eukaryotic cells (eg. Cas9/sgRNA ribonucleoprotein complex).

[0399] P. capsici was grown on V8 medium (20% V8 juice, 4.5 g/L CaCO.sub.3) solidified with 1.8% Bacto Agar at 23.degree. C. in the dark for 3 days. The plate was then placed in the light at 23.degree. C. for an additional 7 days. Plates were chilled at 4.degree. C. for 30 minutes. Water was placed on the plate to just cover the surface and allowed to incubate for 30 minutes at room temperature. Liquid was removed to harvest zoospores. Zoospores were confirmed via microscopic analysis. An equal volume of 2.times. encystment medium (40 g/L Tryptone, 10 g/L Yeast extract, 200 ml/L 10.times.SOC salts [5.84 g/L NaCl, 1.86 g/L KCl, 20.3 g/L MgCl.sub.2-6H.sub.2O, 24.6 g/L MgSO.sub.4.7H.sub.2O, 36 g/L Dextrose], 36.4 g/L Sorbitol, 1.47 g/L CaCl.sub.2.2H.sub.2O) was added to the zoospores and gently mixed. Zoospores in enzystment medium were incubated for 20 minutes at room temperature. Encystment was confirmed microscopically. Spores were pelleted and resuspended in an equal volume of YMA medium (2 g/L Yeast extract, 4 g/L Malt extract) and counted using a hemocytometer. Zoospores were diluted to 3.times.10.sup.7 spores/ml in YMA. 100 .mu.l of Zoospores in YMA were mixed with various dsREDexpress fusion proteins (New example 5, table N1) to a final concentration of 5 .mu.M protein. Mixtures were incubated at 25.degree. C. 400 RPM for 2 hours. Cells were washed twice with phosphate buffered saline (PBS) (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na.sub.2HPO.sub.4.2H.sub.2O, 0.24 g/L KH.sub.2PO.sub.4 pH 6.8) and resuspended in a final volume of 200 .mu.l PBS. Uptake of dsREDexpress fusion proteins was monitored using flow cytometry as for Halobacterium salinarium (Example 9). The percent of cells to which the cargo was successfully delivered was determined by drawing an arbitrary gate in the dsREDexpress treated cells such that 0.1% of the population scored as a false positive red event (1:1000 cells). The results of this treatment can be seen in Table 7. pVEC, pep1, and tp10 produce 5.8, 5.5, and 1.8 fold more red cells than the dsREDexpress treated cells alone suggesting that these CPP moieties might be candidates for delivering other cargo to Oomycetes (eg. Cas9/sgRNA ribonucleoprotein complex)

TABLE-US-00011 TABLE 7 CPP delivery of dsREDexpress to Phytophora capsici. Percent of population Fold increase in in red cell gate .+-. red population over Treatment standard deviation.sup.1 dsREDexpress alone dsREDexpress 0.10 .+-. 0.03 1.00 pVEC-dsREDexpress 0.56 .+-. 0.16 5.79 CFFKDEL-dsREDexpress 0.01 .+-. 0.01 0.07 TLM-dsREDexpress 0.00 .+-. 0.00 0.00 pep1-dsREDexpress 0.53 .+-. 0.29 5.52 Tp10-dsREDexpress 0.17 .+-. 0.14 1.76 MPG-dsREDexpress 0.00 .+-. 0.00 0.00 Zebra-dsREDexpress 0.03 .+-. 0.05 0.34 .sup.1Data represents three biological replicates .+-. standard deviation

[0400] B. cinerea was grown on PDA medium (24 g/L potato dextrose broth) solidified with 1.8% Bacto agar in the dark for 5 to 10 days. Conidia were harvested in water with a sterile plastic spreader and filtered through 2 layers of cheesecloth. Conidia were counted on a hemocytometer and diluted to 3.times.10.sup.7conidia per ml in YMA medium. 100 .mu.l of conidia in YMA were mixed with various dsREDexpress fusion proteins (New example 5, table N1) to a final concentration of 5 .mu.M protein. Mixtures were incubated at 25.degree. C. 400 RPM for 2 hours. Cells were washed twice with phosphate buffered saline (PBS) (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na.sub.2HPO.sub.4.2H.sub.2O, 0.24 g/L KH.sub.2PO.sub.4 pH 6.8) and resuspended in a final volume of 200 .mu.l PBS. Uptake of dsREDexpress fusion proteins was monitored using flow cytometry as for Halobacterium salinarium (Example 8). The percent of cells to which the cargo was successfully delivered was determined by drawing an arbitrary gate in the dsREDexpress treated cells such that 0.1% of the population scored as a false positive red event (1:1000 cells). The results of this treatment can be seen in Table 8.

TABLE-US-00012 TABLE 8 CPP delivery of dsREDexpress to Botrytis cinerea Percent of population Fold increase in in red cell gate .+-. red population over Treatment standard deviation.sup.1 dsREDexpress alone dsREDexpress 0.12 .+-. 0.04 1.00 pVEC-dsREDexpress 0.08 .+-. 0.10 0.68 CFFKDEL-dsREDexpress 0.03 .+-. 0.01 0.22 TLM-dsREDexpress 0.01 .+-. 0.01 0.05 pep1-dsREDexpress 0.01 .+-. 0.01 0.05 Tp10-dsREDexpress 0.03 .+-. 0.02 0.24 MPG-dsREDexpress 0.01 .+-. 0.02 0.11 Zebra-dsREDexpress 0.01 .+-. 0.02 0.11 .sup.1Data represents three biological replicates .+-. standard deviation

[0401] S. tritici was grown on YMA medium solidified with 1.8% Bacto agar at 23.degree. C. in light. Conidia were harvested after 5 to 10 days with a sterile plastic spreader and water. Conidia was counted on a hemocytometer and diluted to 3.times.10.sup.7 conidia in YMA medium. 100 .mu.l of conidia in YMA were mixed with various dsREDexpress fusion proteins (New example 5, table N1) to a final concentration of 5 .mu.M protein. Mixtures were incubated at 25.degree. C. 400 RPM for 2 hours. Cells were washed twice with phosphate buffered saline (PBS) (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na.sub.2HPO.sub.4.2H.sub.2O, 0.24 g/L KH.sub.2PO.sub.4 pH 6.8) and resuspended in a final volume of 200 .mu.l PBS. Uptake of dsREDexpress fusion proteins was monitored using flow cytometry as for Halobacterium salinarium (Example 9). The percent of cells to which the cargo was successfully delivered was determined by drawing an arbitrary gate in the dsREDexpress treated cells such that 0.1% of the population scored as a false positive red event (1:1000 cells). The results of this treatment can be seen in Table 9. pVEC, TLM, pep1, and tp10 increased the delivery of dsREDexpress 25, 4, 3, and 5 fold respectively compared to dsREDexpress alone. This suggests that these CPPs would be good candidates for the delivery of other cargo to True fungi (eg. Cas9/sgRNA ribonucleoprotein complex).

TABLE-US-00013 TABLE 9 CPP delivery of dsREDexpress to Septoria tritici Percent of population Fold increase in in red cell gate .+-. red population over Treatment standard deviation.sup.1 dsREDexpress alone dsREDexpress 0.12 .+-. 0.03 1.00 pVEC-dsREDexpress 3.02 .+-. 0.91 25.2 CFFKDEL-dsREDexpress 0.00 .+-. 0.01 0.03 TLM-dsREDexpress 0.48 .+-. 0.14 4.03 pep1-dsREDexpress 0.37 .+-. 0.21 3.06 Tp10-dsREDexpress 0.71 .+-. 0.69 5.94 MPG-dsREDexpress 0.14 .+-. 0.05 1.17 Zebra-dsREDexpress 0.00 .+-. 0.00 0.00 .sup.1Data represents three biological replicates .+-. standard deviation

Example 11

Delivery of Seven CPPs-dsRED and Two CPPs-tagRFP into Seven Gut Bacteria

[0402] In this example, the efficiency of CPPs in delivering two cargo proteins, dsRED and tag RFP, into 7 gut bacterial species (whose beneficial effects on host physiology have been demonstrated) was tested.

Bacterial cells were grown in appropriate media (see Table 10) overnight at 37.degree. C. in a rotary shaker at 150 rpm in an anaerobic tent (80% N2, 15% CO.sub.2, and 5% H.sub.2). For the assay, 1.times.10.sup.8 bacterial cells were mixed with a final concentration of 5 uM of CPPs-dsRED and CPPs-tagRFP proteins in a 96 well plate, followed by two hours outgrowth at 37.degree. C. To measure the dsRED and RFP fluorescence signals in cells, bacterial cells were harvested by centrifugation (3,500.times.g, 4.degree. C., 20 min) and washed twice in phosphate buffered saline (100 ul per well). Fluorescence intensities were quantitated with Tecan Spark 10M plate reader (Tecan, Mannedorf, Switzerland) equipped with 554 nm excitation and 586 nm emission filters with 10 nm bandwidth. Raw fluorescence values were subtracted from that of the untreated cells (background). The fluorescence intensity values of 7000 as a minimum cutoff was taken for delivery of CPPs inside the cells.

TABLE-US-00014 TABLE 10 Culture medium of 7 bacterial species Bacteria Phylum Culture medium Bacteroides Bacteroidetes Brain and Heart thetaiotaomicron Infusion supplemented with 10% bovine blood (Blood BHI) Eubacterium hallii Firmicutes Blood BHI Faecalibacterium Firmicutes Blood BHI prausnitzii Blautia Firmicutes YCFA hydrogenotrophica Bacteroides fragilis Bacteroidetes Blood BHI Prevotella histicola Bacteroidetes Blood BHI Clostridium scindens Firmicutes YCFA

As shown in Table 11, these results indicate that five CPPs including MPG, pVEC, TLM, ZEBRA, and pep1 were effectively delivered into the anaerobic gut bacteria belonging to the phyla Firmicutes and Bacteroidetes, thereby indicating that the CPP's can traverse through the cell membrane of these (Table 9).

TABLE-US-00015 TABLE 11 Differential delivery efficiencies of CPPs in different bacterial strains as demonstrated by the fluorescence intensity above the cutoff value of 7000 MPG-1- pVEC- TLM- ZEBRA- pep1- dsRED dsRED dsRED dsRED dsRED Bacteroides -- -- -- 10230 16657 thetaiotaomicron Eubacterium hallii 10015 17156 -- 16894 7004 Faecalibacterium -- 40525 14998 17014 12696 prausnitzii Blautia -- 11770 14612 9623 -- hydrogenotrophica Bacteroides fragilis -- 14783 -- 15026 -- Prevotella histicola -- -- -- 22416 -- Clostridium scindens -- 17677 32492 -- --

Sequence CWU 1

1

14414107DNAartificial sequenceS. pyogenes Cas9 1atggacaaga aatactccat cggcctggac attggaacca actctgtcgg ctgggctgtc 60atcaccgacg agtacaaggt gccctccaag aaattcaagg tcctcggaaa caccgatcga 120cactccatca agaaaaacct cattggtgcc ctgttgttcg attctggcga gactgccgaa 180gctaccagac tcaagcgaac tgctcggcga cgttacaccc gacggaagaa ccgaatctgc 240tacctgcagg agatcttttc caacgagatg gccaaggtgg acgattcgtt ctttcatcga 300ctggaggaat ccttcctcgt cgaggaagac aagaaacacg agcgtcatcc catctttggc 360aacattgtgg acgaggttgc ttaccacgag aagtatccta ccatctacca cctgcgaaag 420aaactcgtcg attccaccga caaggcggat ctcagactta tctacctcgc tctggcacac 480atgatcaagt ttcgaggtca tttcctcatc gagggcgatc tcaatcccga caacagcgat 540gtggacaagc tgttcattca gctcgttcag acctacaacc agctgttcga ggaaaacccc 600atcaatgcct ccggagtcga tgcaaaggcc atcttgtctg ctcgactctc gaagagcaga 660cgactggaga acctcattgc ccaacttcct ggcgagaaaa agaacggact gtttggcaac 720ctcattgccc tttctcttgg tctcacaccc aacttcaagt ccaacttcga tctggcggag 780gacgccaagc tccagctgtc caaggacacc tacgacgatg acctcgacaa cctgcttgca 840cagattggcg atcagtacgc cgacctgttt ctcgctgcca agaacctttc ggatgctatt 900ctcttgtctg acattctgcg agtcaacacc gagatcacaa aggctcccct ttctgcctcc 960atgatcaagc gatacgacga gcaccatcag gatctcacac tgctcaaggc tcttgtccga 1020cagcaactgc ccgagaagta caaggagatc tttttcgatc agtcgaagaa cggctacgct 1080ggatacatcg acggcggagc ctctcaggaa gagttctaca agttcatcaa gccaattctc 1140gagaagatgg acggaaccga ggaactgctt gtcaagctca atcgagagga tctgcttcgg 1200aagcaacgaa ccttcgacaa cggcagcatt cctcatcaga tccacctcgg tgagctgcac 1260gccattcttc gacgtcagga agacttctac ccctttctca aggacaaccg agagaagatc 1320gagaagattc ttacctttcg aatcccctac tatgttggtc ctcttgccag aggaaactct 1380cgatttgctt ggatgactcg aaagtccgag gaaaccatca ctccctggaa cttcgaggaa 1440gtcgtggaca agggtgcctc tgcacagtcc ttcatcgagc gaatgaccaa cttcgacaag 1500aatctgccca acgagaaggt tcttcccaag cattcgctgc tctacgagta ctttacagtc 1560tacaacgaac tcaccaaagt caagtacgtt accgagggaa tgcgaaagcc tgccttcttg 1620tctggcgaac agaagaaagc cattgtcgat ctcctgttca agaccaaccg aaaggtcact 1680gttaagcagc tcaaggagga ctacttcaag aaaatcgagt gtttcgacag cgtcgagatt 1740tccggagttg aggaccgatt caacgcctct ttgggcacct atcacgatct gctcaagatt 1800atcaaggaca aggattttct cgacaacgag gaaaacgagg acattctgga ggacatcgtg 1860ctcactctta ccctgttcga agatcgggag atgatcgagg aacgactcaa gacatacgct 1920cacctgttcg acgacaaggt catgaaacaa ctcaagcgac gtagatacac cggctgggga 1980agactttcgc gaaagctcat caacggcatc agagacaagc agtccggaaa gaccattctg 2040gactttctca agtccgatgg ctttgccaac cgaaacttca tgcagctcat tcacgacgat 2100tctcttacct tcaaggagga catccagaag gcacaagtgt ccggtcaggg cgacagcttg 2160cacgaacata ttgccaacct ggctggttcg ccagccatca agaaaggcat tctccagact 2220gtcaaggttg tcgacgagct ggtgaaggtc atgggacgtc acaagcccga gaacattgtg 2280atcgagatgg ccagagagaa ccagacaact caaaagggtc agaaaaactc gcgagagcgg 2340atgaagcgaa tcgaggaagg catcaaggag ctgggatccc agattctcaa ggagcatccc 2400gtcgagaaca ctcaactgca gaacgagaag ctgtatctct actatctgca gaatggtcga 2460gacatgtacg tggatcagga actggacatc aatcgtctca gcgactacga tgtggaccac 2520attgtccctc aatcctttct caaggacgat tctatcgaca acaaggtcct tacacgatcc 2580gacaagaaca gaggcaagtc ggacaacgtt cccagcgaag aggtggtcaa aaagatgaag 2640aactactggc gacagctgct caacgccaag ctcattaccc agcgaaagtt cgacaatctt 2700accaaggccg agcgaggcgg tctgtccgag ctcgacaagg ctggcttcat caagcgtcaa 2760ctcgtcgaga ccagacagat cacaaagcac gtcgcacaga ttctcgattc tcggatgaac 2820accaagtacg acgagaacga caagctcatc cgagaggtca aggtgattac tctcaagtcc 2880aaactggtct ccgatttccg aaaggacttt cagttctaca aggtgcgaga gatcaacaat 2940taccaccatg cccacgatgc ttacctcaac gccgtcgttg gcactgcgct catcaagaaa 3000taccccaagc tcgaaagcga gttcgtttac ggcgattaca aggtctacga cgttcgaaag 3060atgattgcca agtccgaaca ggagattggc aaggctactg ccaagtactt cttttactcc 3120aacatcatga actttttcaa gaccgagatc accttggcca acggagagat tcgaaagaga 3180ccacttatcg agaccaacgg cgaaactgga gagatcgtgt gggacaaggg tcgagacttt 3240gcaaccgtgc gaaaggttct gtcgatgcct caggtcaaca tcgtcaagaa aaccgaggtt 3300cagactggcg gattctccaa ggagtcgatt ctgcccaagc gaaactccga caagctcatc 3360gctcgaaaga aagactggga tcccaagaaa tacggtggct tcgattctcc taccgtcgcc 3420tattccgtgc ttgtcgttgc gaaggtcgag aagggcaagt ccaaaaagct caagtccgtc 3480aaggagctgc tcggaattac catcatggag cgatcgagct tcgagaagaa tcccatcgac 3540ttcttggaag ccaagggtta caaggaggtc aagaaagacc tcattatcaa gctgcccaag 3600tactctctgt tcgaactgga gaacggtcga aagcgtatgc tcgcctccgc tggcgagctg 3660cagaagggaa acgagcttgc cttgccttcg aagtacgtca actttctcta tctggcttct 3720cactacgaga agctcaaggg ttctcccgag gacaacgaac agaagcaact cttcgttgag 3780cagcacaaac attacctcga cgagattatc gagcagattt ccgagttttc gaagcgagtc 3840atcctggctg atgccaactt ggacaaggtg ctctctgcct acaacaagca tcgggacaaa 3900cccattcgag aacaggcgga gaacatcatt cacctgttta ctcttaccaa cctgggtgct 3960cctgcagctt tcaagtactt cgataccact atcgaccgaa agcggtacac atccaccaag 4020gaggttctcg atgccaccct gattcaccag tccatcactg gcctgtacga gacccgaatc 4080gacctgtctc agcttggtgg cgactaa 410724140DNAartificial sequenceS. pyogenes Cas9 with NLS 2atggacaaga aatactccat cggcctggac attggaacca actctgtcgg ctgggctgtc 60atcaccgacg agtacaaggt gccctccaag aaattcaagg tcctcggaaa caccgatcga 120cactccatca agaaaaacct cattggtgcc ctgttgttcg attctggcga gactgccgaa 180gctaccagac tcaagcgaac tgctcggcga cgttacaccc gacggaagaa ccgaatctgc 240tacctgcagg agatcttttc caacgagatg gccaaggtgg acgattcgtt ctttcatcga 300ctggaggaat ccttcctcgt cgaggaagac aagaaacacg agcgtcatcc catctttggc 360aacattgtgg acgaggttgc ttaccacgag aagtatccta ccatctacca cctgcgaaag 420aaactcgtcg attccaccga caaggcggat ctcagactta tctacctcgc tctggcacac 480atgatcaagt ttcgaggtca tttcctcatc gagggcgatc tcaatcccga caacagcgat 540gtggacaagc tgttcattca gctcgttcag acctacaacc agctgttcga ggaaaacccc 600atcaatgcct ccggagtcga tgcaaaggcc atcttgtctg ctcgactctc gaagagcaga 660cgactggaga acctcattgc ccaacttcct ggcgagaaaa agaacggact gtttggcaac 720ctcattgccc tttctcttgg tctcacaccc aacttcaagt ccaacttcga tctggcggag 780gacgccaagc tccagctgtc caaggacacc tacgacgatg acctcgacaa cctgcttgca 840cagattggcg atcagtacgc cgacctgttt ctcgctgcca agaacctttc ggatgctatt 900ctcttgtctg acattctgcg agtcaacacc gagatcacaa aggctcccct ttctgcctcc 960atgatcaagc gatacgacga gcaccatcag gatctcacac tgctcaaggc tcttgtccga 1020cagcaactgc ccgagaagta caaggagatc tttttcgatc agtcgaagaa cggctacgct 1080ggatacatcg acggcggagc ctctcaggaa gagttctaca agttcatcaa gccaattctc 1140gagaagatgg acggaaccga ggaactgctt gtcaagctca atcgagagga tctgcttcgg 1200aagcaacgaa ccttcgacaa cggcagcatt cctcatcaga tccacctcgg tgagctgcac 1260gccattcttc gacgtcagga agacttctac ccctttctca aggacaaccg agagaagatc 1320gagaagattc ttacctttcg aatcccctac tatgttggtc ctcttgccag aggaaactct 1380cgatttgctt ggatgactcg aaagtccgag gaaaccatca ctccctggaa cttcgaggaa 1440gtcgtggaca agggtgcctc tgcacagtcc ttcatcgagc gaatgaccaa cttcgacaag 1500aatctgccca acgagaaggt tcttcccaag cattcgctgc tctacgagta ctttacagtc 1560tacaacgaac tcaccaaagt caagtacgtt accgagggaa tgcgaaagcc tgccttcttg 1620tctggcgaac agaagaaagc cattgtcgat ctcctgttca agaccaaccg aaaggtcact 1680gttaagcagc tcaaggagga ctacttcaag aaaatcgagt gtttcgacag cgtcgagatt 1740tccggagttg aggaccgatt caacgcctct ttgggcacct atcacgatct gctcaagatt 1800atcaaggaca aggattttct cgacaacgag gaaaacgagg acattctgga ggacatcgtg 1860ctcactctta ccctgttcga agatcgggag atgatcgagg aacgactcaa gacatacgct 1920cacctgttcg acgacaaggt catgaaacaa ctcaagcgac gtagatacac cggctgggga 1980agactttcgc gaaagctcat caacggcatc agagacaagc agtccggaaa gaccattctg 2040gactttctca agtccgatgg ctttgccaac cgaaacttca tgcagctcat tcacgacgat 2100tctcttacct tcaaggagga catccagaag gcacaagtgt ccggtcaggg cgacagcttg 2160cacgaacata ttgccaacct ggctggttcg ccagccatca agaaaggcat tctccagact 2220gtcaaggttg tcgacgagct ggtgaaggtc atgggacgtc acaagcccga gaacattgtg 2280atcgagatgg ccagagagaa ccagacaact caaaagggtc agaaaaactc gcgagagcgg 2340atgaagcgaa tcgaggaagg catcaaggag ctgggatccc agattctcaa ggagcatccc 2400gtcgagaaca ctcaactgca gaacgagaag ctgtatctct actatctgca gaatggtcga 2460gacatgtacg tggatcagga actggacatc aatcgtctca gcgactacga tgtggaccac 2520attgtccctc aatcctttct caaggacgat tctatcgaca acaaggtcct tacacgatcc 2580gacaagaaca gaggcaagtc ggacaacgtt cccagcgaag aggtggtcaa aaagatgaag 2640aactactggc gacagctgct caacgccaag ctcattaccc agcgaaagtt cgacaatctt 2700accaaggccg agcgaggcgg tctgtccgag ctcgacaagg ctggcttcat caagcgtcaa 2760ctcgtcgaga ccagacagat cacaaagcac gtcgcacaga ttctcgattc tcggatgaac 2820accaagtacg acgagaacga caagctcatc cgagaggtca aggtgattac tctcaagtcc 2880aaactggtct ccgatttccg aaaggacttt cagttctaca aggtgcgaga gatcaacaat 2940taccaccatg cccacgatgc ttacctcaac gccgtcgttg gcactgcgct catcaagaaa 3000taccccaagc tcgaaagcga gttcgtttac ggcgattaca aggtctacga cgttcgaaag 3060atgattgcca agtccgaaca ggagattggc aaggctactg ccaagtactt cttttactcc 3120aacatcatga actttttcaa gaccgagatc accttggcca acggagagat tcgaaagaga 3180ccacttatcg agaccaacgg cgaaactgga gagatcgtgt gggacaaggg tcgagacttt 3240gcaaccgtgc gaaaggttct gtcgatgcct caggtcaaca tcgtcaagaa aaccgaggtt 3300cagactggcg gattctccaa ggagtcgatt ctgcccaagc gaaactccga caagctcatc 3360gctcgaaaga aagactggga tcccaagaaa tacggtggct tcgattctcc taccgtcgcc 3420tattccgtgc ttgtcgttgc gaaggtcgag aagggcaagt ccaaaaagct caagtccgtc 3480aaggagctgc tcggaattac catcatggag cgatcgagct tcgagaagaa tcccatcgac 3540ttcttggaag ccaagggtta caaggaggtc aagaaagacc tcattatcaa gctgcccaag 3600tactctctgt tcgaactgga gaacggtcga aagcgtatgc tcgcctccgc tggcgagctg 3660cagaagggaa acgagcttgc cttgccttcg aagtacgtca actttctcta tctggcttct 3720cactacgaga agctcaaggg ttctcccgag gacaacgaac agaagcaact cttcgttgag 3780cagcacaaac attacctcga cgagattatc gagcagattt ccgagttttc gaagcgagtc 3840atcctggctg atgccaactt ggacaaggtg ctctctgcct acaacaagca tcgggacaaa 3900cccattcgag aacaggcgga gaacatcatt cacctgttta ctcttaccaa cctgggtgct 3960cctgcagctt tcaagtactt cgataccact atcgaccgaa agcggtacac atccaccaag 4020gaggttctcg atgccaccct gattcaccag tccatcactg gcctgtacga gacccgaatc 4080gacctgtctc agcttggtgg cgactccaga gccgatccca agaaaaagcg aaaggtctaa 414031379PRTartificial sequenceS. pyogenes Cas9 with NLS 3Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val1 5 10 15Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys65 70 75 80Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His145 150 155 160Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn225 230 235 240Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser305 310 315 320Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg385 390 395 400Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu465 470 475 480Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr545 550 555 560Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala625 630 635 640His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu705 710 715 720His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro785 790 795 800Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys865 870 875 880Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser945 950 955 960Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile

Val Lys Lys Thr 1085 1090 1095Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val 1370 13754543DNAYarrowia lipolytica 4tcgacgttta aaccatcatc taagggcctc aaaactacct cggaactgct gcgctgatct 60ggacaccaca gaggttccga gcactttagg ttgcaccaaa tgtcccacca ggtgcaggca 120gaaaacgctg gaacagcgtg tacagtttgt cttaacaaaa agtgagggcg ctgaggtcga 180gcagggtggt gtgacttgtt atagccttta gagctgcgaa agcgcgtatg gatttggctc 240atcaggccag attgagggtc tgtggacaca tgtcatgtta gtgtacttca atcgccccct 300ggatatagcc ccgacaatag gccgtggcct catttttttg ccttccgcac atttccattg 360ctcggtaccc acaccttgct tctcctgcac ttgccaacct taatactggt ttacattgac 420caacatctta caagcggggg gcttgtctag ggtatatata aacagtggct ctcccaatcg 480gttgccagtc tcttttttcc tttctttccc cacagattcg aaatctaaac tacacatcac 540acc 54354683DNAartificial sequenceCas9-NLS expression cassette (FBA1 promoter and Cas9-NLS open reading frame) 5tcgacgttta aaccatcatc taagggcctc aaaactacct cggaactgct gcgctgatct 60ggacaccaca gaggttccga gcactttagg ttgcaccaaa tgtcccacca ggtgcaggca 120gaaaacgctg gaacagcgtg tacagtttgt cttaacaaaa agtgagggcg ctgaggtcga 180gcagggtggt gtgacttgtt atagccttta gagctgcgaa agcgcgtatg gatttggctc 240atcaggccag attgagggtc tgtggacaca tgtcatgtta gtgtacttca atcgccccct 300ggatatagcc ccgacaatag gccgtggcct catttttttg ccttccgcac atttccattg 360ctcggtaccc acaccttgct tctcctgcac ttgccaacct taatactggt ttacattgac 420caacatctta caagcggggg gcttgtctag ggtatatata aacagtggct ctcccaatcg 480gttgccagtc tcttttttcc tttctttccc cacagattcg aaatctaaac tacacatcac 540accatggaca agaaatactc catcggcctg gacattggaa ccaactctgt cggctgggct 600gtcatcaccg acgagtacaa ggtgccctcc aagaaattca aggtcctcgg aaacaccgat 660cgacactcca tcaagaaaaa cctcattggt gccctgttgt tcgattctgg cgagactgcc 720gaagctacca gactcaagcg aactgctcgg cgacgttaca cccgacggaa gaaccgaatc 780tgctacctgc aggagatctt ttccaacgag atggccaagg tggacgattc gttctttcat 840cgactggagg aatccttcct cgtcgaggaa gacaagaaac acgagcgtca tcccatcttt 900ggcaacattg tggacgaggt tgcttaccac gagaagtatc ctaccatcta ccacctgcga 960aagaaactcg tcgattccac cgacaaggcg gatctcagac ttatctacct cgctctggca 1020cacatgatca agtttcgagg tcatttcctc atcgagggcg atctcaatcc cgacaacagc 1080gatgtggaca agctgttcat tcagctcgtt cagacctaca accagctgtt cgaggaaaac 1140cccatcaatg cctccggagt cgatgcaaag gccatcttgt ctgctcgact ctcgaagagc 1200agacgactgg agaacctcat tgcccaactt cctggcgaga aaaagaacgg actgtttggc 1260aacctcattg ccctttctct tggtctcaca cccaacttca agtccaactt cgatctggcg 1320gaggacgcca agctccagct gtccaaggac acctacgacg atgacctcga caacctgctt 1380gcacagattg gcgatcagta cgccgacctg tttctcgctg ccaagaacct ttcggatgct 1440attctcttgt ctgacattct gcgagtcaac accgagatca caaaggctcc cctttctgcc 1500tccatgatca agcgatacga cgagcaccat caggatctca cactgctcaa ggctcttgtc 1560cgacagcaac tgcccgagaa gtacaaggag atctttttcg atcagtcgaa gaacggctac 1620gctggataca tcgacggcgg agcctctcag gaagagttct acaagttcat caagccaatt 1680ctcgagaaga tggacggaac cgaggaactg cttgtcaagc tcaatcgaga ggatctgctt 1740cggaagcaac gaaccttcga caacggcagc attcctcatc agatccacct cggtgagctg 1800cacgccattc ttcgacgtca ggaagacttc tacccctttc tcaaggacaa ccgagagaag 1860atcgagaaga ttcttacctt tcgaatcccc tactatgttg gtcctcttgc cagaggaaac 1920tctcgatttg cttggatgac tcgaaagtcc gaggaaacca tcactccctg gaacttcgag 1980gaagtcgtgg acaagggtgc ctctgcacag tccttcatcg agcgaatgac caacttcgac 2040aagaatctgc ccaacgagaa ggttcttccc aagcattcgc tgctctacga gtactttaca 2100gtctacaacg aactcaccaa agtcaagtac gttaccgagg gaatgcgaaa gcctgccttc 2160ttgtctggcg aacagaagaa agccattgtc gatctcctgt tcaagaccaa ccgaaaggtc 2220actgttaagc agctcaagga ggactacttc aagaaaatcg agtgtttcga cagcgtcgag 2280atttccggag ttgaggaccg attcaacgcc tctttgggca cctatcacga tctgctcaag 2340attatcaagg acaaggattt tctcgacaac gaggaaaacg aggacattct ggaggacatc 2400gtgctcactc ttaccctgtt cgaagatcgg gagatgatcg aggaacgact caagacatac 2460gctcacctgt tcgacgacaa ggtcatgaaa caactcaagc gacgtagata caccggctgg 2520ggaagacttt cgcgaaagct catcaacggc atcagagaca agcagtccgg aaagaccatt 2580ctggactttc tcaagtccga tggctttgcc aaccgaaact tcatgcagct cattcacgac 2640gattctctta ccttcaagga ggacatccag aaggcacaag tgtccggtca gggcgacagc 2700ttgcacgaac atattgccaa cctggctggt tcgccagcca tcaagaaagg cattctccag 2760actgtcaagg ttgtcgacga gctggtgaag gtcatgggac gtcacaagcc cgagaacatt 2820gtgatcgaga tggccagaga gaaccagaca actcaaaagg gtcagaaaaa ctcgcgagag 2880cggatgaagc gaatcgagga aggcatcaag gagctgggat cccagattct caaggagcat 2940cccgtcgaga acactcaact gcagaacgag aagctgtatc tctactatct gcagaatggt 3000cgagacatgt acgtggatca ggaactggac atcaatcgtc tcagcgacta cgatgtggac 3060cacattgtcc ctcaatcctt tctcaaggac gattctatcg acaacaaggt ccttacacga 3120tccgacaaga acagaggcaa gtcggacaac gttcccagcg aagaggtggt caaaaagatg 3180aagaactact ggcgacagct gctcaacgcc aagctcatta cccagcgaaa gttcgacaat 3240cttaccaagg ccgagcgagg cggtctgtcc gagctcgaca aggctggctt catcaagcgt 3300caactcgtcg agaccagaca gatcacaaag cacgtcgcac agattctcga ttctcggatg 3360aacaccaagt acgacgagaa cgacaagctc atccgagagg tcaaggtgat tactctcaag 3420tccaaactgg tctccgattt ccgaaaggac tttcagttct acaaggtgcg agagatcaac 3480aattaccacc atgcccacga tgcttacctc aacgccgtcg ttggcactgc gctcatcaag 3540aaatacccca agctcgaaag cgagttcgtt tacggcgatt acaaggtcta cgacgttcga 3600aagatgattg ccaagtccga acaggagatt ggcaaggcta ctgccaagta cttcttttac 3660tccaacatca tgaacttttt caagaccgag atcaccttgg ccaacggaga gattcgaaag 3720agaccactta tcgagaccaa cggcgaaact ggagagatcg tgtgggacaa gggtcgagac 3780tttgcaaccg tgcgaaaggt tctgtcgatg cctcaggtca acatcgtcaa gaaaaccgag 3840gttcagactg gcggattctc caaggagtcg attctgccca agcgaaactc cgacaagctc 3900atcgctcgaa agaaagactg ggatcccaag aaatacggtg gcttcgattc tcctaccgtc 3960gcctattccg tgcttgtcgt tgcgaaggtc gagaagggca agtccaaaaa gctcaagtcc 4020gtcaaggagc tgctcggaat taccatcatg gagcgatcga gcttcgagaa gaatcccatc 4080gacttcttgg aagccaaggg ttacaaggag gtcaagaaag acctcattat caagctgccc 4140aagtactctc tgttcgaact ggagaacggt cgaaagcgta tgctcgcctc cgctggcgag 4200ctgcagaagg gaaacgagct tgccttgcct tcgaagtacg tcaactttct ctatctggct 4260tctcactacg agaagctcaa gggttctccc gaggacaacg aacagaagca actcttcgtt 4320gagcagcaca aacattacct cgacgagatt atcgagcaga tttccgagtt ttcgaagcga 4380gtcatcctgg ctgatgccaa cttggacaag gtgctctctg cctacaacaa gcatcgggac 4440aaacccattc gagaacaggc ggagaacatc attcacctgt ttactcttac caacctgggt 4500gctcctgcag ctttcaagta cttcgatacc actatcgacc gaaagcggta cacatccacc 4560aaggaggttc tcgatgccac cctgattcac cagtccatca ctggcctgta cgagacccga 4620atcgacctgt ctcagcttgg tggcgactcc agagccgatc ccaagaaaaa gcgaaaggtc 4680taa 4683610706DNAartificial sequencepZUFCas9 plasmid 6catggacaag aaatactcca tcggcctgga cattggaacc aactctgtcg gctgggctgt 60catcaccgac gagtacaagg tgccctccaa gaaattcaag gtcctcggaa acaccgatcg 120acactccatc aagaaaaacc tcattggtgc cctgttgttc gattctggcg agactgccga 180agctaccaga ctcaagcgaa ctgctcggcg acgttacacc cgacggaaga accgaatctg 240ctacctgcag gagatctttt ccaacgagat ggccaaggtg gacgattcgt tctttcatcg 300actggaggaa tccttcctcg tcgaggaaga caagaaacac gagcgtcatc ccatctttgg 360caacattgtg gacgaggttg cttaccacga gaagtatcct accatctacc acctgcgaaa 420gaaactcgtc gattccaccg acaaggcgga tctcagactt atctacctcg ctctggcaca 480catgatcaag tttcgaggtc atttcctcat cgagggcgat ctcaatcccg acaacagcga 540tgtggacaag ctgttcattc agctcgttca gacctacaac cagctgttcg aggaaaaccc 600catcaatgcc tccggagtcg atgcaaaggc catcttgtct gctcgactct cgaagagcag 660acgactggag aacctcattg cccaacttcc tggcgagaaa aagaacggac tgtttggcaa 720cctcattgcc ctttctcttg gtctcacacc caacttcaag tccaacttcg atctggcgga 780ggacgccaag ctccagctgt ccaaggacac ctacgacgat gacctcgaca acctgcttgc 840acagattggc gatcagtacg ccgacctgtt tctcgctgcc aagaaccttt cggatgctat 900tctcttgtct gacattctgc gagtcaacac cgagatcaca aaggctcccc tttctgcctc 960catgatcaag cgatacgacg agcaccatca ggatctcaca ctgctcaagg ctcttgtccg 1020acagcaactg cccgagaagt acaaggagat ctttttcgat cagtcgaaga acggctacgc 1080tggatacatc gacggcggag cctctcagga agagttctac aagttcatca agccaattct 1140cgagaagatg gacggaaccg aggaactgct tgtcaagctc aatcgagagg atctgcttcg 1200gaagcaacga accttcgaca acggcagcat tcctcatcag atccacctcg gtgagctgca 1260cgccattctt cgacgtcagg aagacttcta cccctttctc aaggacaacc gagagaagat 1320cgagaagatt cttacctttc gaatccccta ctatgttggt cctcttgcca gaggaaactc 1380tcgatttgct tggatgactc gaaagtccga ggaaaccatc actccctgga acttcgagga 1440agtcgtggac aagggtgcct ctgcacagtc cttcatcgag cgaatgacca acttcgacaa 1500gaatctgccc aacgagaagg ttcttcccaa gcattcgctg ctctacgagt actttacagt 1560ctacaacgaa ctcaccaaag tcaagtacgt taccgaggga atgcgaaagc ctgccttctt 1620gtctggcgaa cagaagaaag ccattgtcga tctcctgttc aagaccaacc gaaaggtcac 1680tgttaagcag ctcaaggagg actacttcaa gaaaatcgag tgtttcgaca gcgtcgagat 1740ttccggagtt gaggaccgat tcaacgcctc tttgggcacc tatcacgatc tgctcaagat 1800tatcaaggac aaggattttc tcgacaacga ggaaaacgag gacattctgg aggacatcgt 1860gctcactctt accctgttcg aagatcggga gatgatcgag gaacgactca agacatacgc 1920tcacctgttc gacgacaagg tcatgaaaca actcaagcga cgtagataca ccggctgggg 1980aagactttcg cgaaagctca tcaacggcat cagagacaag cagtccggaa agaccattct 2040ggactttctc aagtccgatg gctttgccaa ccgaaacttc atgcagctca ttcacgacga 2100ttctcttacc ttcaaggagg acatccagaa ggcacaagtg tccggtcagg gcgacagctt 2160gcacgaacat attgccaacc tggctggttc gccagccatc aagaaaggca ttctccagac 2220tgtcaaggtt gtcgacgagc tggtgaaggt catgggacgt cacaagcccg agaacattgt 2280gatcgagatg gccagagaga accagacaac tcaaaagggt cagaaaaact cgcgagagcg 2340gatgaagcga atcgaggaag gcatcaagga gctgggatcc cagattctca aggagcatcc 2400cgtcgagaac actcaactgc agaacgagaa gctgtatctc tactatctgc agaatggtcg 2460agacatgtac gtggatcagg aactggacat caatcgtctc agcgactacg atgtggacca 2520cattgtccct caatcctttc tcaaggacga ttctatcgac aacaaggtcc ttacacgatc 2580cgacaagaac agaggcaagt cggacaacgt tcccagcgaa gaggtggtca aaaagatgaa 2640gaactactgg cgacagctgc tcaacgccaa gctcattacc cagcgaaagt tcgacaatct 2700taccaaggcc gagcgaggcg gtctgtccga gctcgacaag gctggcttca tcaagcgtca 2760actcgtcgag accagacaga tcacaaagca cgtcgcacag attctcgatt ctcggatgaa 2820caccaagtac gacgagaacg acaagctcat ccgagaggtc aaggtgatta ctctcaagtc 2880caaactggtc tccgatttcc gaaaggactt tcagttctac aaggtgcgag agatcaacaa 2940ttaccaccat gcccacgatg cttacctcaa cgccgtcgtt ggcactgcgc tcatcaagaa 3000ataccccaag ctcgaaagcg agttcgttta cggcgattac aaggtctacg acgttcgaaa 3060gatgattgcc aagtccgaac aggagattgg caaggctact gccaagtact tcttttactc 3120caacatcatg aactttttca agaccgagat caccttggcc aacggagaga ttcgaaagag 3180accacttatc gagaccaacg gcgaaactgg agagatcgtg tgggacaagg gtcgagactt 3240tgcaaccgtg cgaaaggttc tgtcgatgcc tcaggtcaac atcgtcaaga aaaccgaggt 3300tcagactggc ggattctcca aggagtcgat tctgcccaag cgaaactccg acaagctcat 3360cgctcgaaag aaagactggg atcccaagaa atacggtggc ttcgattctc ctaccgtcgc 3420ctattccgtg cttgtcgttg cgaaggtcga gaagggcaag tccaaaaagc tcaagtccgt 3480caaggagctg ctcggaatta ccatcatgga gcgatcgagc ttcgagaaga atcccatcga 3540cttcttggaa gccaagggtt acaaggaggt caagaaagac ctcattatca agctgcccaa 3600gtactctctg ttcgaactgg agaacggtcg aaagcgtatg ctcgcctccg ctggcgagct 3660gcagaaggga aacgagcttg ccttgccttc gaagtacgtc aactttctct atctggcttc 3720tcactacgag aagctcaagg gttctcccga ggacaacgaa cagaagcaac tcttcgttga 3780gcagcacaaa cattacctcg acgagattat cgagcagatt tccgagtttt cgaagcgagt 3840catcctggct gatgccaact tggacaaggt gctctctgcc tacaacaagc atcgggacaa 3900acccattcga gaacaggcgg agaacatcat tcacctgttt actcttacca acctgggtgc 3960tcctgcagct ttcaagtact tcgataccac tatcgaccga aagcggtaca catccaccaa 4020ggaggttctc gatgccaccc tgattcacca gtccatcact ggcctgtacg agacccgaat 4080cgacctgtct cagcttggtg gcgactccag agccgatccc aagaaaaagc gaaaggtcta 4140agcggccgca agtgtggatg gggaagtgag tgcccggttc tgtgtgcaca attggcaatc 4200caagatggat ggattcaaca cagggatata gcgagctacg tggtggtgcg aggatatagc 4260aacggatatt tatgtttgac acttgagaat gtacgataca agcactgtcc aagtacaata 4320ctaaacatac tgtacatact catactcgta cccgggcaac ggtttcactt gagtgcagtg 4380gctagtgctc ttactcgtac agtgtgcaat actgcgtatc atagtctttg atgtatatcg 4440tattcattca tgttagttgc gtacgagccg gaagcataaa gtgtaaagcc tggggtgcct 4500aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 4560acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 4620ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 4680gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 4740caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 4800tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 4860gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 4920ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 4980cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 5040tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 5100tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 5160cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 5220agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga 5280agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 5340gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 5400aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 5460ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 5520gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 5580taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 5640tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 5700tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 5760gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 5820gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 5880ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 5940cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 6000tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 6060cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 6120agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 6180cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 6240aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 6300aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 6360gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 6420gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 6480tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 6540ttccccgaaa agtgccacct gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 6600tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 6660tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 6720tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 6780gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 6840agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 6900cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 6960agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttcca 7020ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt 7080acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt 7140ttcccagtca cgacgttgta aaacgacggc cagtgaattg taatacgact cactataggg 7200cgaattgggt accgggcccc ccctcgaggt cgatggtgtc gataagcttg atatcgaatt 7260catgtcacac aaaccgatct tcgcctcaag gaaacctaat tctacatccg agagactgcc 7320gagatccagt ctacactgat taattttcgg gccaataatt taaaaaaatc gtgttatata 7380atattatatg tattatatat atacatcatg atgatactga cagtcatgtc ccattgctaa 7440atagacagac tccatctgcc gcctccaact gatgttctca atatttaagg ggtcatctcg 7500cattgtttaa taataaacag actccatcta ccgcctccaa atgatgttct caaaatatat 7560tgtatgaact tatttttatt acttagtatt attagacaac ttacttgctt tatgaaaaac 7620acttcctatt taggaaacaa tttataatgg cagttcgttc atttaacaat ttatgtagaa 7680taaatgttat aaatgcgtat gggaaatctt aaatatggat agcataaatg atatctgcat 7740tgcctaattc gaaatcaaca

gcaacgaaaa aaatcccttg tacaacataa atagtcatcg 7800agaaatatca actatcaaag aacagctatt cacacgttac tattgagatt attattggac 7860gagaatcaca cactcaactg tctttctctc ttctagaaat acaggtacaa gtatgtacta 7920ttctcattgt tcatacttct agtcatttca tcccacatat tccttggatt tctctccaat 7980gaatgacatt ctatcttgca aattcaacaa ttataataag atataccaaa gtagcggtat 8040agtggcaatc aaaaagcttc tctggtgtgc ttctcgtatt tatttttatt ctaatgatcc 8100attaaaggta tatatttatt tcttgttata taatcctttt gtttattaca tgggctggat 8160acataaaggt attttgattt aattttttgc ttaaattcaa tcccccctcg ttcagtgtca 8220actgtaatgg taggaaatta ccatactttt gaagaagcaa aaaaaatgaa agaaaaaaaa 8280aatcgtattt ccaggttaga cgttccgcag aatctagaat gcggtatgcg gtacattgtt 8340cttcgaacgt aaaagttgcg ctccctgaga tattgtacat ttttgctttt acaagtacaa 8400gtacatcgta caactatgta ctactgttga tgcatccaca acagtttgtt ttgttttttt 8460ttgttttttt tttttctaat gattcattac cgctatgtat acctacttgt acttgtagta 8520agccgggtta ttggcgttca attaatcata gacttatgaa tctgcacggt gtgcgctgcg 8580agttactttt agcttatgca tgctacttgg gtgtaatatt gggatctgtt cggaaatcaa 8640cggatgctca atcgatttcg acagtaatta attaagtcat acacaagtca gctttcttcg 8700agcctcatat aagtataagt agttcaacgt attagcactg tacccagcat ctccgtatcg 8760agaaacacaa caacatgccc cattggacag atcatgcgga tacacaggtt gtgcagtatc 8820atacatactc gatcagacag gtcgtctgac catcatacaa gctgaacaag cgctccatac 8880ttgcacgctc tctatataca cagttaaatt acatatccat agtctaacct ctaacagtta 8940atcttctggt aagcctccca gccagccttc tggtatcgct tggcctcctc aataggatct 9000cggttctggc cgtacagacc tcggccgaca attatgatat ccgttccggt agacatgaca 9060tcctcaacag ttcggtactg ctgtccgaga gcgtctccct tgtcgtcaag acccaccccg 9120ggggtcagaa taagccagtc ctcagagtcg cccttaggtc ggttctgggc aatgaagcca 9180accacaaact cggggtcgga tcgggcaagc tcaatggtct gcttggagta ctcgccagtg 9240gccagagagc ccttgcaaga cagctcggcc agcatgagca gacctctggc cagcttctcg 9300ttgggagagg ggactaggaa ctccttgtac tgggagttct cgtagtcaga gacgtcctcc 9360ttcttctgtt cagagacagt ttcctcggca ccagctcgca ggccagcaat gattccggtt 9420ccgggtacac cgtgggcgtt ggtgatatcg gaccactcgg cgattcggtg acaccggtac 9480tggtgcttga cagtgttgcc aatatctgcg aactttctgt cctcgaacag gaagaaaccg 9540tgcttaagag caagttcctt gagggggagc acagtgccgg cgtaggtgaa gtcgtcaatg 9600atgtcgatat gggttttgat catgcacaca taaggtccga ccttatcggc aagctcaatg 9660agctccttgg tggtggtaac atccagagaa gcacacaggt tggttttctt ggctgccacg 9720agcttgagca ctcgagcggc aaaggcggac ttgtggacgt tagctcgagc ttcgtaggag 9780ggcattttgg tggtgaagag gagactgaaa taaatttagt ctgcagaact ttttatcgga 9840accttatctg gggcagtgaa gtatatgtta tggtaatagt tacgagttag ttgaacttat 9900agatagactg gactatacgg ctatcggtcc aaattagaaa gaacgtcaat ggctctctgg 9960gcgtcgcctt tgccgacaaa aatgtgatca tgatgaaagc cagcaatgac gttgcagctg 10020atattgttgt cggccaaccg cgccgaaaac gcagctgtca gacccacagc ctccaacgaa 10080gaatgtatcg tcaaagtgat ccaagcacac tcatagttgg agtcgtactc caaaggcggc 10140aatgacgagt cagacagata ctcgtcgacg tttaaaccat catctaaggg cctcaaaact 10200acctcggaac tgctgcgctg atctggacac cacagaggtt ccgagcactt taggttgcac 10260caaatgtccc accaggtgca ggcagaaaac gctggaacag cgtgtacagt ttgtcttaac 10320aaaaagtgag ggcgctgagg tcgagcaggg tggtgtgact tgttatagcc tttagagctg 10380cgaaagcgcg tatggatttg gctcatcagg ccagattgag ggtctgtgga cacatgtcat 10440gttagtgtac ttcaatcgcc ccctggatat agccccgaca ataggccgtg gcctcatttt 10500tttgccttcc gcacatttcc attgctcggt acccacacct tgcttctcct gcacttgcca 10560accttaatac tggtttacat tgaccaacat cttacaagcg gggggcttgt ctagggtata 10620tataaacagt ggctctccca atcggttgcc agtctctttt ttcctttctt tccccacaga 10680ttcgaaatct aaactacaca tcacac 10706735DNAartificial sequenceCas9-NLS forward PCR primer 7gggggaattc gacaagaaat actccatcgg cctgg 35831DNAartificial sequenceCas9-NLS reverse PCR primer 8ccccaagctt agcggccgct tagacctttc g 3194166DNAartificial sequenceEcoRI-Cas9-NLS-HinDIII PCR product 9gggggaattc gacaagaaat actccatcgg cctggacatt ggaaccaact ctgtcggctg 60ggctgtcatc accgacgagt acaaggtgcc ctccaagaaa ttcaaggtcc tcggaaacac 120cgatcgacac tccatcaaga aaaacctcat tggtgccctg ttgttcgatt ctggcgagac 180tgccgaagct accagactca agcgaactgc tcggcgacgt tacacccgac ggaagaaccg 240aatctgctac ctgcaggaga tcttttccaa cgagatggcc aaggtggacg attcgttctt 300tcatcgactg gaggaatcct tcctcgtcga ggaagacaag aaacacgagc gtcatcccat 360ctttggcaac attgtggacg aggttgctta ccacgagaag tatcctacca tctaccacct 420gcgaaagaaa ctcgtcgatt ccaccgacaa ggcggatctc agacttatct acctcgctct 480ggcacacatg atcaagtttc gaggtcattt cctcatcgag ggcgatctca atcccgacaa 540cagcgatgtg gacaagctgt tcattcagct cgttcagacc tacaaccagc tgttcgagga 600aaaccccatc aatgcctccg gagtcgatgc aaaggccatc ttgtctgctc gactctcgaa 660gagcagacga ctggagaacc tcattgccca acttcctggc gagaaaaaga acggactgtt 720tggcaacctc attgcccttt ctcttggtct cacacccaac ttcaagtcca acttcgatct 780ggcggaggac gccaagctcc agctgtccaa ggacacctac gacgatgacc tcgacaacct 840gcttgcacag attggcgatc agtacgccga cctgtttctc gctgccaaga acctttcgga 900tgctattctc ttgtctgaca ttctgcgagt caacaccgag atcacaaagg ctcccctttc 960tgcctccatg atcaagcgat acgacgagca ccatcaggat ctcacactgc tcaaggctct 1020tgtccgacag caactgcccg agaagtacaa ggagatcttt ttcgatcagt cgaagaacgg 1080ctacgctgga tacatcgacg gcggagcctc tcaggaagag ttctacaagt tcatcaagcc 1140aattctcgag aagatggacg gaaccgagga actgcttgtc aagctcaatc gagaggatct 1200gcttcggaag caacgaacct tcgacaacgg cagcattcct catcagatcc acctcggtga 1260gctgcacgcc attcttcgac gtcaggaaga cttctacccc tttctcaagg acaaccgaga 1320gaagatcgag aagattctta cctttcgaat cccctactat gttggtcctc ttgccagagg 1380aaactctcga tttgcttgga tgactcgaaa gtccgaggaa accatcactc cctggaactt 1440cgaggaagtc gtggacaagg gtgcctctgc acagtccttc atcgagcgaa tgaccaactt 1500cgacaagaat ctgcccaacg agaaggttct tcccaagcat tcgctgctct acgagtactt 1560tacagtctac aacgaactca ccaaagtcaa gtacgttacc gagggaatgc gaaagcctgc 1620cttcttgtct ggcgaacaga agaaagccat tgtcgatctc ctgttcaaga ccaaccgaaa 1680ggtcactgtt aagcagctca aggaggacta cttcaagaaa atcgagtgtt tcgacagcgt 1740cgagatttcc ggagttgagg accgattcaa cgcctctttg ggcacctatc acgatctgct 1800caagattatc aaggacaagg attttctcga caacgaggaa aacgaggaca ttctggagga 1860catcgtgctc actcttaccc tgttcgaaga tcgggagatg atcgaggaac gactcaagac 1920atacgctcac ctgttcgacg acaaggtcat gaaacaactc aagcgacgta gatacaccgg 1980ctggggaaga ctttcgcgaa agctcatcaa cggcatcaga gacaagcagt ccggaaagac 2040cattctggac tttctcaagt ccgatggctt tgccaaccga aacttcatgc agctcattca 2100cgacgattct cttaccttca aggaggacat ccagaaggca caagtgtccg gtcagggcga 2160cagcttgcac gaacatattg ccaacctggc tggttcgcca gccatcaaga aaggcattct 2220ccagactgtc aaggttgtcg acgagctggt gaaggtcatg ggacgtcaca agcccgagaa 2280cattgtgatc gagatggcca gagagaacca gacaactcaa aagggtcaga aaaactcgcg 2340agagcggatg aagcgaatcg aggaaggcat caaggagctg ggatcccaga ttctcaagga 2400gcatcccgtc gagaacactc aactgcagaa cgagaagctg tatctctact atctgcagaa 2460tggtcgagac atgtacgtgg atcaggaact ggacatcaat cgtctcagcg actacgatgt 2520ggaccacatt gtccctcaat cctttctcaa ggacgattct atcgacaaca aggtccttac 2580acgatccgac aagaacagag gcaagtcgga caacgttccc agcgaagagg tggtcaaaaa 2640gatgaagaac tactggcgac agctgctcaa cgccaagctc attacccagc gaaagttcga 2700caatcttacc aaggccgagc gaggcggtct gtccgagctc gacaaggctg gcttcatcaa 2760gcgtcaactc gtcgagacca gacagatcac aaagcacgtc gcacagattc tcgattctcg 2820gatgaacacc aagtacgacg agaacgacaa gctcatccga gaggtcaagg tgattactct 2880caagtccaaa ctggtctccg atttccgaaa ggactttcag ttctacaagg tgcgagagat 2940caacaattac caccatgccc acgatgctta cctcaacgcc gtcgttggca ctgcgctcat 3000caagaaatac cccaagctcg aaagcgagtt cgtttacggc gattacaagg tctacgacgt 3060tcgaaagatg attgccaagt ccgaacagga gattggcaag gctactgcca agtacttctt 3120ttactccaac atcatgaact ttttcaagac cgagatcacc ttggccaacg gagagattcg 3180aaagagacca cttatcgaga ccaacggcga aactggagag atcgtgtggg acaagggtcg 3240agactttgca accgtgcgaa aggttctgtc gatgcctcag gtcaacatcg tcaagaaaac 3300cgaggttcag actggcggat tctccaagga gtcgattctg cccaagcgaa actccgacaa 3360gctcatcgct cgaaagaaag actgggatcc caagaaatac ggtggcttcg attctcctac 3420cgtcgcctat tccgtgcttg tcgttgcgaa ggtcgagaag ggcaagtcca aaaagctcaa 3480gtccgtcaag gagctgctcg gaattaccat catggagcga tcgagcttcg agaagaatcc 3540catcgacttc ttggaagcca agggttacaa ggaggtcaag aaagacctca ttatcaagct 3600gcccaagtac tctctgttcg aactggagaa cggtcgaaag cgtatgctcg cctccgctgg 3660cgagctgcag aagggaaacg agcttgcctt gccttcgaag tacgtcaact ttctctatct 3720ggcttctcac tacgagaagc tcaagggttc tcccgaggac aacgaacaga agcaactctt 3780cgttgagcag cacaaacatt acctcgacga gattatcgag cagatttccg agttttcgaa 3840gcgagtcatc ctggctgatg ccaacttgga caaggtgctc tctgcctaca acaagcatcg 3900ggacaaaccc attcgagaac aggcggagaa catcattcac ctgtttactc ttaccaacct 3960gggtgctcct gcagctttca agtacttcga taccactatc gaccgaaagc ggtacacatc 4020caccaaggag gttctcgatg ccaccctgat tcaccagtcc atcactggcc tgtacgagac 4080ccgaatcgac ctgtctcagc ttggtggcga ctccagagcc gatcccaaga aaaagcgaaa 4140ggtctaagcg gccgctaagc ttgggg 4166104092DNAartificial sequencepBAD/HisB plasmid 10aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 120aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc 300taacaggagg aattaaccat ggggggttct catcatcatc atcatcatgg tatggctagc 360atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatccgagc 420tcgagatctg cagctggtac catatgggaa ttcgaagctt ggctgttttg gcggatgaga 480gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa 540tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa 600acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc 660atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt 720cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc 780aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc 840agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttttg tttatttttc 900taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 960tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 1020gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 1080gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 1140cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 1200tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac 1260tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 1320atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 1380ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 1440gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 1500gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 1560gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 1620gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 1680gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 1740cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 1800atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 1860tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 1920ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 1980gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 2040tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 2100ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 2160ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 2220gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 2280ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 2340tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 2400ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 2460agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 2520agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 2580gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 2640tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 2700accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 2760gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt 2820atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 2880cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa 2940cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 3000tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 3060ggcagcagat caattcgcgc gcgaaggcga agcggcatgc ataatgtgcc tgtcaaatgg 3120acgaagcagg gattctgcaa accctatgct actccgtcaa gccgtcaatt gtctgattcg 3180ttaccaatta tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga 3240actcgctcgg gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt 3300caaaaccaac attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct 3360tcgcctggct gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc 3420ggaaaagatg tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat 3480caaaattgct gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat 3540ccatcggtgg atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa 3600gcagatttat cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt 3660gcccaaacag gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat 3720tggcaaatat tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa 3780cccactggtg ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg 3840gcgggaacag caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca 3900ccccctgacc gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga 3960taaaaaaatc gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat 4020taaacgagta tcccggcagc aggggatcat tttgcgcttc agccatactt ttcatactcc 4080cgccattcag ag 4092118237DNAartificial sequencepRF48 plasmid 11aattcgacaa gaaatactcc atcggcctgg acattggaac caactctgtc ggctgggctg 60tcatcaccga cgagtacaag gtgccctcca agaaattcaa ggtcctcgga aacaccgatc 120gacactccat caagaaaaac ctcattggtg ccctgttgtt cgattctggc gagactgccg 180aagctaccag actcaagcga actgctcggc gacgttacac ccgacggaag aaccgaatct 240gctacctgca ggagatcttt tccaacgaga tggccaaggt ggacgattcg ttctttcatc 300gactggagga atccttcctc gtcgaggaag acaagaaaca cgagcgtcat cccatctttg 360gcaacattgt ggacgaggtt gcttaccacg agaagtatcc taccatctac cacctgcgaa 420agaaactcgt cgattccacc gacaaggcgg atctcagact tatctacctc gctctggcac 480acatgatcaa gtttcgaggt catttcctca tcgagggcga tctcaatccc gacaacagcg 540atgtggacaa gctgttcatt cagctcgttc agacctacaa ccagctgttc gaggaaaacc 600ccatcaatgc ctccggagtc gatgcaaagg ccatcttgtc tgctcgactc tcgaagagca 660gacgactgga gaacctcatt gcccaacttc ctggcgagaa aaagaacgga ctgtttggca 720acctcattgc cctttctctt ggtctcacac ccaacttcaa gtccaacttc gatctggcgg 780aggacgccaa gctccagctg tccaaggaca cctacgacga tgacctcgac aacctgcttg 840cacagattgg cgatcagtac gccgacctgt ttctcgctgc caagaacctt tcggatgcta 900ttctcttgtc tgacattctg cgagtcaaca ccgagatcac aaaggctccc ctttctgcct 960ccatgatcaa gcgatacgac gagcaccatc aggatctcac actgctcaag gctcttgtcc 1020gacagcaact gcccgagaag tacaaggaga tctttttcga tcagtcgaag aacggctacg 1080ctggatacat cgacggcgga gcctctcagg aagagttcta caagttcatc aagccaattc 1140tcgagaagat ggacggaacc gaggaactgc ttgtcaagct caatcgagag gatctgcttc 1200ggaagcaacg aaccttcgac aacggcagca ttcctcatca gatccacctc ggtgagctgc 1260acgccattct tcgacgtcag gaagacttct acccctttct caaggacaac cgagagaaga 1320tcgagaagat tcttaccttt cgaatcccct actatgttgg tcctcttgcc agaggaaact 1380ctcgatttgc ttggatgact cgaaagtccg aggaaaccat cactccctgg aacttcgagg 1440aagtcgtgga caagggtgcc tctgcacagt ccttcatcga gcgaatgacc aacttcgaca 1500agaatctgcc caacgagaag gttcttccca agcattcgct gctctacgag tactttacag 1560tctacaacga actcaccaaa gtcaagtacg ttaccgaggg aatgcgaaag cctgccttct 1620tgtctggcga acagaagaaa gccattgtcg atctcctgtt caagaccaac cgaaaggtca 1680ctgttaagca gctcaaggag gactacttca agaaaatcga gtgtttcgac agcgtcgaga 1740tttccggagt tgaggaccga ttcaacgcct ctttgggcac ctatcacgat ctgctcaaga 1800ttatcaagga caaggatttt ctcgacaacg aggaaaacga ggacattctg gaggacatcg 1860tgctcactct taccctgttc gaagatcggg agatgatcga ggaacgactc aagacatacg 1920ctcacctgtt cgacgacaag gtcatgaaac aactcaagcg acgtagatac accggctggg 1980gaagactttc gcgaaagctc atcaacggca tcagagacaa gcagtccgga aagaccattc 2040tggactttct caagtccgat ggctttgcca accgaaactt catgcagctc attcacgacg 2100attctcttac cttcaaggag gacatccaga aggcacaagt gtccggtcag ggcgacagct 2160tgcacgaaca tattgccaac ctggctggtt cgccagccat caagaaaggc attctccaga 2220ctgtcaaggt tgtcgacgag ctggtgaagg tcatgggacg tcacaagccc gagaacattg 2280tgatcgagat ggccagagag aaccagacaa ctcaaaaggg tcagaaaaac tcgcgagagc 2340ggatgaagcg aatcgaggaa ggcatcaagg agctgggatc ccagattctc aaggagcatc 2400ccgtcgagaa cactcaactg cagaacgaga agctgtatct ctactatctg cagaatggtc 2460gagacatgta cgtggatcag gaactggaca tcaatcgtct cagcgactac gatgtggacc 2520acattgtccc tcaatccttt ctcaaggacg attctatcga caacaaggtc cttacacgat 2580ccgacaagaa cagaggcaag tcggacaacg ttcccagcga agaggtggtc aaaaagatga 2640agaactactg gcgacagctg ctcaacgcca agctcattac ccagcgaaag ttcgacaatc 2700ttaccaaggc cgagcgaggc ggtctgtccg agctcgacaa ggctggcttc atcaagcgtc 2760aactcgtcga gaccagacag atcacaaagc acgtcgcaca gattctcgat tctcggatga 2820acaccaagta cgacgagaac gacaagctca tccgagaggt caaggtgatt actctcaagt 2880ccaaactggt ctccgatttc cgaaaggact ttcagttcta caaggtgcga gagatcaaca 2940attaccacca tgcccacgat gcttacctca acgccgtcgt tggcactgcg ctcatcaaga 3000aataccccaa gctcgaaagc gagttcgttt acggcgatta caaggtctac gacgttcgaa 3060agatgattgc caagtccgaa caggagattg gcaaggctac tgccaagtac ttcttttact 3120ccaacatcat gaactttttc aagaccgaga tcaccttggc caacggagag attcgaaaga 3180gaccacttat cgagaccaac ggcgaaactg gagagatcgt gtgggacaag ggtcgagact 3240ttgcaaccgt gcgaaaggtt ctgtcgatgc ctcaggtcaa catcgtcaag aaaaccgagg 3300ttcagactgg cggattctcc aaggagtcga ttctgcccaa gcgaaactcc gacaagctca 3360tcgctcgaaa gaaagactgg

gatcccaaga aatacggtgg cttcgattct cctaccgtcg 3420cctattccgt gcttgtcgtt gcgaaggtcg agaagggcaa gtccaaaaag ctcaagtccg 3480tcaaggagct gctcggaatt accatcatgg agcgatcgag cttcgagaag aatcccatcg 3540acttcttgga agccaagggt tacaaggagg tcaagaaaga cctcattatc aagctgccca 3600agtactctct gttcgaactg gagaacggtc gaaagcgtat gctcgcctcc gctggcgagc 3660tgcagaaggg aaacgagctt gccttgcctt cgaagtacgt caactttctc tatctggctt 3720ctcactacga gaagctcaag ggttctcccg aggacaacga acagaagcaa ctcttcgttg 3780agcagcacaa acattacctc gacgagatta tcgagcagat ttccgagttt tcgaagcgag 3840tcatcctggc tgatgccaac ttggacaagg tgctctctgc ctacaacaag catcgggaca 3900aacccattcg agaacaggcg gagaacatca ttcacctgtt tactcttacc aacctgggtg 3960ctcctgcagc tttcaagtac ttcgatacca ctatcgaccg aaagcggtac acatccacca 4020aggaggttct cgatgccacc ctgattcacc agtccatcac tggcctgtac gagacccgaa 4080tcgacctgtc tcagcttggt ggcgactcca gagccgatcc caagaaaaag cgaaaggtct 4140aagcggccgc taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga 4200ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 4260tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 4320tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 4380tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg 4440acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca 4500ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc 4560ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc 4620gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 4680tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 4740tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4800gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4860acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 4920tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4980gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 5040tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 5100accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 5160ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 5220agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 5280gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 5340ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 5400tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 5460ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 5520gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 5580acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 5640aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 5700atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 5760gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5820tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5880ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5940ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 6000ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 6060aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 6120cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 6180gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 6240ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 6300cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 6360tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 6420cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 6480cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 6540tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 6600cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6660gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6720tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg 6780aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc 6840ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg 6900gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg 6960cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg 7020gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc 7080tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac 7140ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga 7200tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg 7260ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc 7320gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc 7380ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta 7440agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga 7500gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc 7560ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga 7620ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg 7680gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg 7740ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat 7800tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc 7860aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca 7920aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac 7980ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga 8040cgctttttat cgcaactctc tactgtttct ccatacccgt tttttgggct aacaggagga 8100attaaccatg gggggttctc atcatcatca tcatcatggt atggctagca tgactggtgg 8160acagcaaatg ggtcgggatc tgtacgacga tgacgataag gatccgagct cgagatctgc 8220agctggtacc atatggg 82371254PRTEpstein-Barr virus 12Glu Cys Asp Ser Glu Leu Glu Ile Lys Arg Tyr Lys Arg Val Arg Val1 5 10 15Ala Ser Arg Lys Cys Arg Ala Lys Phe Lys Gln Leu Leu Gln His Tyr 20 25 30Arg Glu Val Ala Ala Ala Lys Ser Ser Glu Asn Asp Arg Leu Arg Leu 35 40 45Leu Leu Lys Gln Met Cys 501318PRTmus musculus 13Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His1 5 10 15Ser Lys1421PRTunknownTP10 CPP 14Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu Lys Ala Cys Ala Ala Cys1 5 10 15Ala Lys Lys Ile Leu 201517PRTartificial sequencePolyR CPP 15Gly Gly Gly Gly Arg Arg Arg Arg Arg Arg Arg Arg Arg Leu Leu Leu1 5 10 15Leu16194DNAartificial sequenceNcoI-6xHis-ZEBRA CPP-EcoRI 16ccatggggca tcaccaccat caccacgaat gcgactcaga actggaaatc aaacgctata 60aacgtgtgcg tgtggcatcc cgtaaatgtc gcgcaaagtt taaacagctg ctgcaacatt 120atcgtgaagt agcggctgcg aaaagctccg aaaacgaccg tttacgcctc ctcctgaagc 180aaatgtgcga attc 1941786DNAartificial sequenceNcoI-6xHis-pVEC CPP-EcoRI 17ccatggggca tcaccaccat caccacttat tgattatctt gcgtcgtcgc atccgcaaac 60aggcgcacgc acatagcaag gaattc 861895DNAartificial sequenceNcoI-6xHis-TP10 CPP-EcoRI 18ccatggggca tcaccaccat caccacgcgg gttacctgct gggcaagatt aatcttaaag 60cctgcgccgc gtgtgctaag aaaattttgg aattc 951983DNAartificial sequenceNcoI-6xHis-PolyR CPP-EcoRI 19ccatggggca tcaccaccat caccacggcg ggggtggtcg tcgtcgccgt cgccgccgtc 60gtcgcctcct gctgctggaa ttc 83208294DNAartificial sequencepRF144 plasmid 20ccatggggca tcaccaccat caccacgaat gcgactcaga actggaaatc aaacgctata 60aacgtgtgcg tgtggcatcc cgtaaatgtc gcgcaaagtt taaacagctg ctgcaacatt 120atcgtgaagt agcggctgcg aaaagctccg aaaacgaccg tttacgcctc ctcctgaagc 180aaatgtgcga attcgacaag aaatactcca tcggcctgga cattggaacc aactctgtcg 240gctgggctgt catcaccgac gagtacaagg tgccctccaa gaaattcaag gtcctcggaa 300acaccgatcg acactccatc aagaaaaacc tcattggtgc cctgttgttc gattctggcg 360agactgccga agctaccaga ctcaagcgaa ctgctcggcg acgttacacc cgacggaaga 420accgaatctg ctacctgcag gagatctttt ccaacgagat ggccaaggtg gacgattcgt 480tctttcatcg actggaggaa tccttcctcg tcgaggaaga caagaaacac gagcgtcatc 540ccatctttgg caacattgtg gacgaggttg cttaccacga gaagtatcct accatctacc 600acctgcgaaa gaaactcgtc gattccaccg acaaggcgga tctcagactt atctacctcg 660ctctggcaca catgatcaag tttcgaggtc atttcctcat cgagggcgat ctcaatcccg 720acaacagcga tgtggacaag ctgttcattc agctcgttca gacctacaac cagctgttcg 780aggaaaaccc catcaatgcc tccggagtcg atgcaaaggc catcttgtct gctcgactct 840cgaagagcag acgactggag aacctcattg cccaacttcc tggcgagaaa aagaacggac 900tgtttggcaa cctcattgcc ctttctcttg gtctcacacc caacttcaag tccaacttcg 960atctggcgga ggacgccaag ctccagctgt ccaaggacac ctacgacgat gacctcgaca 1020acctgcttgc acagattggc gatcagtacg ccgacctgtt tctcgctgcc aagaaccttt 1080cggatgctat tctcttgtct gacattctgc gagtcaacac cgagatcaca aaggctcccc 1140tttctgcctc catgatcaag cgatacgacg agcaccatca ggatctcaca ctgctcaagg 1200ctcttgtccg acagcaactg cccgagaagt acaaggagat ctttttcgat cagtcgaaga 1260acggctacgc tggatacatc gacggcggag cctctcagga agagttctac aagttcatca 1320agccaattct cgagaagatg gacggaaccg aggaactgct tgtcaagctc aatcgagagg 1380atctgcttcg gaagcaacga accttcgaca acggcagcat tcctcatcag atccacctcg 1440gtgagctgca cgccattctt cgacgtcagg aagacttcta cccctttctc aaggacaacc 1500gagagaagat cgagaagatt cttacctttc gaatccccta ctatgttggt cctcttgcca 1560gaggaaactc tcgatttgct tggatgactc gaaagtccga ggaaaccatc actccctgga 1620acttcgagga agtcgtggac aagggtgcct ctgcacagtc cttcatcgag cgaatgacca 1680acttcgacaa gaatctgccc aacgagaagg ttcttcccaa gcattcgctg ctctacgagt 1740actttacagt ctacaacgaa ctcaccaaag tcaagtacgt taccgaggga atgcgaaagc 1800ctgccttctt gtctggcgaa cagaagaaag ccattgtcga tctcctgttc aagaccaacc 1860gaaaggtcac tgttaagcag ctcaaggagg actacttcaa gaaaatcgag tgtttcgaca 1920gcgtcgagat ttccggagtt gaggaccgat tcaacgcctc tttgggcacc tatcacgatc 1980tgctcaagat tatcaaggac aaggattttc tcgacaacga ggaaaacgag gacattctgg 2040aggacatcgt gctcactctt accctgttcg aagatcggga gatgatcgag gaacgactca 2100agacatacgc tcacctgttc gacgacaagg tcatgaaaca actcaagcga cgtagataca 2160ccggctgggg aagactttcg cgaaagctca tcaacggcat cagagacaag cagtccggaa 2220agaccattct ggactttctc aagtccgatg gctttgccaa ccgaaacttc atgcagctca 2280ttcacgacga ttctcttacc ttcaaggagg acatccagaa ggcacaagtg tccggtcagg 2340gcgacagctt gcacgaacat attgccaacc tggctggttc gccagccatc aagaaaggca 2400ttctccagac tgtcaaggtt gtcgacgagc tggtgaaggt catgggacgt cacaagcccg 2460agaacattgt gatcgagatg gccagagaga accagacaac tcaaaagggt cagaaaaact 2520cgcgagagcg gatgaagcga atcgaggaag gcatcaagga gctgggatcc cagattctca 2580aggagcatcc cgtcgagaac actcaactgc agaacgagaa gctgtatctc tactatctgc 2640agaatggtcg agacatgtac gtggatcagg aactggacat caatcgtctc agcgactacg 2700atgtggacca cattgtccct caatcctttc tcaaggacga ttctatcgac aacaaggtcc 2760ttacacgatc cgacaagaac agaggcaagt cggacaacgt tcccagcgaa gaggtggtca 2820aaaagatgaa gaactactgg cgacagctgc tcaacgccaa gctcattacc cagcgaaagt 2880tcgacaatct taccaaggcc gagcgaggcg gtctgtccga gctcgacaag gctggcttca 2940tcaagcgtca actcgtcgag accagacaga tcacaaagca cgtcgcacag attctcgatt 3000ctcggatgaa caccaagtac gacgagaacg acaagctcat ccgagaggtc aaggtgatta 3060ctctcaagtc caaactggtc tccgatttcc gaaaggactt tcagttctac aaggtgcgag 3120agatcaacaa ttaccaccat gcccacgatg cttacctcaa cgccgtcgtt ggcactgcgc 3180tcatcaagaa ataccccaag ctcgaaagcg agttcgttta cggcgattac aaggtctacg 3240acgttcgaaa gatgattgcc aagtccgaac aggagattgg caaggctact gccaagtact 3300tcttttactc caacatcatg aactttttca agaccgagat caccttggcc aacggagaga 3360ttcgaaagag accacttatc gagaccaacg gcgaaactgg agagatcgtg tgggacaagg 3420gtcgagactt tgcaaccgtg cgaaaggttc tgtcgatgcc tcaggtcaac atcgtcaaga 3480aaaccgaggt tcagactggc ggattctcca aggagtcgat tctgcccaag cgaaactccg 3540acaagctcat cgctcgaaag aaagactggg atcccaagaa atacggtggc ttcgattctc 3600ctaccgtcgc ctattccgtg cttgtcgttg cgaaggtcga gaagggcaag tccaaaaagc 3660tcaagtccgt caaggagctg ctcggaatta ccatcatgga gcgatcgagc ttcgagaaga 3720atcccatcga cttcttggaa gccaagggtt acaaggaggt caagaaagac ctcattatca 3780agctgcccaa gtactctctg ttcgaactgg agaacggtcg aaagcgtatg ctcgcctccg 3840ctggcgagct gcagaaggga aacgagcttg ccttgccttc gaagtacgtc aactttctct 3900atctggcttc tcactacgag aagctcaagg gttctcccga ggacaacgaa cagaagcaac 3960tcttcgttga gcagcacaaa cattacctcg acgagattat cgagcagatt tccgagtttt 4020cgaagcgagt catcctggct gatgccaact tggacaaggt gctctctgcc tacaacaagc 4080atcgggacaa acccattcga gaacaggcgg agaacatcat tcacctgttt actcttacca 4140acctgggtgc tcctgcagct ttcaagtact tcgataccac tatcgaccga aagcggtaca 4200catccaccaa ggaggttctc gatgccaccc tgattcacca gtccatcact ggcctgtacg 4260agacccgaat cgacctgtct cagcttggtg gcgactccag agccgatccc aagaaaaagc 4320gaaaggtcta agcggccgct aagcttggct gttttggcgg atgagagaag attttcagcc 4380tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca 4440gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg 4500atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga 4560aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 4620ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg 4680tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg 4740acggatggcc tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa 4800tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 4860gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 4920tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 4980tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 5040ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 5100atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 5160cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 5220attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 5280gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 5340ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 5400gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 5460agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 5520gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 5580gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 5640ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 5700tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 5760tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 5820catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 5880gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 5940aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 6000gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 6060gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 6120gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 6180atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 6240cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 6300cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 6360agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 6420tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 6480gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 6540catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 6600agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 6660ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 6720atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg 6780ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg 6840ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 6900agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat 6960tcgcgcgcga aggcgaagcg gcatgcataa tgtgcctgtc aaatggacga agcagggatt 7020ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct gattcgttac caattatgac 7080aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg 7140gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg 7200cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata 7260cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac 7320agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct 7380gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg 7440agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc 7500agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg 7560ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac 7620ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac 7680cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa 7740atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga 7800atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga 7860taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc 7920ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa 7980gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc 8040tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa 8100gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat 8160tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag attagcggat 8220cctacctgac gctttttatc gcaactctct actgtttctc catacccgtt ttttgggcta 8280acaggaggaa ttaa 8294218183DNAartificial sequencepRF145 plasmid 21aattcgacaa gaaatactcc atcggcctgg acattggaac caactctgtc ggctgggctg 60tcatcaccga cgagtacaag gtgccctcca agaaattcaa ggtcctcgga aacaccgatc 120gacactccat caagaaaaac ctcattggtg ccctgttgtt

cgattctggc gagactgccg 180aagctaccag actcaagcga actgctcggc gacgttacac ccgacggaag aaccgaatct 240gctacctgca ggagatcttt tccaacgaga tggccaaggt ggacgattcg ttctttcatc 300gactggagga atccttcctc gtcgaggaag acaagaaaca cgagcgtcat cccatctttg 360gcaacattgt ggacgaggtt gcttaccacg agaagtatcc taccatctac cacctgcgaa 420agaaactcgt cgattccacc gacaaggcgg atctcagact tatctacctc gctctggcac 480acatgatcaa gtttcgaggt catttcctca tcgagggcga tctcaatccc gacaacagcg 540atgtggacaa gctgttcatt cagctcgttc agacctacaa ccagctgttc gaggaaaacc 600ccatcaatgc ctccggagtc gatgcaaagg ccatcttgtc tgctcgactc tcgaagagca 660gacgactgga gaacctcatt gcccaacttc ctggcgagaa aaagaacgga ctgtttggca 720acctcattgc cctttctctt ggtctcacac ccaacttcaa gtccaacttc gatctggcgg 780aggacgccaa gctccagctg tccaaggaca cctacgacga tgacctcgac aacctgcttg 840cacagattgg cgatcagtac gccgacctgt ttctcgctgc caagaacctt tcggatgcta 900ttctcttgtc tgacattctg cgagtcaaca ccgagatcac aaaggctccc ctttctgcct 960ccatgatcaa gcgatacgac gagcaccatc aggatctcac actgctcaag gctcttgtcc 1020gacagcaact gcccgagaag tacaaggaga tctttttcga tcagtcgaag aacggctacg 1080ctggatacat cgacggcgga gcctctcagg aagagttcta caagttcatc aagccaattc 1140tcgagaagat ggacggaacc gaggaactgc ttgtcaagct caatcgagag gatctgcttc 1200ggaagcaacg aaccttcgac aacggcagca ttcctcatca gatccacctc ggtgagctgc 1260acgccattct tcgacgtcag gaagacttct acccctttct caaggacaac cgagagaaga 1320tcgagaagat tcttaccttt cgaatcccct actatgttgg tcctcttgcc agaggaaact 1380ctcgatttgc ttggatgact cgaaagtccg aggaaaccat cactccctgg aacttcgagg 1440aagtcgtgga caagggtgcc tctgcacagt ccttcatcga gcgaatgacc aacttcgaca 1500agaatctgcc caacgagaag gttcttccca agcattcgct gctctacgag tactttacag 1560tctacaacga actcaccaaa gtcaagtacg ttaccgaggg aatgcgaaag cctgccttct 1620tgtctggcga acagaagaaa gccattgtcg atctcctgtt caagaccaac cgaaaggtca 1680ctgttaagca gctcaaggag gactacttca agaaaatcga gtgtttcgac agcgtcgaga 1740tttccggagt tgaggaccga ttcaacgcct ctttgggcac ctatcacgat ctgctcaaga 1800ttatcaagga caaggatttt ctcgacaacg aggaaaacga ggacattctg gaggacatcg 1860tgctcactct taccctgttc gaagatcggg agatgatcga ggaacgactc aagacatacg 1920ctcacctgtt cgacgacaag gtcatgaaac aactcaagcg acgtagatac accggctggg 1980gaagactttc gcgaaagctc atcaacggca tcagagacaa gcagtccgga aagaccattc 2040tggactttct caagtccgat ggctttgcca accgaaactt catgcagctc attcacgacg 2100attctcttac cttcaaggag gacatccaga aggcacaagt gtccggtcag ggcgacagct 2160tgcacgaaca tattgccaac ctggctggtt cgccagccat caagaaaggc attctccaga 2220ctgtcaaggt tgtcgacgag ctggtgaagg tcatgggacg tcacaagccc gagaacattg 2280tgatcgagat ggccagagag aaccagacaa ctcaaaaggg tcagaaaaac tcgcgagagc 2340ggatgaagcg aatcgaggaa ggcatcaagg agctgggatc ccagattctc aaggagcatc 2400ccgtcgagaa cactcaactg cagaacgaga agctgtatct ctactatctg cagaatggtc 2460gagacatgta cgtggatcag gaactggaca tcaatcgtct cagcgactac gatgtggacc 2520acattgtccc tcaatccttt ctcaaggacg attctatcga caacaaggtc cttacacgat 2580ccgacaagaa cagaggcaag tcggacaacg ttcccagcga agaggtggtc aaaaagatga 2640agaactactg gcgacagctg ctcaacgcca agctcattac ccagcgaaag ttcgacaatc 2700ttaccaaggc cgagcgaggc ggtctgtccg agctcgacaa ggctggcttc atcaagcgtc 2760aactcgtcga gaccagacag atcacaaagc acgtcgcaca gattctcgat tctcggatga 2820acaccaagta cgacgagaac gacaagctca tccgagaggt caaggtgatt actctcaagt 2880ccaaactggt ctccgatttc cgaaaggact ttcagttcta caaggtgcga gagatcaaca 2940attaccacca tgcccacgat gcttacctca acgccgtcgt tggcactgcg ctcatcaaga 3000aataccccaa gctcgaaagc gagttcgttt acggcgatta caaggtctac gacgttcgaa 3060agatgattgc caagtccgaa caggagattg gcaaggctac tgccaagtac ttcttttact 3120ccaacatcat gaactttttc aagaccgaga tcaccttggc caacggagag attcgaaaga 3180gaccacttat cgagaccaac ggcgaaactg gagagatcgt gtgggacaag ggtcgagact 3240ttgcaaccgt gcgaaaggtt ctgtcgatgc ctcaggtcaa catcgtcaag aaaaccgagg 3300ttcagactgg cggattctcc aaggagtcga ttctgcccaa gcgaaactcc gacaagctca 3360tcgctcgaaa gaaagactgg gatcccaaga aatacggtgg cttcgattct cctaccgtcg 3420cctattccgt gcttgtcgtt gcgaaggtcg agaagggcaa gtccaaaaag ctcaagtccg 3480tcaaggagct gctcggaatt accatcatgg agcgatcgag cttcgagaag aatcccatcg 3540acttcttgga agccaagggt tacaaggagg tcaagaaaga cctcattatc aagctgccca 3600agtactctct gttcgaactg gagaacggtc gaaagcgtat gctcgcctcc gctggcgagc 3660tgcagaaggg aaacgagctt gccttgcctt cgaagtacgt caactttctc tatctggctt 3720ctcactacga gaagctcaag ggttctcccg aggacaacga acagaagcaa ctcttcgttg 3780agcagcacaa acattacctc gacgagatta tcgagcagat ttccgagttt tcgaagcgag 3840tcatcctggc tgatgccaac ttggacaagg tgctctctgc ctacaacaag catcgggaca 3900aacccattcg agaacaggcg gagaacatca ttcacctgtt tactcttacc aacctgggtg 3960ctcctgcagc tttcaagtac ttcgatacca ctatcgaccg aaagcggtac acatccacca 4020aggaggttct cgatgccacc ctgattcacc agtccatcac tggcctgtac gagacccgaa 4080tcgacctgtc tcagcttggt ggcgactcca gagccgatcc caagaaaaag cgaaaggtct 4140aagcggccgc taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga 4200ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 4260tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 4320tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 4380tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg 4440acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca 4500ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc 4560ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc 4620gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 4680tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 4740tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4800gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4860acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 4920tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4980gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 5040tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 5100accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 5160ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 5220agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 5280gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 5340ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 5400tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 5460ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 5520gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 5580acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 5640aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 5700atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 5760gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5820tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5880ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5940ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 6000ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 6060aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 6120cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 6180gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 6240ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 6300cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 6360tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 6420cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 6480cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 6540tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 6600cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6660gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6720tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg 6780aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc 6840ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg 6900gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg 6960cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg 7020gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc 7080tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac 7140ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga 7200tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg 7260ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc 7320gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc 7380ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta 7440agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga 7500gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc 7560ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga 7620ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg 7680gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg 7740ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat 7800tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc 7860aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca 7920aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac 7980ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga 8040cgctttttat cgcaactctc tactgtttct ccatacccgt tttttgggct aacaggagga 8100attaaccatg gggcatcacc accatcacca cggcgggggt ggtcgtcgtc gccgtcgccg 8160ccgtcgtcgc ctcctgctgc tgg 8183228195DNAartificial sequencepRF146 plasmid 22aattcgacaa gaaatactcc atcggcctgg acattggaac caactctgtc ggctgggctg 60tcatcaccga cgagtacaag gtgccctcca agaaattcaa ggtcctcgga aacaccgatc 120gacactccat caagaaaaac ctcattggtg ccctgttgtt cgattctggc gagactgccg 180aagctaccag actcaagcga actgctcggc gacgttacac ccgacggaag aaccgaatct 240gctacctgca ggagatcttt tccaacgaga tggccaaggt ggacgattcg ttctttcatc 300gactggagga atccttcctc gtcgaggaag acaagaaaca cgagcgtcat cccatctttg 360gcaacattgt ggacgaggtt gcttaccacg agaagtatcc taccatctac cacctgcgaa 420agaaactcgt cgattccacc gacaaggcgg atctcagact tatctacctc gctctggcac 480acatgatcaa gtttcgaggt catttcctca tcgagggcga tctcaatccc gacaacagcg 540atgtggacaa gctgttcatt cagctcgttc agacctacaa ccagctgttc gaggaaaacc 600ccatcaatgc ctccggagtc gatgcaaagg ccatcttgtc tgctcgactc tcgaagagca 660gacgactgga gaacctcatt gcccaacttc ctggcgagaa aaagaacgga ctgtttggca 720acctcattgc cctttctctt ggtctcacac ccaacttcaa gtccaacttc gatctggcgg 780aggacgccaa gctccagctg tccaaggaca cctacgacga tgacctcgac aacctgcttg 840cacagattgg cgatcagtac gccgacctgt ttctcgctgc caagaacctt tcggatgcta 900ttctcttgtc tgacattctg cgagtcaaca ccgagatcac aaaggctccc ctttctgcct 960ccatgatcaa gcgatacgac gagcaccatc aggatctcac actgctcaag gctcttgtcc 1020gacagcaact gcccgagaag tacaaggaga tctttttcga tcagtcgaag aacggctacg 1080ctggatacat cgacggcgga gcctctcagg aagagttcta caagttcatc aagccaattc 1140tcgagaagat ggacggaacc gaggaactgc ttgtcaagct caatcgagag gatctgcttc 1200ggaagcaacg aaccttcgac aacggcagca ttcctcatca gatccacctc ggtgagctgc 1260acgccattct tcgacgtcag gaagacttct acccctttct caaggacaac cgagagaaga 1320tcgagaagat tcttaccttt cgaatcccct actatgttgg tcctcttgcc agaggaaact 1380ctcgatttgc ttggatgact cgaaagtccg aggaaaccat cactccctgg aacttcgagg 1440aagtcgtgga caagggtgcc tctgcacagt ccttcatcga gcgaatgacc aacttcgaca 1500agaatctgcc caacgagaag gttcttccca agcattcgct gctctacgag tactttacag 1560tctacaacga actcaccaaa gtcaagtacg ttaccgaggg aatgcgaaag cctgccttct 1620tgtctggcga acagaagaaa gccattgtcg atctcctgtt caagaccaac cgaaaggtca 1680ctgttaagca gctcaaggag gactacttca agaaaatcga gtgtttcgac agcgtcgaga 1740tttccggagt tgaggaccga ttcaacgcct ctttgggcac ctatcacgat ctgctcaaga 1800ttatcaagga caaggatttt ctcgacaacg aggaaaacga ggacattctg gaggacatcg 1860tgctcactct taccctgttc gaagatcggg agatgatcga ggaacgactc aagacatacg 1920ctcacctgtt cgacgacaag gtcatgaaac aactcaagcg acgtagatac accggctggg 1980gaagactttc gcgaaagctc atcaacggca tcagagacaa gcagtccgga aagaccattc 2040tggactttct caagtccgat ggctttgcca accgaaactt catgcagctc attcacgacg 2100attctcttac cttcaaggag gacatccaga aggcacaagt gtccggtcag ggcgacagct 2160tgcacgaaca tattgccaac ctggctggtt cgccagccat caagaaaggc attctccaga 2220ctgtcaaggt tgtcgacgag ctggtgaagg tcatgggacg tcacaagccc gagaacattg 2280tgatcgagat ggccagagag aaccagacaa ctcaaaaggg tcagaaaaac tcgcgagagc 2340ggatgaagcg aatcgaggaa ggcatcaagg agctgggatc ccagattctc aaggagcatc 2400ccgtcgagaa cactcaactg cagaacgaga agctgtatct ctactatctg cagaatggtc 2460gagacatgta cgtggatcag gaactggaca tcaatcgtct cagcgactac gatgtggacc 2520acattgtccc tcaatccttt ctcaaggacg attctatcga caacaaggtc cttacacgat 2580ccgacaagaa cagaggcaag tcggacaacg ttcccagcga agaggtggtc aaaaagatga 2640agaactactg gcgacagctg ctcaacgcca agctcattac ccagcgaaag ttcgacaatc 2700ttaccaaggc cgagcgaggc ggtctgtccg agctcgacaa ggctggcttc atcaagcgtc 2760aactcgtcga gaccagacag atcacaaagc acgtcgcaca gattctcgat tctcggatga 2820acaccaagta cgacgagaac gacaagctca tccgagaggt caaggtgatt actctcaagt 2880ccaaactggt ctccgatttc cgaaaggact ttcagttcta caaggtgcga gagatcaaca 2940attaccacca tgcccacgat gcttacctca acgccgtcgt tggcactgcg ctcatcaaga 3000aataccccaa gctcgaaagc gagttcgttt acggcgatta caaggtctac gacgttcgaa 3060agatgattgc caagtccgaa caggagattg gcaaggctac tgccaagtac ttcttttact 3120ccaacatcat gaactttttc aagaccgaga tcaccttggc caacggagag attcgaaaga 3180gaccacttat cgagaccaac ggcgaaactg gagagatcgt gtgggacaag ggtcgagact 3240ttgcaaccgt gcgaaaggtt ctgtcgatgc ctcaggtcaa catcgtcaag aaaaccgagg 3300ttcagactgg cggattctcc aaggagtcga ttctgcccaa gcgaaactcc gacaagctca 3360tcgctcgaaa gaaagactgg gatcccaaga aatacggtgg cttcgattct cctaccgtcg 3420cctattccgt gcttgtcgtt gcgaaggtcg agaagggcaa gtccaaaaag ctcaagtccg 3480tcaaggagct gctcggaatt accatcatgg agcgatcgag cttcgagaag aatcccatcg 3540acttcttgga agccaagggt tacaaggagg tcaagaaaga cctcattatc aagctgccca 3600agtactctct gttcgaactg gagaacggtc gaaagcgtat gctcgcctcc gctggcgagc 3660tgcagaaggg aaacgagctt gccttgcctt cgaagtacgt caactttctc tatctggctt 3720ctcactacga gaagctcaag ggttctcccg aggacaacga acagaagcaa ctcttcgttg 3780agcagcacaa acattacctc gacgagatta tcgagcagat ttccgagttt tcgaagcgag 3840tcatcctggc tgatgccaac ttggacaagg tgctctctgc ctacaacaag catcgggaca 3900aacccattcg agaacaggcg gagaacatca ttcacctgtt tactcttacc aacctgggtg 3960ctcctgcagc tttcaagtac ttcgatacca ctatcgaccg aaagcggtac acatccacca 4020aggaggttct cgatgccacc ctgattcacc agtccatcac tggcctgtac gagacccgaa 4080tcgacctgtc tcagcttggt ggcgactcca gagccgatcc caagaaaaag cgaaaggtct 4140aagcggccgc taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga 4200ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 4260tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 4320tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 4380tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg 4440acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca 4500ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc 4560ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc 4620gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 4680tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 4740tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4800gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4860acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 4920tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4980gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 5040tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 5100accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 5160ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 5220agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 5280gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 5340ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 5400tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 5460ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 5520gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 5580acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 5640aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 5700atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 5760gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5820tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5880ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5940ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 6000ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 6060aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 6120cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 6180gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 6240ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 6300cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 6360tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 6420cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 6480cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 6540tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 6600cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6660gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6720tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg 6780aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc 6840ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg 6900gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg

6960cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg 7020gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc 7080tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac 7140ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga 7200tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg 7260ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc 7320gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc 7380ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta 7440agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga 7500gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc 7560ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga 7620ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg 7680gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg 7740ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat 7800tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc 7860aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca 7920aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac 7980ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga 8040cgctttttat cgcaactctc tactgtttct ccatacccgt tttttgggct aacaggagga 8100attaaccatg gggcatcacc accatcacca cgcgggttac ctgctgggca agattaatct 8160taaagcctgc gccgcgtgtg ctaagaaaat tttgg 8195238186DNAartificial sequencepRF162 plasmid 23aattcgacaa gaaatactcc atcggcctgg acattggaac caactctgtc ggctgggctg 60tcatcaccga cgagtacaag gtgccctcca agaaattcaa ggtcctcgga aacaccgatc 120gacactccat caagaaaaac ctcattggtg ccctgttgtt cgattctggc gagactgccg 180aagctaccag actcaagcga actgctcggc gacgttacac ccgacggaag aaccgaatct 240gctacctgca ggagatcttt tccaacgaga tggccaaggt ggacgattcg ttctttcatc 300gactggagga atccttcctc gtcgaggaag acaagaaaca cgagcgtcat cccatctttg 360gcaacattgt ggacgaggtt gcttaccacg agaagtatcc taccatctac cacctgcgaa 420agaaactcgt cgattccacc gacaaggcgg atctcagact tatctacctc gctctggcac 480acatgatcaa gtttcgaggt catttcctca tcgagggcga tctcaatccc gacaacagcg 540atgtggacaa gctgttcatt cagctcgttc agacctacaa ccagctgttc gaggaaaacc 600ccatcaatgc ctccggagtc gatgcaaagg ccatcttgtc tgctcgactc tcgaagagca 660gacgactgga gaacctcatt gcccaacttc ctggcgagaa aaagaacgga ctgtttggca 720acctcattgc cctttctctt ggtctcacac ccaacttcaa gtccaacttc gatctggcgg 780aggacgccaa gctccagctg tccaaggaca cctacgacga tgacctcgac aacctgcttg 840cacagattgg cgatcagtac gccgacctgt ttctcgctgc caagaacctt tcggatgcta 900ttctcttgtc tgacattctg cgagtcaaca ccgagatcac aaaggctccc ctttctgcct 960ccatgatcaa gcgatacgac gagcaccatc aggatctcac actgctcaag gctcttgtcc 1020gacagcaact gcccgagaag tacaaggaga tctttttcga tcagtcgaag aacggctacg 1080ctggatacat cgacggcgga gcctctcagg aagagttcta caagttcatc aagccaattc 1140tcgagaagat ggacggaacc gaggaactgc ttgtcaagct caatcgagag gatctgcttc 1200ggaagcaacg aaccttcgac aacggcagca ttcctcatca gatccacctc ggtgagctgc 1260acgccattct tcgacgtcag gaagacttct acccctttct caaggacaac cgagagaaga 1320tcgagaagat tcttaccttt cgaatcccct actatgttgg tcctcttgcc agaggaaact 1380ctcgatttgc ttggatgact cgaaagtccg aggaaaccat cactccctgg aacttcgagg 1440aagtcgtgga caagggtgcc tctgcacagt ccttcatcga gcgaatgacc aacttcgaca 1500agaatctgcc caacgagaag gttcttccca agcattcgct gctctacgag tactttacag 1560tctacaacga actcaccaaa gtcaagtacg ttaccgaggg aatgcgaaag cctgccttct 1620tgtctggcga acagaagaaa gccattgtcg atctcctgtt caagaccaac cgaaaggtca 1680ctgttaagca gctcaaggag gactacttca agaaaatcga gtgtttcgac agcgtcgaga 1740tttccggagt tgaggaccga ttcaacgcct ctttgggcac ctatcacgat ctgctcaaga 1800ttatcaagga caaggatttt ctcgacaacg aggaaaacga ggacattctg gaggacatcg 1860tgctcactct taccctgttc gaagatcggg agatgatcga ggaacgactc aagacatacg 1920ctcacctgtt cgacgacaag gtcatgaaac aactcaagcg acgtagatac accggctggg 1980gaagactttc gcgaaagctc atcaacggca tcagagacaa gcagtccgga aagaccattc 2040tggactttct caagtccgat ggctttgcca accgaaactt catgcagctc attcacgacg 2100attctcttac cttcaaggag gacatccaga aggcacaagt gtccggtcag ggcgacagct 2160tgcacgaaca tattgccaac ctggctggtt cgccagccat caagaaaggc attctccaga 2220ctgtcaaggt tgtcgacgag ctggtgaagg tcatgggacg tcacaagccc gagaacattg 2280tgatcgagat ggccagagag aaccagacaa ctcaaaaggg tcagaaaaac tcgcgagagc 2340ggatgaagcg aatcgaggaa ggcatcaagg agctgggatc ccagattctc aaggagcatc 2400ccgtcgagaa cactcaactg cagaacgaga agctgtatct ctactatctg cagaatggtc 2460gagacatgta cgtggatcag gaactggaca tcaatcgtct cagcgactac gatgtggacc 2520acattgtccc tcaatccttt ctcaaggacg attctatcga caacaaggtc cttacacgat 2580ccgacaagaa cagaggcaag tcggacaacg ttcccagcga agaggtggtc aaaaagatga 2640agaactactg gcgacagctg ctcaacgcca agctcattac ccagcgaaag ttcgacaatc 2700ttaccaaggc cgagcgaggc ggtctgtccg agctcgacaa ggctggcttc atcaagcgtc 2760aactcgtcga gaccagacag atcacaaagc acgtcgcaca gattctcgat tctcggatga 2820acaccaagta cgacgagaac gacaagctca tccgagaggt caaggtgatt actctcaagt 2880ccaaactggt ctccgatttc cgaaaggact ttcagttcta caaggtgcga gagatcaaca 2940attaccacca tgcccacgat gcttacctca acgccgtcgt tggcactgcg ctcatcaaga 3000aataccccaa gctcgaaagc gagttcgttt acggcgatta caaggtctac gacgttcgaa 3060agatgattgc caagtccgaa caggagattg gcaaggctac tgccaagtac ttcttttact 3120ccaacatcat gaactttttc aagaccgaga tcaccttggc caacggagag attcgaaaga 3180gaccacttat cgagaccaac ggcgaaactg gagagatcgt gtgggacaag ggtcgagact 3240ttgcaaccgt gcgaaaggtt ctgtcgatgc ctcaggtcaa catcgtcaag aaaaccgagg 3300ttcagactgg cggattctcc aaggagtcga ttctgcccaa gcgaaactcc gacaagctca 3360tcgctcgaaa gaaagactgg gatcccaaga aatacggtgg cttcgattct cctaccgtcg 3420cctattccgt gcttgtcgtt gcgaaggtcg agaagggcaa gtccaaaaag ctcaagtccg 3480tcaaggagct gctcggaatt accatcatgg agcgatcgag cttcgagaag aatcccatcg 3540acttcttgga agccaagggt tacaaggagg tcaagaaaga cctcattatc aagctgccca 3600agtactctct gttcgaactg gagaacggtc gaaagcgtat gctcgcctcc gctggcgagc 3660tgcagaaggg aaacgagctt gccttgcctt cgaagtacgt caactttctc tatctggctt 3720ctcactacga gaagctcaag ggttctcccg aggacaacga acagaagcaa ctcttcgttg 3780agcagcacaa acattacctc gacgagatta tcgagcagat ttccgagttt tcgaagcgag 3840tcatcctggc tgatgccaac ttggacaagg tgctctctgc ctacaacaag catcgggaca 3900aacccattcg agaacaggcg gagaacatca ttcacctgtt tactcttacc aacctgggtg 3960ctcctgcagc tttcaagtac ttcgatacca ctatcgaccg aaagcggtac acatccacca 4020aggaggttct cgatgccacc ctgattcacc agtccatcac tggcctgtac gagacccgaa 4080tcgacctgtc tcagcttggt ggcgactcca gagccgatcc caagaaaaag cgaaaggtct 4140aagcggccgc taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga 4200ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 4260tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 4320tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 4380tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg 4440acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca 4500ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc 4560ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc 4620gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 4680tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 4740tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4800gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4860acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 4920tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4980gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 5040tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 5100accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 5160ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 5220agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 5280gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 5340ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 5400tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 5460ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 5520gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 5580acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 5640aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 5700atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 5760gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5820tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5880ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5940ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 6000ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 6060aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 6120cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 6180gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 6240ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 6300cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 6360tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 6420cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 6480cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 6540tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 6600cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6660gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6720tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg 6780aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc 6840ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg 6900gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg 6960cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg 7020gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc 7080tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac 7140ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga 7200tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg 7260ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc 7320gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc 7380ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta 7440agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga 7500gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc 7560ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga 7620ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg 7680gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg 7740ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat 7800tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc 7860aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca 7920aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac 7980ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga 8040cgctttttat cgcaactctc tactgtttct ccatacccgt tttttgggct aacaggagga 8100attaaccatg gggcatcacc accatcacca cttattgatt atcttgcgtc gtcgcatccg 8160caaacaggcg cacgcacata gcaagg 81862480RNAartificial sequenceCas9 endonuclease recognition (CER) domain 24guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc cguuaucaac uugaaaaagu 60ggcaccgagu cggugcuuuu 802520DNAYarrowia lipolytica 25tcaaacgatt acccaccctc 202643RNAartificial sequenceHammerhead (HH) ribozymemisc_feature(1)..(6)n is a, c, g, or u 26nnnnnncuga ugaguccgug aggacgaaac gaguaagcuc guc 432768RNAhepatitis delta virus 27ggccggcaug gucccagccu ccucgcuggc gccggcuggg caacaugcuu cggcauggcg 60aaugggac 6828211DNAartificial sequenceHH-sgRNA-HDV (RGR) pre-sgRNA expression cassette 28gtttgactga tgagtccgtg aggacgaaac gagtaagctc gtctcaaacg attacccacc 60ctcgttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa 120agtggcaccg agtcggtgct tttggccggc atggtcccag cctcctcgct ggcgccggct 180gggcaacatg cttcggcatg gcgaatggga c 2112920DNABacteriophage T7 29taatacgact cactataggg 20302875DNAartificial sequencepRF46 plasmid 30agcttgtccc attcgccatg ccgaagcatg ttgcccagcc ggcgccagcg aggaggctgg 60gaccatgccg gccaaaagca ccaccgactc ggtgccactt tttcaagttg ataacggact 120agccttattt taacttgcta tttctagctc taaaacgagg gtgggtaatc gtttgagacg 180agcttactcg tttcgtcctc acggactcat cagtcaaacc cctatagtga gtcgtattag 240aattcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 300cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 360ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 420ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 480gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 540cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 600tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 660cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 720aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 780cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 840gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 900ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 960cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 1020aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 1080tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 1140ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 1200tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 1260ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 1320agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 1380atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 1440cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 1500ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 1560ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1620agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 1680agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 1740gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 1800cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 1860gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 1920tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 1980tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 2040aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 2100cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 2160cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 2220aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 2280ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 2340tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 2400ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 2460acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag 2520ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag 2580ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag 2640attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 2700taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg 2760cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt 2820tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgcca 28753120DNAartificial sequenceT7 forward PCR primer 31ccggctcgta tgttgtgtgg 203220DNAartificial sequencegRNArev1 reverse primer 32aaaagcaccg actcggtgcc 203321DNAartificial sequenceIV-up forward primer 33ccacgaaacg acgtttcgac c 213420DNAartificial sequenceIV-down reverse primer 34gcaaagactc ggttgatggc 2035982DNAYarrowia lipolytica 35ccacgaaacg acgtttcgac cttaacgacc ctgccgtctc catccatccg accacaatgg 60aaaagacatt ttcaaacgat tacccaccct ccgggactga ggcccacatc cacatcaacc 120acacggccca ctcggatgac tcagaggagg tgccctcgca caaggaaaat tacaacacca 180gtggccacga cctggaggag tccgacccgg ataaccatgt cggtgagacc ctcgaggtca 240agcgaggtct caagatgcga cacatctcca tgatctcgct tggaggaacc attggtaccg 300gtctcttcat tggtaccgga ggagctctcc agcaggccgg tccctgtggc gccctcgtcg 360cctacgtgtt catggccacc attgtctact ctgttgccga gtctcttgga gaactggcta 420cgtacattcc catcaccggc tcctttgccg tctttactac ccgatatctg tcacagtcgt 480ttggtgcctc catgggctgg ctatactggt tctcgtgggc gatcaccttc gccatcgagc 540tcaacaccat tggtcccgtg attgagtact ggactgacgc cgttcctact gctgcctgga 600ttgccatctt cttcgtcatc ctcactacca tcaacttctt ccccgtgggc ttctatggcg 660aagtcgagtt ctgggtggcc tccgtgaagg tcattgccat cattggatgg ctcatctacg 720cgctctgcat gacgtgtgga gcaggtgtaa caggtcctgt gggattcaga tactggaacc 780accccggacc catgggagac ggaatctgga ccgacggcgt gcccattgtg cgaaacgcgc 840ccggtcgacg attcatggga tggctcaatt cgctcgttaa cgccgccttc acctaccagg 900gctgtgagct ggtcggagtc actgccggtg aggcccagaa ccccagaaag tccgtccctc 960gagccatcaa ccgagtcttt gc 982364RNAunknownRNA loop-forming sequence (GAAA) 36gaaa 4374RNAunknownRNA loop-forming sequence (CAAA) 37caaa

4384RNAunknownRNA loop-forming sequence (AAAG) 38aaag 4391434PRTartificial sequenceZebra CPP-Cas9-NLS fusion protein 39Glu Cys Asp Ser Glu Leu Glu Ile Lys Arg Tyr Lys Arg Val Arg Val1 5 10 15Ala Ser Arg Lys Cys Arg Ala Lys Phe Lys Gln Leu Leu Gln His Tyr 20 25 30Arg Glu Val Ala Ala Ala Lys Ser Ser Glu Asn Asp Arg Leu Arg Leu 35 40 45Leu Leu Lys Gln Met Cys Glu Phe Asp Lys Lys Tyr Ser Ile Gly Leu 50 55 60Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr65 70 75 80Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His 85 90 95Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu 100 105 110Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr 115 120 125Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu 130 135 140Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe145 150 155 160Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn 165 170 175Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His 180 185 190Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu 195 200 205Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu 210 215 220Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe225 230 235 240Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile 245 250 255Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser 260 265 270Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys 275 280 285Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr 290 295 300Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln305 310 315 320Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln 325 330 335Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser 340 345 350Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr 355 360 365Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His 370 375 380Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu385 390 395 400Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly 405 410 415Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys 420 425 430Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu 435 440 445Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser 450 455 460Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg465 470 475 480Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu 485 490 495Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg 500 505 510Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile 515 520 525Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln 530 535 540Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu545 550 555 560Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr 565 570 575Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro 580 585 590Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe 595 600 605Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe 610 615 620Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp625 630 635 640Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile 645 650 655Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu 660 665 670Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu 675 680 685Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys 690 695 700Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys705 710 715 720Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp 725 730 735Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile 740 745 750His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val 755 760 765Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly 770 775 780Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp785 790 795 800Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile 805 810 815Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser 820 825 830Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser 835 840 845Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu 850 855 860Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp865 870 875 880Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile 885 890 895Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu 900 905 910Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu 915 920 925Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala 930 935 940Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg945 950 955 960Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu 965 970 975Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser 980 985 990Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val 995 1000 1005Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys 1010 1015 1020Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His 1025 1030 1035Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile 1040 1045 1050Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr 1055 1060 1065Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu 1070 1075 1080Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met 1085 1090 1095Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg 1100 1105 1110Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val 1115 1120 1125Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser 1130 1135 1140Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly 1145 1150 1155Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys 1160 1165 1170Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly 1175 1180 1185Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys 1190 1195 1200Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu 1205 1210 1215Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro 1220 1225 1230Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp 1235 1240 1245Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn 1250 1255 1260Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly 1265 1270 1275Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu 1280 1285 1290Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu 1295 1300 1305Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu 1310 1315 1320Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala 1325 1330 1335Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg 1340 1345 1350Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe 1355 1360 1365Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp 1370 1375 1380Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu 1385 1390 1395Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr 1400 1405 1410Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro 1415 1420 1425Lys Lys Lys Arg Lys Val 1430401397PRTartificial sequencePolyR CPP-Cas9-NLS fusion protein 40Gly Gly Gly Gly Arg Arg Arg Arg Arg Arg Arg Arg Arg Leu Leu Leu1 5 10 15Leu Glu Phe Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn 20 25 30Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys 35 40 45Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn 50 55 60Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr65 70 75 80Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg 85 90 95Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp 100 105 110Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp 115 120 125Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val 130 135 140Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu145 150 155 160Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu 165 170 175Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu 180 185 190Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln 195 200 205Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val 210 215 220Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu225 230 235 240Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe 245 250 255Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser 260 265 270Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr 275 280 285Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr 290 295 300Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu305 310 315 320Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser 325 330 335Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu 340 345 350Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile 355 360 365Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly 370 375 380Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys385 390 395 400Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu 405 410 415Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile 420 425 430His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr 435 440 445Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe 450 455 460Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe465 470 475 480Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe 485 490 495Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg 500 505 510Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys 515 520 525His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys 530 535 540Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly545 550 555 560Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys 565 570 575Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys 580 585 590Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser 595 600 605Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe 610 615 620Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr625 630 635 640Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr 645 650 655Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg 660 665 670Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile 675 680 685Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp 690 695 700Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu705 710 715 720Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp 725 730 735Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys 740 745 750Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val 755 760 765Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu 770 775 780Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys785 790 795 800Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu 805 810 815His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr 820 825 830Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile 835 840 845Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe 850 855 860Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys865 870 875 880Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys 885 890 895Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln 900 905 910Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu 915 920 925Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln 930 935 940Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys945 950 955 960Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu 965 970 975Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys 980 985 990Val Arg Glu Ile Asn Asn

Tyr His His Ala His Asp Ala Tyr Leu Asn 995 1000 1005Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu 1010 1015 1020Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys 1025 1030 1035Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys 1040 1045 1050Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile 1055 1060 1065Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr 1070 1075 1080Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe 1085 1090 1095Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val 1100 1105 1110Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile 1115 1120 1125Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp 1130 1135 1140Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala 1145 1150 1155Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1160 1165 1170Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu 1175 1180 1185Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys 1190 1195 1200Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 1205 1210 1215Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala 1220 1225 1230Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser 1235 1240 1245Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu 1250 1255 1260Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu 1265 1270 1275Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu 1280 1285 1290Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val 1295 1300 1305Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln 1310 1315 1320Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala 1325 1330 1335Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg 1340 1345 1350Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln 1355 1360 1365Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu 1370 1375 1380Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val 1385 1390 1395411401PRTartificial sequenceTP10 CPP-Cas9-NLS fusion protein 41Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu Lys Ala Cys Ala Ala Cys1 5 10 15Ala Lys Lys Ile Leu Glu Phe Asp Lys Lys Tyr Ser Ile Gly Leu Asp 20 25 30Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys 35 40 45Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser 50 55 60Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr65 70 75 80Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg 85 90 95Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met 100 105 110Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu 115 120 125Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile 130 135 140Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu145 150 155 160Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile 165 170 175Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile 180 185 190Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile 195 200 205Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn 210 215 220Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys225 230 235 240Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys 245 250 255Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro 260 265 270Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu 275 280 285Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile 290 295 300Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp305 310 315 320Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys 325 330 335Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln 340 345 350Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys 355 360 365Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr 370 375 380Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro385 390 395 400Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn 405 410 415Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile 420 425 430Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln 435 440 445Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys 450 455 460Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly465 470 475 480Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr 485 490 495Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser 500 505 510Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys 515 520 525Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn 530 535 540Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala545 550 555 560Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys 565 570 575Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys 580 585 590Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg 595 600 605Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys 610 615 620Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp625 630 635 640Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu 645 650 655Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln 660 665 670Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu 675 680 685Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe 690 695 700Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His705 710 715 720Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser 725 730 735Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser 740 745 750Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu 755 760 765Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu 770 775 780Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg785 790 795 800Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln 805 810 815Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys 820 825 830Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln 835 840 845Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val 850 855 860Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr865 870 875 880Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu 885 890 895Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys 900 905 910Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly 915 920 925Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val 930 935 940Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg945 950 955 960Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys 965 970 975Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe 980 985 990Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp 995 1000 1005Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr 1010 1015 1020Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr 1025 1030 1035Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys 1040 1045 1050Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe 1055 1060 1065Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro 1070 1075 1080Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys 1085 1090 1095Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln 1100 1105 1110Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser 1115 1120 1125Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala 1130 1135 1140Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser 1145 1150 1155Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys 1160 1165 1170Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile 1175 1180 1185Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe 1190 1195 1200Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile 1205 1210 1215Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys 1220 1225 1230Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu 1235 1240 1245Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His 1250 1255 1260Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln 1265 1270 1275Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu 1280 1285 1290Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn 1295 1300 1305Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro 1310 1315 1320Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr 1325 1330 1335Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile 1340 1345 1350Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr 1355 1360 1365Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp 1370 1375 1380Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys 1385 1390 1395Arg Lys Val 1400421398PRTartificial sequencepVEC CPP-Cas9-NLS fusion protein 42Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His1 5 10 15Ser Lys Glu Phe Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr 20 25 30Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser 35 40 45Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys 50 55 60Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala65 70 75 80Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn 85 90 95Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val 100 105 110Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu 115 120 125Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu 130 135 140Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys145 150 155 160Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala 165 170 175Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp 180 185 190Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val 195 200 205Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly 210 215 220Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg225 230 235 240Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu 245 250 255Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys 260 265 270Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp 275 280 285Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln 290 295 300Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu305 310 315 320Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu 325 330 335Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr 340 345 350Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu 355 360 365Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly 370 375 380Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu385 390 395 400Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp 405 410 415Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln 420 425 430Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe 435 440 445Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr 450 455 460Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg465 470 475 480Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn 485 490 495Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu 500 505 510Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro 515 520 525Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr 530 535 540Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser545 550 555 560Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg 565 570 575Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu 580 585 590Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala 595 600 605Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp 610 615 620Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu625 630 635 640Thr Leu

Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys 645 650 655Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg 660 665 670Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly 675 680 685Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser 690 695 700Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser705 710 715 720Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly 725 730 735Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile 740 745 750Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys 755 760 765Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg 770 775 780Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met785 790 795 800Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys 805 810 815Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu 820 825 830Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp 835 840 845Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser 850 855 860Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp865 870 875 880Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys 885 890 895Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr 900 905 910Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser 915 920 925Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg 930 935 940Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr945 950 955 960Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr 965 970 975Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr 980 985 990Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu 995 1000 1005Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu 1010 1015 1020Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg 1025 1030 1035Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala 1040 1045 1050Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu 1055 1060 1065Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu 1070 1075 1080Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp 1085 1090 1095Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile 1100 1105 1110Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser 1115 1120 1125Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys 1130 1135 1140Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val 1145 1150 1155Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser 1160 1165 1170Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met 1175 1180 1185Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala 1190 1195 1200Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro 1205 1210 1215Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu 1220 1225 1230Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro 1235 1240 1245Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys 1250 1255 1260Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val 1265 1270 1275Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser 1280 1285 1290Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys 1295 1300 1305Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu 1310 1315 1320Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly 1325 1330 1335Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys 1340 1345 1350Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His 1355 1360 1365Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln 1370 1375 1380Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val 1385 1390 13954323DNAunknownExample of a Cas9 target sitePAM sequencemisc_feature(1)..(20)n = A, C, T, or Gmisc_feature(21)..(21)n = A, C, T, or G (indicated as an "X" in Specification) 43nnnnnnnnnn nnnnnnnnnn ngg 23443DNAunknownPAM sequence NGGmisc_feature(1)..(1)n = A, C, T, or G 44ngg 3456DNAunknownPAM sequence NNAGAAmisc_feature(1)..(2)n = A, C, T, or G 45nnagaa 6467DNAunknownPAM sequence NNAGAAWmisc_feature(1)..(2)n = A, C, T, or Gmisc_feature(7)..(7)w = A or T 46nnagaaw 7475DNAunknownPAM sequence NGGNGmisc_feature(1)..(1)n = A, C, T, or Gmisc_feature(4)..(4)n = A, C, T, or G 47nggng 5488DNAunknownPAM sequence NNNNGATTmisc_feature(1)..(4)n = A, C, T, or G 48nnnngatt 8496DNAunknownPAM sequence NAAAACmisc_feature(1)..(1)n = A, C, T, or G 49naaaac 6502DNAunknownPAM sequence NGmisc_feature(1)..(1)n = A, C, T, or G 50ng 25122RNAunknownTracrRNA mate sequence example 1 51guuuuuguac ucucaagauu ua 225215RNAunknownTracrRNA mate sequence example 2 52guuuuuguac ucuca 155312RNAunknownTracrRNA mate sequence example 3 53guuuuagagc ua 125413RNAunknownTracrRNA mate sequence example 4 54guuuuagagc uag 135560RNAStreptococcus pyogenes 55uagcaaguua aaauaaggcu aguccguuau caacuugaaa aaguggcacc gagucggugc 605645RNAStreptococcus pyogenes 56uagcaaguua aaauaaggcu aguccguuau caacuugaaa aagug 455732RNAStreptococcus pyogenes 57uagcaaguua aaauaaggcu aguccguuau ca 325885RNAStreptococcus thermophilus 58uaaaucuugc agaagcuaca aagauaaggc uucaugccga aaucaacacc cugucauuuu 60auggcagggu guuuucguua uuuaa 855977RNAStreptococcus thermophilus 59ugcagaagcu acaaagauaa ggcuucaugc cgaaaucaac acccugucau uuuauggcag 60gguguuuucg uuauuua 776065RNAStreptococcus thermophilus 60ugcagaagcu acaaagauaa ggcuucaugc cgaaaucaac acccugucau uuuauggcag 60ggugu 6561131RNAartificial sequencegRNA example 1misc_feature(1)..(20)n = A, C, U, or G 61nnnnnnnnnn nnnnnnnnnn guuuuuguac ucucaagauu uagaaauaaa ucuugcagaa 60gcuacaaaga uaaggcuuca ugccgaaauc aacacccugu cauuuuaugg caggguguuu 120ucguuauuua a 13162117RNAartificial sequencegRNA example 2misc_feature(1)..(20)n = A, C, U, or G 62nnnnnnnnnn nnnnnnnnnn guuuuuguac ucucagaaau gcagaagcua caaagauaag 60gcuucaugcc gaaaucaaca cccugucauu uuauggcagg guguuuucgu uauuuaa 11763104RNAartificial sequencegRNA example 3misc_feature(1)..(20)n = A, C, U, or G 63nnnnnnnnnn nnnnnnnnnn guuuuuguac ucucagaaau gcagaagcua caaagauaag 60gcuucaugcc gaaaucaaca cccugucauu uuauggcagg gugu 1046499RNAartificial sequencegRNA example 4misc_feature(1)..(20)n = A, C, U, or G 64nnnnnnnnnn nnnnnnnnnn guuuuuguac ucucagaaau agcaaguuaa aauaaggcua 60guccguuauc aacuugaaaa aguggcaccg agucggugc 996581RNAartificial sequencegRNA example 5misc_feature(1)..(20)n = A, C, U, or G 65nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu g 816668RNAartificial sequencegRNA example 6misc_feature(1)..(20)n = A, C, U, or G 66nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuauca 6867100RNAartificial sequencegRNA example 7misc_feature(1)..(20)n = A, C, U, or G 67nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1006810PRTHuman immunodeficiency virus 68Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 10699PRTHuman immunodeficiency virus 69Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5708PRTHuman immunodeficiency virus 70Arg Lys Lys Arg Arg Gln Arg Arg1 57116PRTDrosophila melanogaster 71Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys1 5 10 157211PRTartificial sequencepolyarginine CPP example 2 72Thr His Arg Leu Pro Arg Arg Arg Arg Arg Arg1 5 107311PRTartificial sequencepolyarginine CPP example 3 73Gly Gly Arg Arg Ala Arg Arg Arg Arg Arg Arg1 5 107417PRTmus musculus 74Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His Ser1 5 10 15Lys7510PRTartificial sequence(KFF)3K CPP 75Lys Phe Phe Lys Phe Phe Lys Phe Phe Lys1 5 107618PRTartificial sequenceMAP peptide CPP 76Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala Leu Lys1 5 10 15Leu Ala7712PRTartificial sequenceCPP (RRQRRTSKLMKR) 77Arg Arg Gln Arg Arg Thr Ser Lys Leu Met Lys Arg1 5 107833PRTartificial sequenceCPP (KALAWEAKLAKALAKALAKHLAKALAKALKCEA) 78Lys Ala Leu Ala Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala1 5 10 15Leu Ala Lys His Leu Ala Lys Ala Leu Ala Lys Ala Leu Lys Cys Glu 20 25 30Ala796PRTartificial sequenceProline-rich CPP repeat example 1 79Val His Leu Pro Pro Pro1 5806PRTartificial sequenceProline-rich CPP repeat example 2 80Val His Arg Pro Pro Pro1 58127PRTartificial sequenceMPG peptide CPP 81Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly1 5 10 15Ala Trp Ser Gln Pro Lys Ser Lys Arg Lys Val 20 258221PRTartificial sequencePep-1 peptide CPP 82Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys1 5 10 15Lys Lys Arg Lys Val 208324PRTHomo sapiens 83Leu Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln1 5 10 15Thr Ala Ile Gly Val Gly Ala Pro 208418PRTHomo sapiens 84Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe1 5 10 15Asn Lys85240PRTArtificial sequencehis tagged dsRED 85Met Gly Ser Ser His His His His His His Glu Phe Gly Gly Gly Gly1 5 10 15Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg 20 25 30Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly 35 40 45Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr 50 55 60Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe65 70 75 80Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro Asp 85 90 95Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met 100 105 110Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu 115 120 125Gln Asp Gly Ser Phe Ile Tyr Lys Val Lys Phe Ile Gly Val Asn Phe 130 135 140Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala145 150 155 160Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu Ile 165 170 175His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu Phe 180 185 190Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr Tyr 195 200 205Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr 210 215 220Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His His Leu Phe Leu225 230 235 24086731DNAArtificial sequenceE. coli codon optimized dsRED 86ccatgggctc cagccatcat catcaccatc atgaattcgg aggtggcggt gcatcctcgg 60aggatgtgat taaagaattt atgcggttta aagtacgtat ggaaggatcg gtgaatggcc 120atgaatttga gattgagggt gaaggcgaag gccgcccgta cgaaggaact caaacagcga 180aattaaaagt tacaaaagga ggtcctctgc cgtttgcctg ggacatcttg agcccgcaat 240tccagtacgg ttccaaagtg tatgtaaaac accctgcgga tattccggat tataaaaaac 300tgagttttcc cgaggggttt aaatgggaac gggtgatgaa ttttgaggat ggtggagttg 360tcaccgtgac ccaggactct agcttacaag acggtagttt catctacaaa gtaaaattta 420tcggcgtaaa cttcccatcg gacggccccg tcatgcagaa aaagacgatg ggctgggaag 480ccagcaccga acgtttgtac ccacgggacg gcgttttgaa aggggaaatc cataaggccc 540ttaaactgaa agacggtggt cactatctcg tggagtttaa atcgatttat atggctaaaa 600aaccagtaca gcttccgggt tattattacg ttgactccaa attggacatc acatcgcata 660atgaagatta cacgattgtt gaacagtacg agcgcgccga gggccggcac catctgtttc 720tgtaaaagct t 731874092DNAArtificial sequencepBAD/HisB 87aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 60tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 120aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 180attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 240atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg ttttttgggc 300taacaggagg aattaaccat ggggggttct catcatcatc atcatcatgg tatggctagc 360atgactggtg gacagcaaat gggtcgggat ctgtacgacg atgacgataa ggatccgagc 420tcgagatctg cagctggtac catatgggaa ttcgaagctt ggctgttttg gcggatgaga 480gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa 540tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa 600acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc 660atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt 720cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gttgcgaagc 780aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc 840agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttttg tttatttttc 900taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 960tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 1020gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 1080gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 1140cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 1200tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac 1260tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 1320atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 1380ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 1440gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 1500gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 1560gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 1620gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 1680gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 1740cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 1800atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 1860tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 1920ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 1980gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 2040tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 2100ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 2160ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc

2220gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 2280ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 2340tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 2400ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 2460agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 2520agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 2580gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 2640tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 2700accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 2760gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt 2820atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc 2880cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa 2940cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 3000tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 3060ggcagcagat caattcgcgc gcgaaggcga agcggcatgc ataatgtgcc tgtcaaatgg 3120acgaagcagg gattctgcaa accctatgct actccgtcaa gccgtcaatt gtctgattcg 3180ttaccaatta tgacaacttg acggctacat cattcacttt ttcttcacaa ccggcacgga 3240actcgctcgg gctggccccg gtgcattttt taaatacccg cgagaaatag agttgatcgt 3300caaaaccaac attgcgaccg acggtggcga taggcatccg ggtggtgctc aaaagcagct 3360tcgcctggct gatacgttgg tcctcgcgcc agcttaagac gctaatccct aactgctggc 3420ggaaaagatg tgacagacgc gacggcgaca agcaaacatg ctgtgcgacg ctggcgatat 3480caaaattgct gtctgccagg tgatcgctga tgtactgaca agcctcgcgt acccgattat 3540ccatcggtgg atggagcgac tcgttaatcg cttccatgcg ccgcagtaac aattgctcaa 3600gcagatttat cgccagcagc tccgaatagc gcccttcccc ttgcccggcg ttaatgattt 3660gcccaaacag gtcgctgaaa tgcggctggt gcgcttcatc cgggcgaaag aaccccgtat 3720tggcaaatat tgacggccag ttaagccatt catgccagta ggcgcgcgga cgaaagtaaa 3780cccactggtg ataccattcg cgagcctccg gatgacgacc gtagtgatga atctctcctg 3840gcgggaacag caaaatatca cccggtcggc aaacaaattc tcgtccctga tttttcacca 3900ccccctgacc gcgaatggtg agattgagaa tataaccttt cattcccagc ggtcggtcga 3960taaaaaaatc gagataaccg ttggcctcaa tcggcgttaa acccgccacc agatgggcat 4020taaacgagta tcccggcagc aggggatcat tttgcgcttc agccatactt ttcatactcc 4080cgccattcag ag 4092884679DNAArtificial sequencepRF161 88catgggctcc agccatcatc atcaccatca tgaattcgga ggtggcggtg catcctcgga 60ggatgtgatt aaagaattta tgcggtttaa agtacgtatg gaaggatcgg tgaatggcca 120tgaatttgag attgagggtg aaggcgaagg ccgcccgtac gaaggaactc aaacagcgaa 180attaaaagtt acaaaaggag gtcctctgcc gtttgcctgg gacatcttga gcccgcaatt 240ccagtacggt tccaaagtgt atgtaaaaca ccctgcggat attccggatt ataaaaaact 300gagttttccc gaggggttta aatgggaacg ggtgatgaat tttgaggatg gtggagttgt 360caccgtgacc caggactcta gcttacaaga cggtagtttc atctacaaag taaaatttat 420cggcgtaaac ttcccatcgg acggccccgt catgcagaaa aagacgatgg gctgggaagc 480cagcaccgaa cgtttgtacc cacgggacgg cgttttgaaa ggggaaatcc ataaggccct 540taaactgaaa gacggtggtc actatctcgt ggagtttaaa tcgatttata tggctaaaaa 600accagtacag cttccgggtt attattacgt tgactccaaa ttggacatca catcgcataa 660tgaagattac acgattgttg aacagtacga gcgcgccgag ggccggcacc atctgtttct 720gtaaaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc 780agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc 840acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc 900tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag 960actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc 1020cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc 1080cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg 1140cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg 1200agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 1260catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 1320ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 1380atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 1440ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc 1500gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 1560ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 1620ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1680gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1740ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1800gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1860ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1920gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 1980gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 2040caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 2100cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 2160ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 2220taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 2280tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 2340gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 2400agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 2460aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 2520gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 2580gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 2640tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 2700agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2760cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2820gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2880gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 2940ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 3000cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 3060cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt 3120acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 3180gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 3240tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 3300ggttttcacc gtcatcaccg aaacgcgcga ggcagcagat caattcgcgc gcgaaggcga 3360agcggcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa accctatgct 3420actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg acggctacat 3480cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg gtgcattttt 3540taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg acggtggcga 3600taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg tcctcgcgcc 3660agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc gacggcgaca 3720agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg tgatcgctga 3780tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac tcgttaatcg 3840cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc tccgaatagc 3900gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa tgcggctggt 3960gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag ttaagccatt 4020catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg cgagcctccg 4080gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca cccggtcggc 4140aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg agattgagaa 4200tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg ttggcctcaa 4260tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc aggggatcat 4320tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc aattgtccat 4380attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta accaaaccgg 4440taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg acaaaaacgc 4500gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg cacggcgtca 4560cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc tgacgctttt 4620tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga ggaattaac 46798920PRTArtificial sequenceTAT 89Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Pro Lys Lys1 5 10 15Lys Arg Lys Val 209018PRTArtificial sequenceTLM 90Pro Leu Ser Ser Ile Phe Ser Arg Ile Gly Asp Pro Lys Lys Lys Arg1 5 10 15Lys Val9123PRTArtificial sequenceMPG1 91Ala Leu Phe Leu Gly Gln Leu Gly Ala Ala Gly Ser Thr Met Gly Ala1 5 10 15Pro Lys Lys Lys Arg Lys Val 209221PRTArtificial sequencepep1 92Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys1 5 10 15Lys Lys Arg Lys Val 20937PRTArtificial sequenceCFFKDEL 93Cys Phe Phe Lys Asp Glu Leu1 59498DNAArtificial sequencehis-TAT E.coli optimized 94ccatggggca tcaccatcat catcacggcc gcaaaaaacg tcgtcagcgc cggcgtccgc 60cccagccgaa aaaacggaaa gtgggcggcg gcgaattc 989589DNAArtificial sequencehis-TLM E.coli optimized 95ccatggggca tcaccatcat catcatccgt taagctcgat cttttctcgt atcggtgatc 60cgccaaaaaa gaaacgcaaa gtagaattc 8996104DNAArtificial sequencehis-MPG1 E. coli optimized 96ccatggggca tcatcatcac catcacggcg ccctgttctt aggccagctg ggcgccgcgg 60gatccacgat gggtgcgccg aagaaaaagc gcaaagttga attc 1049795DNAArtificial sequencehis-pep1 E. coli optimized 97ccatggggca ccatcaccat caccataaag aaacttggtg ggagacttgg tggaccgaat 60ggtcccagcc gaagaaaaaa cgcaaggttg aattc 959851DNAArtificial sequencehis-CFFKDEL E. coli optimized 98ccatggggca tcaccatcac caccattgtt ttttcaaaga cgaactggaa t 51994739DNAArtificial sequencepRF224; 99catggggcat caccatcatc atcacggccg caaaaaacgt cgtcagcgcc ggcgtccgcc 60ccagccgaaa aaacggaaag tgggcggcgg cgaattcgga ggtggcggtg catcctcgga 120ggatgtgatt aaagaattta tgcggtttaa agtacgtatg gaaggatcgg tgaatggcca 180tgaatttgag attgagggtg aaggcgaagg ccgcccgtac gaaggaactc aaacagcgaa 240attaaaagtt acaaaaggag gtcctctgcc gtttgcctgg gacatcttga gcccgcaatt 300ccagtacggt tccaaagtgt atgtaaaaca ccctgcggat attccggatt ataaaaaact 360gagttttccc gaggggttta aatgggaacg ggtgatgaat tttgaggatg gtggagttgt 420caccgtgacc caggactcta gcttacaaga cggtagtttc atctacaaag taaaatttat 480cggcgtaaac ttcccatcgg acggccccgt catgcagaaa aagacgatgg gctgggaagc 540cagcaccgaa cgtttgtacc cacgggacgg cgttttgaaa ggggaaatcc ataaggccct 600taaactgaaa gacggtggtc actatctcgt ggagtttaaa tcgatttata tggctaaaaa 660accagtacag cttccgggtt attattacgt tgactccaaa ttggacatca catcgcataa 720tgaagattac acgattgttg aacagtacga gcgcgccgag ggccggcacc atctgtttct 780gtaaaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc 840agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc 900acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc 960tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag 1020actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc 1080cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc 1140cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg 1200cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg 1260agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 1320catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 1380ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 1440atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 1500ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc 1560gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 1620ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 1680ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1740gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1800ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1860gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1920ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1980gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 2040gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 2100caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 2160cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 2220ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 2280taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 2340tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 2400gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 2460agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 2520aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 2580gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 2640gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 2700tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 2760agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2820cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2880gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2940gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 3000ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 3060cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 3120cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt 3180acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact 3240gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 3300tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 3360ggttttcacc gtcatcaccg aaacgcgcga ggcagcagat caattcgcgc gcgaaggcga 3420agcggcatgc ataatgtgcc tgtcaaatgg acgaagcagg gattctgcaa accctatgct 3480actccgtcaa gccgtcaatt gtctgattcg ttaccaatta tgacaacttg acggctacat 3540cattcacttt ttcttcacaa ccggcacgga actcgctcgg gctggccccg gtgcattttt 3600taaatacccg cgagaaatag agttgatcgt caaaaccaac attgcgaccg acggtggcga 3660taggcatccg ggtggtgctc aaaagcagct tcgcctggct gatacgttgg tcctcgcgcc 3720agcttaagac gctaatccct aactgctggc ggaaaagatg tgacagacgc gacggcgaca 3780agcaaacatg ctgtgcgacg ctggcgatat caaaattgct gtctgccagg tgatcgctga 3840tgtactgaca agcctcgcgt acccgattat ccatcggtgg atggagcgac tcgttaatcg 3900cttccatgcg ccgcagtaac aattgctcaa gcagatttat cgccagcagc tccgaatagc 3960gcccttcccc ttgcccggcg ttaatgattt gcccaaacag gtcgctgaaa tgcggctggt 4020gcgcttcatc cgggcgaaag aaccccgtat tggcaaatat tgacggccag ttaagccatt 4080catgccagta ggcgcgcgga cgaaagtaaa cccactggtg ataccattcg cgagcctccg 4140gatgacgacc gtagtgatga atctctcctg gcgggaacag caaaatatca cccggtcggc 4200aaacaaattc tcgtccctga tttttcacca ccccctgacc gcgaatggtg agattgagaa 4260tataaccttt cattcccagc ggtcggtcga taaaaaaatc gagataaccg ttggcctcaa 4320tcggcgttaa acccgccacc agatgggcat taaacgagta tcccggcagc aggggatcat 4380tttgcgcttc agccatactt ttcatactcc cgccattcag agaagaaacc aattgtccat 4440attgcatcag acattgccgt cactgcgtct tttactggct cttctcgcta accaaaccgg 4500taaccccgct tattaaaagc attctgtaac aaagcgggac caaagccatg acaaaaacgc 4560gtaacaaaag tgtctataat cacggcagaa aagtccacat tgattatttg cacggcgtca 4620cactttgcta tgccatagca tttttatcca taagattagc ggatcctacc tgacgctttt 4680tatcgcaact ctctactgtt tctccatacc cgttttttgg gctaacagga ggaattaac 47391004730DNAArtificial sequencepRF214 100catggggcat caccatcatc atcatccgtt aagctcgatc ttttctcgta tcggtgatcc 60gccaaaaaag aaacgcaaag tagaattcgg aggtggcggt gcatcctcgg aggatgtgat 120taaagaattt atgcggttta aagtacgtat ggaaggatcg gtgaatggcc atgaatttga 180gattgagggt gaaggcgaag gccgcccgta cgaaggaact caaacagcga aattaaaagt 240tacaaaagga ggtcctctgc cgtttgcctg ggacatcttg agcccgcaat tccagtacgg 300ttccaaagtg tatgtaaaac accctgcgga tattccggat tataaaaaac tgagttttcc 360cgaggggttt aaatgggaac gggtgatgaa ttttgaggat ggtggagttg tcaccgtgac 420ccaggactct agcttacaag acggtagttt catctacaaa gtaaaattta tcggcgtaaa 480cttcccatcg gacggccccg tcatgcagaa aaagacgatg ggctgggaag ccagcaccga 540acgtttgtac ccacgggacg gcgttttgaa aggggaaatc cataaggccc ttaaactgaa 600agacggtggt cactatctcg tggagtttaa atcgatttat atggctaaaa aaccagtaca 660gcttccgggt tattattacg ttgactccaa attggacatc acatcgcata atgaagatta 720cacgattgtt gaacagtacg agcgcgccga gggccggcac catctgtttc tgtaaaagct 780tggctgtttt ggcggatgag agaagatttt cagcctgata cagattaaat cagaacgcag 840aagcggtctg ataaaacaga atttgcctgg cggcagtagc gcggtggtcc cacctgaccc 900catgccgaac tcagaagtga aacgccgtag cgccgatggt agtgtggggt ctccccatgc 960gagagtaggg aactgccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct 1020ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag 1080cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa 1140ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac 1200aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 1260ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 1320gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 1380ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 1440gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 1500agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag 1560caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 1620gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 1680agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 1740gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 1800aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 1860ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 1920tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 1980tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 2040gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag

tcaggcaact 2100atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 2160ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 2220aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 2280ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 2340ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 2400tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 2460cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 2520gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 2580gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 2640tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 2700ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 2760gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 2820ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 2880tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 2940ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 3000gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 3060acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt 3120ctccttacgc atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc 3180tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg 3240ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg 3300catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac 3360cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg cgcgaaggcg aagcggcatg 3420cataatgtgc ctgtcaaatg gacgaagcag ggattctgca aaccctatgc tactccgtca 3480agccgtcaat tgtctgattc gttaccaatt atgacaactt gacggctaca tcattcactt 3540tttcttcaca accggcacgg aactcgctcg ggctggcccc ggtgcatttt ttaaataccc 3600gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc 3660gggtggtgct caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga 3720cgctaatccc taactgctgg cggaaaagat gtgacagacg cgacggcgac aagcaaacat 3780gctgtgcgac gctggcgata tcaaaattgc tgtctgccag gtgatcgctg atgtactgac 3840aagcctcgcg tacccgatta tccatcggtg gatggagcga ctcgttaatc gcttccatgc 3900gccgcagtaa caattgctca agcagattta tcgccagcag ctccgaatag cgcccttccc 3960cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat 4020ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca gttaagccat tcatgccagt 4080aggcgcgcgg acgaaagtaa acccactggt gataccattc gcgagcctcc ggatgacgac 4140cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt 4200ctcgtccctg atttttcacc accccctgac cgcgaatggt gagattgaga atataacctt 4260tcattcccag cggtcggtcg ataaaaaaat cgagataacc gttggcctca atcggcgtta 4320aacccgccac cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt 4380cagccatact tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca 4440gacattgccg tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaaccccgc 4500ttattaaaag cattctgtaa caaagcggga ccaaagccat gacaaaaacg cgtaacaaaa 4560gtgtctataa tcacggcaga aaagtccaca ttgattattt gcacggcgtc acactttgct 4620atgccatagc atttttatcc ataagattag cggatcctac ctgacgcttt ttatcgcaac 4680tctctactgt ttctccatac ccgttttttg ggctaacagg aggaattaac 47301014745DNAArtificial sequencepRF213 101catggggcat catcatcacc atcacggcgc cctgttctta ggccagctgg gcgccgcggg 60atccacgatg ggtgcgccga agaaaaagcg caaagttgaa ttcggaggtg gcggtgcatc 120ctcggaggat gtgattaaag aatttatgcg gtttaaagta cgtatggaag gatcggtgaa 180tggccatgaa tttgagattg agggtgaagg cgaaggccgc ccgtacgaag gaactcaaac 240agcgaaatta aaagttacaa aaggaggtcc tctgccgttt gcctgggaca tcttgagccc 300gcaattccag tacggttcca aagtgtatgt aaaacaccct gcggatattc cggattataa 360aaaactgagt tttcccgagg ggtttaaatg ggaacgggtg atgaattttg aggatggtgg 420agttgtcacc gtgacccagg actctagctt acaagacggt agtttcatct acaaagtaaa 480atttatcggc gtaaacttcc catcggacgg ccccgtcatg cagaaaaaga cgatgggctg 540ggaagccagc accgaacgtt tgtacccacg ggacggcgtt ttgaaagggg aaatccataa 600ggcccttaaa ctgaaagacg gtggtcacta tctcgtggag tttaaatcga tttatatggc 660taaaaaacca gtacagcttc cgggttatta ttacgttgac tccaaattgg acatcacatc 720gcataatgaa gattacacga ttgttgaaca gtacgagcgc gccgagggcc ggcaccatct 780gtttctgtaa aagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat 840taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt 900ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt 960ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt 1020cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga 1080caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag 1140gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc 1200tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg 1260ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt 1320attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt 1380gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg 1440ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa 1500cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt 1560gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag 1620tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt 1680gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga 1740ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt 1800tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta 1860gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 1920caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 1980cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt 2040atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 2100gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 2160attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 2220cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 2280atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 2340tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 2400ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 2460ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 2520cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 2580gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2640gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2700acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2760gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 2820agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2880tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2940agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 3000cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc 3060gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc 3120ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact 3180ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac 3240gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 3300cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 3360gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga 3420aggcgaagcg gcatgcataa tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc 3480tatgctactc cgtcaagccg tcaattgtct gattcgttac caattatgac aacttgacgg 3540ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg gccccggtgc 3600attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg cgaccgacgg 3660tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct 3720cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac agacgcgacg 3780gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct gccaggtgat 3840cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg agcgactcgt 3900taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc agcagctccg 3960aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg 4020gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac ggccagttaa 4080gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac cattcgcgag 4140cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa atatcacccg 4200gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga atggtgagat 4260tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg 4320cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc ggcagcaggg 4380gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa gaaaccaatt 4440gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc tcgctaacca 4500aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa gccatgacaa 4560aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat tatttgcacg 4620gcgtcacact ttgctatgcc atagcatttt tatccataag attagcggat cctacctgac 4680gctttttatc gcaactctct actgtttctc catacccgtt ttttgggcta acaggaggaa 4740ttaac 47451024736DNAArtificial sequencepRF217 102catggggcac catcaccatc accataaaga aacttggtgg gagacttggt ggaccgaatg 60gtcccagccg aagaaaaaac gcaaggttga attcggaggt ggcggtgcat cctcggagga 120tgtgattaaa gaatttatgc ggtttaaagt acgtatggaa ggatcggtga atggccatga 180atttgagatt gagggtgaag gcgaaggccg cccgtacgaa ggaactcaaa cagcgaaatt 240aaaagttaca aaaggaggtc ctctgccgtt tgcctgggac atcttgagcc cgcaattcca 300gtacggttcc aaagtgtatg taaaacaccc tgcggatatt ccggattata aaaaactgag 360ttttcccgag gggtttaaat gggaacgggt gatgaatttt gaggatggtg gagttgtcac 420cgtgacccag gactctagct tacaagacgg tagtttcatc tacaaagtaa aatttatcgg 480cgtaaacttc ccatcggacg gccccgtcat gcagaaaaag acgatgggct gggaagccag 540caccgaacgt ttgtacccac gggacggcgt tttgaaaggg gaaatccata aggcccttaa 600actgaaagac ggtggtcact atctcgtgga gtttaaatcg atttatatgg ctaaaaaacc 660agtacagctt ccgggttatt attacgttga ctccaaattg gacatcacat cgcataatga 720agattacacg attgttgaac agtacgagcg cgccgagggc cggcaccatc tgtttctgta 780aaagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga 840acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc 900tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc 960ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact 1020gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc 1080cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc 1140cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt 1200ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 1260caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 1320ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 1380gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 1440gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 1500atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg 1560caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 1620gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 1680accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 1740ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 1800gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 1860acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 1920atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 1980ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca 2040gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 2100gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 2160tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 2220taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 2280cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 2340gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 2400gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 2460agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 2520aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 2580agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 2640cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 2700accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 2760aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 2820ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 2880cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 2940gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta 3000tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc 3060agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg 3120tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca 3180atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta cgtgactggg 3240tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 3300tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 3360tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg aaggcgaagc 3420ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc ctatgctact 3480ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg gctacatcat 3540tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg cattttttaa 3600atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag 3660gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc tcgcgccagc 3720ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac ggcgacaagc 3780aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt 3840actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg ttaatcgctt 3900ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc gaatagcgcc 3960cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg 4020cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta agccattcat 4080gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga gcctccggat 4140gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa 4200caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat 4260aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg 4320gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg ggatcatttt 4380gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat tgtccatatt 4440gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc aaaccggtaa 4500ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta 4560acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac ggcgtcacac 4620tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga cgctttttat 4680cgcaactctc tactgtttct ccatacccgt tttttgggct aacaggagga attaac 47361034694DNAArtificial sequencepRF216 103catggggcat caccatcacc accattgttt tttcaaagac gaactggaat tcggaggtgg 60cggtgcatcc tcggaggatg tgattaaaga atttatgcgg tttaaagtac gtatggaagg 120atcggtgaat ggccatgaat ttgagattga gggtgaaggc gaaggccgcc cgtacgaagg 180aactcaaaca gcgaaattaa aagttacaaa aggaggtcct ctgccgtttg cctgggacat 240cttgagcccg caattccagt acggttccaa agtgtatgta aaacaccctg cggatattcc 300ggattataaa aaactgagtt ttcccgaggg gtttaaatgg gaacgggtga tgaattttga 360ggatggtgga gttgtcaccg tgacccagga ctctagctta caagacggta gtttcatcta 420caaagtaaaa tttatcggcg taaacttccc atcggacggc cccgtcatgc agaaaaagac 480gatgggctgg gaagccagca ccgaacgttt gtacccacgg gacggcgttt tgaaagggga 540aatccataag gcccttaaac tgaaagacgg tggtcactat ctcgtggagt ttaaatcgat 600ttatatggct aaaaaaccag tacagcttcc gggttattat tacgttgact ccaaattgga 660catcacatcg cataatgaag attacacgat tgttgaacag tacgagcgcg ccgagggccg 720gcaccatctg tttctgtaaa agcttggctg ttttggcgga tgagagaaga ttttcagcct 780gatacagatt aaatcagaac gcagaagcgg tctgataaaa cagaatttgc ctggcggcag 840tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga 900tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa 960aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc 1020tgagtaggac aaatccgccg ggagcggatt tgaacgttgc gaagcaacgg cccggagggt 1080ggcgggcagg acgcccgcca taaactgcca ggcatcaaat taagcagaag gccatcctga 1140cggatggcct ttttgcgttt ctacaaactc ttttgtttat ttttctaaat acattcaaat 1200atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 1260agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 1320cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 1380gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 1440cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 1500tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 1560ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 1620ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 1680atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 1740cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 1800atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 1860gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 1920cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 1980tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 2040tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 2100gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 2160gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 2220atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 2280atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 2340aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 2400aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 2460ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 2520ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 2580tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 2640ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 2700acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt

cggaacagga 2760gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 2820cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 2880aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 2940atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 3000gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 3060gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 3120tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc 3180tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc 3240cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 3300gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag cagatcaatt 3360cgcgcgcgaa ggcgaagcgg catgcataat gtgcctgtca aatggacgaa gcagggattc 3420tgcaaaccct atgctactcc gtcaagccgt caattgtctg attcgttacc aattatgaca 3480acttgacggc tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg 3540ccccggtgca ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc 3600gaccgacggt ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac 3660gttggtcctc gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca 3720gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg 3780ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg attatccatc ggtggatgga 3840gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga tttatcgcca 3900gcagctccga atagcgccct tccccttgcc cggcgttaat gatttgccca aacaggtcgc 3960tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc cgtattggca aatattgacg 4020gccagttaag ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc 4080attcgcgagc ctccggatga cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa 4140tatcacccgg tcggcaaaca aattctcgtc cctgattttt caccaccccc tgaccgcgaa 4200tggtgagatt gagaatataa cctttcattc ccagcggtcg gtcgataaaa aaatcgagat 4260aaccgttggc ctcaatcggc gttaaacccg ccaccagatg ggcattaaac gagtatcccg 4320gcagcagggg atcattttgc gcttcagcca tacttttcat actcccgcca ttcagagaag 4380aaaccaattg tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct 4440cgctaaccaa accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag 4500ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt 4560atttgcacgg cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggatc 4620ctacctgacg ctttttatcg caactctcta ctgtttctcc atacccgttt tttgggctaa 4680caggaggaat taac 469410423DNAArtificial sequenceoligo 36 104ccataagatt agcggatcct acc 23105331DNAArtificial sequenceHis-Zebra PCR 105ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat 60acccgttttt tgggctaaca ggaggaatta accatggggc atcaccacca tcaccacgaa 120tgcgactcag aactggaaat caaacgctat aaacgtgtgc gtgtggcatc ccgtaaatgt 180cgcgcaaagt ttaaacagct gctgcaacat tatcgtgaag tagcggctgc gaaaagctcc 240gaaaacgacc gtttacgcct cctcctgaag caaatgtgcg aattcgacaa gaaatactcc 300atcggcctgg acattggaac caactctgtc g 331106232DNAArtificial sequenceHis-tp10 PCR 106ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat 60acccgttttt tgggctaaca ggaggaatta accatggggc atcaccacca tcaccacgcg 120ggttacctgc tgggcaagat taatcttaaa gcctgcgccg cgtgtgctaa gaaaattttg 180gaattcgaca agaaatactc catcggcctg gacattggaa ccaactctgt cg 232107223DNAartificial sequenceHis-pVEC PCR 107ccataagatt agcggatcct acctgacgct ttttatcgca actctctact gtttctccat 60acccgttttt tgggctaaca ggaggaatta accatggggc atcaccacca tcaccactta 120ttgattatct tgcgtcgtcg catccgcaaa caggcgcacg cacatagcaa ggaattcgac 180aagaaatact ccatcggcct ggacattgga accaactctg tcg 2231088294DNAArtificial sequencepRF144 108ccatggggca tcaccaccat caccacgaat gcgactcaga actggaaatc aaacgctata 60aacgtgtgcg tgtggcatcc cgtaaatgtc gcgcaaagtt taaacagctg ctgcaacatt 120atcgtgaagt agcggctgcg aaaagctccg aaaacgaccg tttacgcctc ctcctgaagc 180aaatgtgcga attcgacaag aaatactcca tcggcctgga cattggaacc aactctgtcg 240gctgggctgt catcaccgac gagtacaagg tgccctccaa gaaattcaag gtcctcggaa 300acaccgatcg acactccatc aagaaaaacc tcattggtgc cctgttgttc gattctggcg 360agactgccga agctaccaga ctcaagcgaa ctgctcggcg acgttacacc cgacggaaga 420accgaatctg ctacctgcag gagatctttt ccaacgagat ggccaaggtg gacgattcgt 480tctttcatcg actggaggaa tccttcctcg tcgaggaaga caagaaacac gagcgtcatc 540ccatctttgg caacattgtg gacgaggttg cttaccacga gaagtatcct accatctacc 600acctgcgaaa gaaactcgtc gattccaccg acaaggcgga tctcagactt atctacctcg 660ctctggcaca catgatcaag tttcgaggtc atttcctcat cgagggcgat ctcaatcccg 720acaacagcga tgtggacaag ctgttcattc agctcgttca gacctacaac cagctgttcg 780aggaaaaccc catcaatgcc tccggagtcg atgcaaaggc catcttgtct gctcgactct 840cgaagagcag acgactggag aacctcattg cccaacttcc tggcgagaaa aagaacggac 900tgtttggcaa cctcattgcc ctttctcttg gtctcacacc caacttcaag tccaacttcg 960atctggcgga ggacgccaag ctccagctgt ccaaggacac ctacgacgat gacctcgaca 1020acctgcttgc acagattggc gatcagtacg ccgacctgtt tctcgctgcc aagaaccttt 1080cggatgctat tctcttgtct gacattctgc gagtcaacac cgagatcaca aaggctcccc 1140tttctgcctc catgatcaag cgatacgacg agcaccatca ggatctcaca ctgctcaagg 1200ctcttgtccg acagcaactg cccgagaagt acaaggagat ctttttcgat cagtcgaaga 1260acggctacgc tggatacatc gacggcggag cctctcagga agagttctac aagttcatca 1320agccaattct cgagaagatg gacggaaccg aggaactgct tgtcaagctc aatcgagagg 1380atctgcttcg gaagcaacga accttcgaca acggcagcat tcctcatcag atccacctcg 1440gtgagctgca cgccattctt cgacgtcagg aagacttcta cccctttctc aaggacaacc 1500gagagaagat cgagaagatt cttacctttc gaatccccta ctatgttggt cctcttgcca 1560gaggaaactc tcgatttgct tggatgactc gaaagtccga ggaaaccatc actccctgga 1620acttcgagga agtcgtggac aagggtgcct ctgcacagtc cttcatcgag cgaatgacca 1680acttcgacaa gaatctgccc aacgagaagg ttcttcccaa gcattcgctg ctctacgagt 1740actttacagt ctacaacgaa ctcaccaaag tcaagtacgt taccgaggga atgcgaaagc 1800ctgccttctt gtctggcgaa cagaagaaag ccattgtcga tctcctgttc aagaccaacc 1860gaaaggtcac tgttaagcag ctcaaggagg actacttcaa gaaaatcgag tgtttcgaca 1920gcgtcgagat ttccggagtt gaggaccgat tcaacgcctc tttgggcacc tatcacgatc 1980tgctcaagat tatcaaggac aaggattttc tcgacaacga ggaaaacgag gacattctgg 2040aggacatcgt gctcactctt accctgttcg aagatcggga gatgatcgag gaacgactca 2100agacatacgc tcacctgttc gacgacaagg tcatgaaaca actcaagcga cgtagataca 2160ccggctgggg aagactttcg cgaaagctca tcaacggcat cagagacaag cagtccggaa 2220agaccattct ggactttctc aagtccgatg gctttgccaa ccgaaacttc atgcagctca 2280ttcacgacga ttctcttacc ttcaaggagg acatccagaa ggcacaagtg tccggtcagg 2340gcgacagctt gcacgaacat attgccaacc tggctggttc gccagccatc aagaaaggca 2400ttctccagac tgtcaaggtt gtcgacgagc tggtgaaggt catgggacgt cacaagcccg 2460agaacattgt gatcgagatg gccagagaga accagacaac tcaaaagggt cagaaaaact 2520cgcgagagcg gatgaagcga atcgaggaag gcatcaagga gctgggatcc cagattctca 2580aggagcatcc cgtcgagaac actcaactgc agaacgagaa gctgtatctc tactatctgc 2640agaatggtcg agacatgtac gtggatcagg aactggacat caatcgtctc agcgactacg 2700atgtggacca cattgtccct caatcctttc tcaaggacga ttctatcgac aacaaggtcc 2760ttacacgatc cgacaagaac agaggcaagt cggacaacgt tcccagcgaa gaggtggtca 2820aaaagatgaa gaactactgg cgacagctgc tcaacgccaa gctcattacc cagcgaaagt 2880tcgacaatct taccaaggcc gagcgaggcg gtctgtccga gctcgacaag gctggcttca 2940tcaagcgtca actcgtcgag accagacaga tcacaaagca cgtcgcacag attctcgatt 3000ctcggatgaa caccaagtac gacgagaacg acaagctcat ccgagaggtc aaggtgatta 3060ctctcaagtc caaactggtc tccgatttcc gaaaggactt tcagttctac aaggtgcgag 3120agatcaacaa ttaccaccat gcccacgatg cttacctcaa cgccgtcgtt ggcactgcgc 3180tcatcaagaa ataccccaag ctcgaaagcg agttcgttta cggcgattac aaggtctacg 3240acgttcgaaa gatgattgcc aagtccgaac aggagattgg caaggctact gccaagtact 3300tcttttactc caacatcatg aactttttca agaccgagat caccttggcc aacggagaga 3360ttcgaaagag accacttatc gagaccaacg gcgaaactgg agagatcgtg tgggacaagg 3420gtcgagactt tgcaaccgtg cgaaaggttc tgtcgatgcc tcaggtcaac atcgtcaaga 3480aaaccgaggt tcagactggc ggattctcca aggagtcgat tctgcccaag cgaaactccg 3540acaagctcat cgctcgaaag aaagactggg atcccaagaa atacggtggc ttcgattctc 3600ctaccgtcgc ctattccgtg cttgtcgttg cgaaggtcga gaagggcaag tccaaaaagc 3660tcaagtccgt caaggagctg ctcggaatta ccatcatgga gcgatcgagc ttcgagaaga 3720atcccatcga cttcttggaa gccaagggtt acaaggaggt caagaaagac ctcattatca 3780agctgcccaa gtactctctg ttcgaactgg agaacggtcg aaagcgtatg ctcgcctccg 3840ctggcgagct gcagaaggga aacgagcttg ccttgccttc gaagtacgtc aactttctct 3900atctggcttc tcactacgag aagctcaagg gttctcccga ggacaacgaa cagaagcaac 3960tcttcgttga gcagcacaaa cattacctcg acgagattat cgagcagatt tccgagtttt 4020cgaagcgagt catcctggct gatgccaact tggacaaggt gctctctgcc tacaacaagc 4080atcgggacaa acccattcga gaacaggcgg agaacatcat tcacctgttt actcttacca 4140acctgggtgc tcctgcagct ttcaagtact tcgataccac tatcgaccga aagcggtaca 4200catccaccaa ggaggttctc gatgccaccc tgattcacca gtccatcact ggcctgtacg 4260agacccgaat cgacctgtct cagcttggtg gcgactccag agccgatccc aagaaaaagc 4320gaaaggtcta agcggccgct aagcttggct gttttggcgg atgagagaag attttcagcc 4380tgatacagat taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca 4440gtagcgcggt ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg 4500atggtagtgt ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga 4560aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 4620ctgagtagga caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg 4680tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg 4740acggatggcc tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa 4800tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 4860gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 4920tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 4980tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 5040ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 5100atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 5160cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 5220attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 5280gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 5340ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 5400gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 5460agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 5520gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 5580gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 5640ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 5700tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 5760tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 5820catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 5880gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 5940aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 6000gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta 6060gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 6120gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 6180atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 6240cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc 6300cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 6360agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 6420tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 6480gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 6540catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 6600agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 6660ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 6720atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg 6780ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg 6840ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 6900agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat 6960tcgcgcgcga aggcgaagcg gcatgcataa tgtgcctgtc aaatggacga agcagggatt 7020ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct gattcgttac caattatgac 7080aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg 7140gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg 7200cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata 7260cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac 7320agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct 7380gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg 7440agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc 7500agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg 7560ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac 7620ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac 7680cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa 7740atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga 7800atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga 7860taaccgttgg cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc 7920ggcagcaggg gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa 7980gaaaccaatt gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc 8040tcgctaacca aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa 8100gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat 8160tatttgcacg gcgtcacact ttgctatgcc atagcatttt tatccataag attagcggat 8220cctacctgac gctttttatc gcaactctct actgtttctc catacccgtt ttttgggcta 8280acaggaggaa ttaa 82941098195DNAArtificial sequencepRF162 109aattcgacaa gaaatactcc atcggcctgg acattggaac caactctgtc ggctgggctg 60tcatcaccga cgagtacaag gtgccctcca agaaattcaa ggtcctcgga aacaccgatc 120gacactccat caagaaaaac ctcattggtg ccctgttgtt cgattctggc gagactgccg 180aagctaccag actcaagcga actgctcggc gacgttacac ccgacggaag aaccgaatct 240gctacctgca ggagatcttt tccaacgaga tggccaaggt ggacgattcg ttctttcatc 300gactggagga atccttcctc gtcgaggaag acaagaaaca cgagcgtcat cccatctttg 360gcaacattgt ggacgaggtt gcttaccacg agaagtatcc taccatctac cacctgcgaa 420agaaactcgt cgattccacc gacaaggcgg atctcagact tatctacctc gctctggcac 480acatgatcaa gtttcgaggt catttcctca tcgagggcga tctcaatccc gacaacagcg 540atgtggacaa gctgttcatt cagctcgttc agacctacaa ccagctgttc gaggaaaacc 600ccatcaatgc ctccggagtc gatgcaaagg ccatcttgtc tgctcgactc tcgaagagca 660gacgactgga gaacctcatt gcccaacttc ctggcgagaa aaagaacgga ctgtttggca 720acctcattgc cctttctctt ggtctcacac ccaacttcaa gtccaacttc gatctggcgg 780aggacgccaa gctccagctg tccaaggaca cctacgacga tgacctcgac aacctgcttg 840cacagattgg cgatcagtac gccgacctgt ttctcgctgc caagaacctt tcggatgcta 900ttctcttgtc tgacattctg cgagtcaaca ccgagatcac aaaggctccc ctttctgcct 960ccatgatcaa gcgatacgac gagcaccatc aggatctcac actgctcaag gctcttgtcc 1020gacagcaact gcccgagaag tacaaggaga tctttttcga tcagtcgaag aacggctacg 1080ctggatacat cgacggcgga gcctctcagg aagagttcta caagttcatc aagccaattc 1140tcgagaagat ggacggaacc gaggaactgc ttgtcaagct caatcgagag gatctgcttc 1200ggaagcaacg aaccttcgac aacggcagca ttcctcatca gatccacctc ggtgagctgc 1260acgccattct tcgacgtcag gaagacttct acccctttct caaggacaac cgagagaaga 1320tcgagaagat tcttaccttt cgaatcccct actatgttgg tcctcttgcc agaggaaact 1380ctcgatttgc ttggatgact cgaaagtccg aggaaaccat cactccctgg aacttcgagg 1440aagtcgtgga caagggtgcc tctgcacagt ccttcatcga gcgaatgacc aacttcgaca 1500agaatctgcc caacgagaag gttcttccca agcattcgct gctctacgag tactttacag 1560tctacaacga actcaccaaa gtcaagtacg ttaccgaggg aatgcgaaag cctgccttct 1620tgtctggcga acagaagaaa gccattgtcg atctcctgtt caagaccaac cgaaaggtca 1680ctgttaagca gctcaaggag gactacttca agaaaatcga gtgtttcgac agcgtcgaga 1740tttccggagt tgaggaccga ttcaacgcct ctttgggcac ctatcacgat ctgctcaaga 1800ttatcaagga caaggatttt ctcgacaacg aggaaaacga ggacattctg gaggacatcg 1860tgctcactct taccctgttc gaagatcggg agatgatcga ggaacgactc aagacatacg 1920ctcacctgtt cgacgacaag gtcatgaaac aactcaagcg acgtagatac accggctggg 1980gaagactttc gcgaaagctc atcaacggca tcagagacaa gcagtccgga aagaccattc 2040tggactttct caagtccgat ggctttgcca accgaaactt catgcagctc attcacgacg 2100attctcttac cttcaaggag gacatccaga aggcacaagt gtccggtcag ggcgacagct 2160tgcacgaaca tattgccaac ctggctggtt cgccagccat caagaaaggc attctccaga 2220ctgtcaaggt tgtcgacgag ctggtgaagg tcatgggacg tcacaagccc gagaacattg 2280tgatcgagat ggccagagag aaccagacaa ctcaaaaggg tcagaaaaac tcgcgagagc 2340ggatgaagcg aatcgaggaa ggcatcaagg agctgggatc ccagattctc aaggagcatc 2400ccgtcgagaa cactcaactg cagaacgaga agctgtatct ctactatctg cagaatggtc 2460gagacatgta cgtggatcag gaactggaca tcaatcgtct cagcgactac gatgtggacc 2520acattgtccc tcaatccttt ctcaaggacg attctatcga caacaaggtc cttacacgat 2580ccgacaagaa cagaggcaag tcggacaacg ttcccagcga agaggtggtc aaaaagatga 2640agaactactg gcgacagctg ctcaacgcca agctcattac ccagcgaaag ttcgacaatc 2700ttaccaaggc cgagcgaggc ggtctgtccg agctcgacaa ggctggcttc atcaagcgtc 2760aactcgtcga gaccagacag atcacaaagc acgtcgcaca gattctcgat tctcggatga 2820acaccaagta cgacgagaac gacaagctca tccgagaggt caaggtgatt actctcaagt 2880ccaaactggt ctccgatttc cgaaaggact ttcagttcta caaggtgcga gagatcaaca 2940attaccacca tgcccacgat gcttacctca acgccgtcgt tggcactgcg ctcatcaaga 3000aataccccaa gctcgaaagc gagttcgttt acggcgatta caaggtctac gacgttcgaa 3060agatgattgc caagtccgaa caggagattg gcaaggctac tgccaagtac ttcttttact 3120ccaacatcat gaactttttc aagaccgaga tcaccttggc caacggagag attcgaaaga 3180gaccacttat cgagaccaac ggcgaaactg gagagatcgt gtgggacaag ggtcgagact 3240ttgcaaccgt gcgaaaggtt ctgtcgatgc ctcaggtcaa catcgtcaag aaaaccgagg 3300ttcagactgg cggattctcc aaggagtcga ttctgcccaa gcgaaactcc gacaagctca 3360tcgctcgaaa gaaagactgg gatcccaaga aatacggtgg cttcgattct cctaccgtcg 3420cctattccgt gcttgtcgtt gcgaaggtcg agaagggcaa gtccaaaaag ctcaagtccg 3480tcaaggagct gctcggaatt accatcatgg agcgatcgag cttcgagaag aatcccatcg 3540acttcttgga agccaagggt tacaaggagg tcaagaaaga cctcattatc

aagctgccca 3600agtactctct gttcgaactg gagaacggtc gaaagcgtat gctcgcctcc gctggcgagc 3660tgcagaaggg aaacgagctt gccttgcctt cgaagtacgt caactttctc tatctggctt 3720ctcactacga gaagctcaag ggttctcccg aggacaacga acagaagcaa ctcttcgttg 3780agcagcacaa acattacctc gacgagatta tcgagcagat ttccgagttt tcgaagcgag 3840tcatcctggc tgatgccaac ttggacaagg tgctctctgc ctacaacaag catcgggaca 3900aacccattcg agaacaggcg gagaacatca ttcacctgtt tactcttacc aacctgggtg 3960ctcctgcagc tttcaagtac ttcgatacca ctatcgaccg aaagcggtac acatccacca 4020aggaggttct cgatgccacc ctgattcacc agtccatcac tggcctgtac gagacccgaa 4080tcgacctgtc tcagcttggt ggcgactcca gagccgatcc caagaaaaag cgaaaggtct 4140aagcggccgc taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga 4200ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 4260tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 4320tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 4380tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg 4440acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca 4500ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc 4560ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc 4620gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 4680tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 4740tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4800gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4860acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 4920tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4980gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 5040tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 5100accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 5160ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 5220agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 5280gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 5340ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 5400tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 5460ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 5520gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 5580acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 5640aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 5700atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 5760gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5820tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5880ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5940ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 6000ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 6060aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 6120cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 6180gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 6240ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 6300cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 6360tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 6420cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 6480cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 6540tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 6600cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6660gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6720tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg 6780aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc 6840ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg 6900gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg 6960cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg 7020gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc 7080tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac 7140ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga 7200tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg 7260ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc 7320gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc 7380ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta 7440agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga 7500gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc 7560ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga 7620ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg 7680gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg 7740ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat 7800tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc 7860aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca 7920aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac 7980ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga 8040cgctttttat cgcaactctc tactgtttct ccatacccgt tttttgggct aacaggagga 8100attaaccatg gggcatcacc accatcacca cgcgggttac ctgctgggca agattaatct 8160taaagcctgc gccgcgtgtg ctaagaaaat tttgg 81951108186DNAArtificial SequencepRF146 110aattcgacaa gaaatactcc atcggcctgg acattggaac caactctgtc ggctgggctg 60tcatcaccga cgagtacaag gtgccctcca agaaattcaa ggtcctcgga aacaccgatc 120gacactccat caagaaaaac ctcattggtg ccctgttgtt cgattctggc gagactgccg 180aagctaccag actcaagcga actgctcggc gacgttacac ccgacggaag aaccgaatct 240gctacctgca ggagatcttt tccaacgaga tggccaaggt ggacgattcg ttctttcatc 300gactggagga atccttcctc gtcgaggaag acaagaaaca cgagcgtcat cccatctttg 360gcaacattgt ggacgaggtt gcttaccacg agaagtatcc taccatctac cacctgcgaa 420agaaactcgt cgattccacc gacaaggcgg atctcagact tatctacctc gctctggcac 480acatgatcaa gtttcgaggt catttcctca tcgagggcga tctcaatccc gacaacagcg 540atgtggacaa gctgttcatt cagctcgttc agacctacaa ccagctgttc gaggaaaacc 600ccatcaatgc ctccggagtc gatgcaaagg ccatcttgtc tgctcgactc tcgaagagca 660gacgactgga gaacctcatt gcccaacttc ctggcgagaa aaagaacgga ctgtttggca 720acctcattgc cctttctctt ggtctcacac ccaacttcaa gtccaacttc gatctggcgg 780aggacgccaa gctccagctg tccaaggaca cctacgacga tgacctcgac aacctgcttg 840cacagattgg cgatcagtac gccgacctgt ttctcgctgc caagaacctt tcggatgcta 900ttctcttgtc tgacattctg cgagtcaaca ccgagatcac aaaggctccc ctttctgcct 960ccatgatcaa gcgatacgac gagcaccatc aggatctcac actgctcaag gctcttgtcc 1020gacagcaact gcccgagaag tacaaggaga tctttttcga tcagtcgaag aacggctacg 1080ctggatacat cgacggcgga gcctctcagg aagagttcta caagttcatc aagccaattc 1140tcgagaagat ggacggaacc gaggaactgc ttgtcaagct caatcgagag gatctgcttc 1200ggaagcaacg aaccttcgac aacggcagca ttcctcatca gatccacctc ggtgagctgc 1260acgccattct tcgacgtcag gaagacttct acccctttct caaggacaac cgagagaaga 1320tcgagaagat tcttaccttt cgaatcccct actatgttgg tcctcttgcc agaggaaact 1380ctcgatttgc ttggatgact cgaaagtccg aggaaaccat cactccctgg aacttcgagg 1440aagtcgtgga caagggtgcc tctgcacagt ccttcatcga gcgaatgacc aacttcgaca 1500agaatctgcc caacgagaag gttcttccca agcattcgct gctctacgag tactttacag 1560tctacaacga actcaccaaa gtcaagtacg ttaccgaggg aatgcgaaag cctgccttct 1620tgtctggcga acagaagaaa gccattgtcg atctcctgtt caagaccaac cgaaaggtca 1680ctgttaagca gctcaaggag gactacttca agaaaatcga gtgtttcgac agcgtcgaga 1740tttccggagt tgaggaccga ttcaacgcct ctttgggcac ctatcacgat ctgctcaaga 1800ttatcaagga caaggatttt ctcgacaacg aggaaaacga ggacattctg gaggacatcg 1860tgctcactct taccctgttc gaagatcggg agatgatcga ggaacgactc aagacatacg 1920ctcacctgtt cgacgacaag gtcatgaaac aactcaagcg acgtagatac accggctggg 1980gaagactttc gcgaaagctc atcaacggca tcagagacaa gcagtccgga aagaccattc 2040tggactttct caagtccgat ggctttgcca accgaaactt catgcagctc attcacgacg 2100attctcttac cttcaaggag gacatccaga aggcacaagt gtccggtcag ggcgacagct 2160tgcacgaaca tattgccaac ctggctggtt cgccagccat caagaaaggc attctccaga 2220ctgtcaaggt tgtcgacgag ctggtgaagg tcatgggacg tcacaagccc gagaacattg 2280tgatcgagat ggccagagag aaccagacaa ctcaaaaggg tcagaaaaac tcgcgagagc 2340ggatgaagcg aatcgaggaa ggcatcaagg agctgggatc ccagattctc aaggagcatc 2400ccgtcgagaa cactcaactg cagaacgaga agctgtatct ctactatctg cagaatggtc 2460gagacatgta cgtggatcag gaactggaca tcaatcgtct cagcgactac gatgtggacc 2520acattgtccc tcaatccttt ctcaaggacg attctatcga caacaaggtc cttacacgat 2580ccgacaagaa cagaggcaag tcggacaacg ttcccagcga agaggtggtc aaaaagatga 2640agaactactg gcgacagctg ctcaacgcca agctcattac ccagcgaaag ttcgacaatc 2700ttaccaaggc cgagcgaggc ggtctgtccg agctcgacaa ggctggcttc atcaagcgtc 2760aactcgtcga gaccagacag atcacaaagc acgtcgcaca gattctcgat tctcggatga 2820acaccaagta cgacgagaac gacaagctca tccgagaggt caaggtgatt actctcaagt 2880ccaaactggt ctccgatttc cgaaaggact ttcagttcta caaggtgcga gagatcaaca 2940attaccacca tgcccacgat gcttacctca acgccgtcgt tggcactgcg ctcatcaaga 3000aataccccaa gctcgaaagc gagttcgttt acggcgatta caaggtctac gacgttcgaa 3060agatgattgc caagtccgaa caggagattg gcaaggctac tgccaagtac ttcttttact 3120ccaacatcat gaactttttc aagaccgaga tcaccttggc caacggagag attcgaaaga 3180gaccacttat cgagaccaac ggcgaaactg gagagatcgt gtgggacaag ggtcgagact 3240ttgcaaccgt gcgaaaggtt ctgtcgatgc ctcaggtcaa catcgtcaag aaaaccgagg 3300ttcagactgg cggattctcc aaggagtcga ttctgcccaa gcgaaactcc gacaagctca 3360tcgctcgaaa gaaagactgg gatcccaaga aatacggtgg cttcgattct cctaccgtcg 3420cctattccgt gcttgtcgtt gcgaaggtcg agaagggcaa gtccaaaaag ctcaagtccg 3480tcaaggagct gctcggaatt accatcatgg agcgatcgag cttcgagaag aatcccatcg 3540acttcttgga agccaagggt tacaaggagg tcaagaaaga cctcattatc aagctgccca 3600agtactctct gttcgaactg gagaacggtc gaaagcgtat gctcgcctcc gctggcgagc 3660tgcagaaggg aaacgagctt gccttgcctt cgaagtacgt caactttctc tatctggctt 3720ctcactacga gaagctcaag ggttctcccg aggacaacga acagaagcaa ctcttcgttg 3780agcagcacaa acattacctc gacgagatta tcgagcagat ttccgagttt tcgaagcgag 3840tcatcctggc tgatgccaac ttggacaagg tgctctctgc ctacaacaag catcgggaca 3900aacccattcg agaacaggcg gagaacatca ttcacctgtt tactcttacc aacctgggtg 3960ctcctgcagc tttcaagtac ttcgatacca ctatcgaccg aaagcggtac acatccacca 4020aggaggttct cgatgccacc ctgattcacc agtccatcac tggcctgtac gagacccgaa 4080tcgacctgtc tcagcttggt ggcgactcca gagccgatcc caagaaaaag cgaaaggtct 4140aagcggccgc taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga 4200ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 4260tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 4320tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 4380tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg 4440acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca 4500ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc 4560ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc 4620gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 4680tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 4740tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4800gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4860acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 4920tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4980gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 5040tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 5100accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 5160ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 5220agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 5280gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 5340ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 5400tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 5460ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 5520gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 5580acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 5640aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 5700atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 5760gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5820tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5880ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5940ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 6000ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 6060aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 6120cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 6180gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 6240ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 6300cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 6360tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 6420cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 6480cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 6540tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 6600cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6660gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6720tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg 6780aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc 6840ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg 6900gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg 6960cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg 7020gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc 7080tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac 7140ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga 7200tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg 7260ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc 7320gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc 7380ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta 7440agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga 7500gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc 7560ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga 7620ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg 7680gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg 7740ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat 7800tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc 7860aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca 7920aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac 7980ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga 8040cgctttttat cgcaactctc tactgtttct ccatacccgt tttttgggct aacaggagga 8100attaaccatg gggcatcacc accatcacca cttattgatt atcttgcgtc gtcgcatccg 8160caaacaggcg cacgcacata gcaagg 818611120DNAArtificial sequencEoligo 153 111cgacagagtt ggttccaatg 201124835DNAArtificial sequencepRF186 112catggggcat caccaccatc accacgaatg cgactcagaa ctggaaatca aacgctataa 60acgtgtgcgt gtggcatccc gtaaatgtcg cgcaaagttt aaacagctgc tgcaacatta 120tcgtgaagta gcggctgcga aaagctccga aaacgaccgt ttacgcctcc tcctgaagca 180aatgtgcgaa ttcggaggtg gcggtgcatc ctcggaggat gtgattaaag aatttatgcg 240gtttaaagta cgtatggaag gatcggtgaa tggccatgaa tttgagattg agggtgaagg 300cgaaggccgc ccgtacgaag gaactcaaac agcgaaatta aaagttacaa aaggaggtcc 360tctgccgttt gcctgggaca tcttgagccc gcaattccag tacggttcca aagtgtatgt 420aaaacaccct gcggatattc cggattataa aaaactgagt tttcccgagg ggtttaaatg 480ggaacgggtg atgaattttg aggatggtgg agttgtcacc gtgacccagg actctagctt 540acaagacggt agtttcatct acaaagtaaa atttatcggc gtaaacttcc catcggacgg 600ccccgtcatg cagaaaaaga cgatgggctg ggaagccagc accgaacgtt tgtacccacg 660ggacggcgtt ttgaaagggg aaatccataa ggcccttaaa ctgaaagacg gtggtcacta 720tctcgtggag tttaaatcga tttatatggc taaaaaacca gtacagcttc cgggttatta 780ttacgttgac tccaaattgg acatcacatc gcataatgaa gattacacga ttgttgaaca 840gtacgagcgc gccgagggcc ggcaccatct gtttctgtaa aagcttggct gttttggcgg 900atgagagaag attttcagcc tgatacagat taaatcagaa cgcagaagcg gtctgataaa 960acagaatttg cctggcggca gtagcgcggt ggtcccacct gaccccatgc cgaactcaga 1020agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc catgcgagag tagggaactg 1080ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt 1140gtttgtcggt gaacgctctc ctgagtagga caaatccgcc gggagcggat ttgaacgttg 1200cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc ataaactgcc aggcatcaaa 1260ttaagcagaa ggccatcctg acggatggcc tttttgcgtt tctacaaact cttttgttta 1320tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 1380caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 1440ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 1500gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 1560aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 1620ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc 1680atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 1740gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 1800gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 1860atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 1920aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 1980actggcgaac tacttactct agcttcccgg caacaattaa tagactggat

ggaggcggat 2040aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 2100tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 2160ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 2220agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 2280tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 2340aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 2400gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 2460atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 2520gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 2580gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 2640tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 2700accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 2760ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 2820cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 2880agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 2940ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 3000tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 3060ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 3120cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 3180gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg 3240tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga tgccgcatag 3300ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc 3360cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 3420aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 3480gcgcgaggca gcagatcaat tcgcgcgcga aggcgaagcg gcatgcataa tgtgcctgtc 3540aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct 3600gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct tcacaaccgg 3660cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag aaatagagtt 3720gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa 3780gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta atccctaact 3840gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt gcgacgctgg 3900cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc 3960gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc agtaacaatt 4020gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc ccggcgttaa 4080tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc 4140ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg cgcggacgaa 4200agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag tgatgaatct 4260ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt ccctgatttt 4320tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt cccagcggtc 4380ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc gccaccagat 4440gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttcagcc atacttttca 4500tactcccgcc attcagagaa gaaaccaatt gtccatattg catcagacat tgccgtcact 4560gcgtctttta ctggctcttc tcgctaacca aaccggtaac cccgcttatt aaaagcattc 4620tgtaacaaag cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc tataatcacg 4680gcagaaaagt ccacattgat tatttgcacg gcgtcacact ttgctatgcc atagcatttt 4740tatccataag attagcggat cctacctgac gctttttatc gcaactctct actgtttctc 4800catacccgtt ttttgggcta acaggaggaa ttaac 48351134736DNAArtificial sequencepRF192 113aattcggagg tggcggtgca tcctcggagg atgtgattaa agaatttatg cggtttaaag 60tacgtatgga aggatcggtg aatggccatg aatttgagat tgagggtgaa ggcgaaggcc 120gcccgtacga aggaactcaa acagcgaaat taaaagttac aaaaggaggt cctctgccgt 180ttgcctggga catcttgagc ccgcaattcc agtacggttc caaagtgtat gtaaaacacc 240ctgcggatat tccggattat aaaaaactga gttttcccga ggggtttaaa tgggaacggg 300tgatgaattt tgaggatggt ggagttgtca ccgtgaccca ggactctagc ttacaagacg 360gtagtttcat ctacaaagta aaatttatcg gcgtaaactt cccatcggac ggccccgtca 420tgcagaaaaa gacgatgggc tgggaagcca gcaccgaacg tttgtaccca cgggacggcg 480ttttgaaagg ggaaatccat aaggccctta aactgaaaga cggtggtcac tatctcgtgg 540agtttaaatc gatttatatg gctaaaaaac cagtacagct tccgggttat tattacgttg 600actccaaatt ggacatcaca tcgcataatg aagattacac gattgttgaa cagtacgagc 660gcgccgaggg ccggcaccat ctgtttctgt aaaagcttgg ctgttttggc ggatgagaga 720agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 780tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 840gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 900caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 960gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa 1020cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag 1080aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tatttttcta 1140aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1200ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1260ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1320agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1380tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1440tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1500ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1560gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1620acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1680tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1740gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1800actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 1860aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 1920cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 1980tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 2040cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 2100tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 2160ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2220ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2280cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2340aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 2400agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2460tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2520ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2580cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 2640atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2700ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2760tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 2820gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 2880gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 2940cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 3000gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 3060ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 3120gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca 3180cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 3240accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg 3300cagcagatca attcgcgcgc gaaggcgaag cggcatgcat aatgtgcctg tcaaatggac 3360gaagcaggga ttctgcaaac cctatgctac tccgtcaagc cgtcaattgt ctgattcgtt 3420accaattatg acaacttgac ggctacatca ttcacttttt cttcacaacc ggcacggaac 3480tcgctcgggc tggccccggt gcatttttta aatacccgcg agaaatagag ttgatcgtca 3540aaaccaacat tgcgaccgac ggtggcgata ggcatccggg tggtgctcaa aagcagcttc 3600gcctggctga tacgttggtc ctcgcgccag cttaagacgc taatccctaa ctgctggcgg 3660aaaagatgtg acagacgcga cggcgacaag caaacatgct gtgcgacgct ggcgatatca 3720aaattgctgt ctgccaggtg atcgctgatg tactgacaag cctcgcgtac ccgattatcc 3780atcggtggat ggagcgactc gttaatcgct tccatgcgcc gcagtaacaa ttgctcaagc 3840agatttatcg ccagcagctc cgaatagcgc ccttcccctt gcccggcgtt aatgatttgc 3900ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg ggcgaaagaa ccccgtattg 3960gcaaatattg acggccagtt aagccattca tgccagtagg cgcgcggacg aaagtaaacc 4020cactggtgat accattcgcg agcctccgga tgacgaccgt agtgatgaat ctctcctggc 4080gggaacagca aaatatcacc cggtcggcaa acaaattctc gtccctgatt tttcaccacc 4140ccctgaccgc gaatggtgag attgagaata taacctttca ttcccagcgg tcggtcgata 4200aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac ccgccaccag atgggcatta 4260aacgagtatc ccggcagcag gggatcattt tgcgcttcag ccatactttt catactcccg 4320ccattcagag aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt 4380tactggctct tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa 4440agcgggacca aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa 4500gtccacattg attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata 4560agattagcgg atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg 4620ttttttgggc taacaggagg aattaaccat ggggcatcac caccatcacc acgcgggtta 4680cctgctgggc aagattaatc ttaaagcctg cgccgcgtgt gctaagaaaa ttttgg 47361144727DNAArtificial sequencepRF190 114aattcggagg tggcggtgca tcctcggagg atgtgattaa agaatttatg cggtttaaag 60tacgtatgga aggatcggtg aatggccatg aatttgagat tgagggtgaa ggcgaaggcc 120gcccgtacga aggaactcaa acagcgaaat taaaagttac aaaaggaggt cctctgccgt 180ttgcctggga catcttgagc ccgcaattcc agtacggttc caaagtgtat gtaaaacacc 240ctgcggatat tccggattat aaaaaactga gttttcccga ggggtttaaa tgggaacggg 300tgatgaattt tgaggatggt ggagttgtca ccgtgaccca ggactctagc ttacaagacg 360gtagtttcat ctacaaagta aaatttatcg gcgtaaactt cccatcggac ggccccgtca 420tgcagaaaaa gacgatgggc tgggaagcca gcaccgaacg tttgtaccca cgggacggcg 480ttttgaaagg ggaaatccat aaggccctta aactgaaaga cggtggtcac tatctcgtgg 540agtttaaatc gatttatatg gctaaaaaac cagtacagct tccgggttat tattacgttg 600actccaaatt ggacatcaca tcgcataatg aagattacac gattgttgaa cagtacgagc 660gcgccgaggg ccggcaccat ctgtttctgt aaaagcttgg ctgttttggc ggatgagaga 720agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 780tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 840gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 900caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg 960gtgaacgctc tcctgagtag gacaaatccg ccgggagcgg atttgaacgt tgcgaagcaa 1020cggcccggag ggtggcgggc aggacgcccg ccataaactg ccaggcatca aattaagcag 1080aaggccatcc tgacggatgg cctttttgcg tttctacaaa ctcttttgtt tatttttcta 1140aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1200ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1260ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1320agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1380tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 1440tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 1500ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 1560gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 1620acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 1680tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 1740gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 1800actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 1860aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 1920cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 1980tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 2040cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 2100tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 2160ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2220ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2280cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2340aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 2400agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 2460tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 2520ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 2580cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 2640atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 2700ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 2760tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 2820gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 2880gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 2940cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 3000gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 3060ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 3120gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca 3180cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 3240accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg 3300cagcagatca attcgcgcgc gaaggcgaag cggcatgcat aatgtgcctg tcaaatggac 3360gaagcaggga ttctgcaaac cctatgctac tccgtcaagc cgtcaattgt ctgattcgtt 3420accaattatg acaacttgac ggctacatca ttcacttttt cttcacaacc ggcacggaac 3480tcgctcgggc tggccccggt gcatttttta aatacccgcg agaaatagag ttgatcgtca 3540aaaccaacat tgcgaccgac ggtggcgata ggcatccggg tggtgctcaa aagcagcttc 3600gcctggctga tacgttggtc ctcgcgccag cttaagacgc taatccctaa ctgctggcgg 3660aaaagatgtg acagacgcga cggcgacaag caaacatgct gtgcgacgct ggcgatatca 3720aaattgctgt ctgccaggtg atcgctgatg tactgacaag cctcgcgtac ccgattatcc 3780atcggtggat ggagcgactc gttaatcgct tccatgcgcc gcagtaacaa ttgctcaagc 3840agatttatcg ccagcagctc cgaatagcgc ccttcccctt gcccggcgtt aatgatttgc 3900ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg ggcgaaagaa ccccgtattg 3960gcaaatattg acggccagtt aagccattca tgccagtagg cgcgcggacg aaagtaaacc 4020cactggtgat accattcgcg agcctccgga tgacgaccgt agtgatgaat ctctcctggc 4080gggaacagca aaatatcacc cggtcggcaa acaaattctc gtccctgatt tttcaccacc 4140ccctgaccgc gaatggtgag attgagaata taacctttca ttcccagcgg tcggtcgata 4200aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac ccgccaccag atgggcatta 4260aacgagtatc ccggcagcag gggatcattt tgcgcttcag ccatactttt catactcccg 4320ccattcagag aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt 4380tactggctct tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa 4440agcgggacca aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa 4500gtccacattg attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata 4560agattagcgg atcctacctg acgcttttta tcgcaactct ctactgtttc tccatacccg 4620ttttttgggc taacaggagg aattaaccat ggggcatcac caccatcacc acttattgat 4680tatcttgcgt cgtcgcatcc gcaaacaggc gcacgcacat agcaagg 47271151395PRTartificial sequencehis-CFFKDEL-Cas9 115Met Gly His His His His His His Cys Phe Phe Lys Asp Glu Leu Glu1 5 10 15Phe Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 20 25 30Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 35 40 45Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 50 55 60Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu65 70 75 80Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys 85 90 95Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 100 105 110Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 115 120 125His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 130 135 140His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp145 150 155 160Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 165 170 175Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 180 185 190Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 195 200 205Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 210 215 220Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn225 230 235 240Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 245 250 255Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 260 265 270Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 275 280 285Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 290 295 300Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp305 310 315 320Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 325 330 335Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 340 345 350Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 355 360 365Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 370 375 380Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp385 390 395 400Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 405 410 415Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 420 425

430Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 435 440 445Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 450 455 460Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp465 470 475 480Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 485 490 495Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 500 505 510Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 515 520 525Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 530 535 540Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln545 550 555 560Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 565 570 575Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 580 585 590Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 595 600 605Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 610 615 620Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr625 630 635 640Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 645 650 655His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 660 665 670Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 675 680 685Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 690 695 700Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe705 710 715 720Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 725 730 735His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 740 745 750Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 755 760 765Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 770 775 780Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile785 790 795 800Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 805 810 815Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 820 825 830Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 835 840 845Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 850 855 860Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg865 870 875 880Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 885 890 895Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 900 905 910Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 915 920 925Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 930 935 940Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp945 950 955 960Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 965 970 975Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 980 985 990Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 995 1000 1005Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu 1010 1015 1020Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile 1025 1030 1035Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe 1040 1045 1050Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu 1055 1060 1065Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly 1070 1075 1080Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr 1085 1090 1095Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys 1100 1105 1110Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro 1115 1120 1125Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp 1130 1135 1140Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser 1145 1150 1155Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu 1160 1165 1170Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser 1175 1180 1185Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr 1190 1195 1200Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser 1205 1210 1215Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala 1220 1225 1230Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr 1235 1240 1245Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly 1250 1255 1260Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His 1265 1270 1275Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser 1280 1285 1290Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser 1295 1300 1305Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu 1310 1315 1320Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala 1325 1330 1335Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr 1340 1345 1350Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile 1355 1360 1365Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly 1370 1375 1380Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val 1385 1390 13951161412PRTArtificial sequencehis-MPG1-Cas9 116Met Gly His His His His His His Gly Ala Leu Phe Leu Gly Gln Leu1 5 10 15Gly Ala Ala Gly Ser Thr Met Gly Ala Pro Lys Lys Lys Arg Lys Val 20 25 30Glu Phe Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser 35 40 45Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys 50 55 60Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu65 70 75 80Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg 85 90 95Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile 100 105 110Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp 115 120 125Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys 130 135 140Lys His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala145 150 155 160Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val 165 170 175Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala 180 185 190His Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn 195 200 205Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr 210 215 220Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp225 230 235 240Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu 245 250 255Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly 260 265 270Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn 275 280 285Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr 290 295 300Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala305 310 315 320Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser 325 330 335Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala 340 345 350Ser Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu 355 360 365Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe 370 375 380Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala385 390 395 400Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met 405 410 415Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu 420 425 430Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His 435 440 445Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro 450 455 460Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg465 470 475 480Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala 485 490 495Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu 500 505 510Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met 515 520 525Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His 530 535 540Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val545 550 555 560Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu 565 570 575Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val 580 585 590Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe 595 600 605Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu 610 615 620Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu625 630 635 640Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu 645 650 655Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr 660 665 670Ala His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg 675 680 685Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg 690 695 700Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly705 710 715 720Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr 725 730 735Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser 740 745 750Leu His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys 755 760 765Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met 770 775 780Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn785 790 795 800Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg 805 810 815Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His 820 825 830Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr 835 840 845Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn 850 855 860Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu865 870 875 880Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn 885 890 895Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met 900 905 910Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg 915 920 925Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu 930 935 940Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile945 950 955 960Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr 965 970 975Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys 980 985 990Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val 995 1000 1005Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn 1010 1015 1020Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu 1025 1030 1035Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys 1040 1045 1050Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys 1055 1060 1065Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile 1070 1075 1080Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr 1085 1090 1095Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe 1100 1105 1110Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val 1115 1120 1125Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile 1130 1135 1140Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp 1145 1150 1155Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala 1160 1165 1170Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1175 1180 1185Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu 1190 1195 1200Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys 1205 1210 1215Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 1220 1225 1230Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala 1235 1240 1245Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser 1250 1255 1260Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu 1265 1270 1275Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu 1280 1285 1290Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu 1295 1300 1305Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val 1310 1315 1320Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln 1325 1330 1335Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala 1340 1345 1350Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg 1355 1360 1365Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln 1370 1375 1380Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu 1385 1390 1395Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val 1400 1405 14101178237DNAArtificial sequencepRF48 117aattcgacaa gaaatactcc atcggcctgg acattggaac caactctgtc ggctgggctg 60tcatcaccga cgagtacaag gtgccctcca agaaattcaa ggtcctcgga aacaccgatc 120gacactccat caagaaaaac ctcattggtg ccctgttgtt cgattctggc gagactgccg 180aagctaccag actcaagcga actgctcggc gacgttacac ccgacggaag aaccgaatct 240gctacctgca ggagatcttt tccaacgaga tggccaaggt ggacgattcg ttctttcatc 300gactggagga atccttcctc gtcgaggaag acaagaaaca cgagcgtcat cccatctttg 360gcaacattgt ggacgaggtt gcttaccacg agaagtatcc taccatctac cacctgcgaa 420agaaactcgt

cgattccacc gacaaggcgg atctcagact tatctacctc gctctggcac 480acatgatcaa gtttcgaggt catttcctca tcgagggcga tctcaatccc gacaacagcg 540atgtggacaa gctgttcatt cagctcgttc agacctacaa ccagctgttc gaggaaaacc 600ccatcaatgc ctccggagtc gatgcaaagg ccatcttgtc tgctcgactc tcgaagagca 660gacgactgga gaacctcatt gcccaacttc ctggcgagaa aaagaacgga ctgtttggca 720acctcattgc cctttctctt ggtctcacac ccaacttcaa gtccaacttc gatctggcgg 780aggacgccaa gctccagctg tccaaggaca cctacgacga tgacctcgac aacctgcttg 840cacagattgg cgatcagtac gccgacctgt ttctcgctgc caagaacctt tcggatgcta 900ttctcttgtc tgacattctg cgagtcaaca ccgagatcac aaaggctccc ctttctgcct 960ccatgatcaa gcgatacgac gagcaccatc aggatctcac actgctcaag gctcttgtcc 1020gacagcaact gcccgagaag tacaaggaga tctttttcga tcagtcgaag aacggctacg 1080ctggatacat cgacggcgga gcctctcagg aagagttcta caagttcatc aagccaattc 1140tcgagaagat ggacggaacc gaggaactgc ttgtcaagct caatcgagag gatctgcttc 1200ggaagcaacg aaccttcgac aacggcagca ttcctcatca gatccacctc ggtgagctgc 1260acgccattct tcgacgtcag gaagacttct acccctttct caaggacaac cgagagaaga 1320tcgagaagat tcttaccttt cgaatcccct actatgttgg tcctcttgcc agaggaaact 1380ctcgatttgc ttggatgact cgaaagtccg aggaaaccat cactccctgg aacttcgagg 1440aagtcgtgga caagggtgcc tctgcacagt ccttcatcga gcgaatgacc aacttcgaca 1500agaatctgcc caacgagaag gttcttccca agcattcgct gctctacgag tactttacag 1560tctacaacga actcaccaaa gtcaagtacg ttaccgaggg aatgcgaaag cctgccttct 1620tgtctggcga acagaagaaa gccattgtcg atctcctgtt caagaccaac cgaaaggtca 1680ctgttaagca gctcaaggag gactacttca agaaaatcga gtgtttcgac agcgtcgaga 1740tttccggagt tgaggaccga ttcaacgcct ctttgggcac ctatcacgat ctgctcaaga 1800ttatcaagga caaggatttt ctcgacaacg aggaaaacga ggacattctg gaggacatcg 1860tgctcactct taccctgttc gaagatcggg agatgatcga ggaacgactc aagacatacg 1920ctcacctgtt cgacgacaag gtcatgaaac aactcaagcg acgtagatac accggctggg 1980gaagactttc gcgaaagctc atcaacggca tcagagacaa gcagtccgga aagaccattc 2040tggactttct caagtccgat ggctttgcca accgaaactt catgcagctc attcacgacg 2100attctcttac cttcaaggag gacatccaga aggcacaagt gtccggtcag ggcgacagct 2160tgcacgaaca tattgccaac ctggctggtt cgccagccat caagaaaggc attctccaga 2220ctgtcaaggt tgtcgacgag ctggtgaagg tcatgggacg tcacaagccc gagaacattg 2280tgatcgagat ggccagagag aaccagacaa ctcaaaaggg tcagaaaaac tcgcgagagc 2340ggatgaagcg aatcgaggaa ggcatcaagg agctgggatc ccagattctc aaggagcatc 2400ccgtcgagaa cactcaactg cagaacgaga agctgtatct ctactatctg cagaatggtc 2460gagacatgta cgtggatcag gaactggaca tcaatcgtct cagcgactac gatgtggacc 2520acattgtccc tcaatccttt ctcaaggacg attctatcga caacaaggtc cttacacgat 2580ccgacaagaa cagaggcaag tcggacaacg ttcccagcga agaggtggtc aaaaagatga 2640agaactactg gcgacagctg ctcaacgcca agctcattac ccagcgaaag ttcgacaatc 2700ttaccaaggc cgagcgaggc ggtctgtccg agctcgacaa ggctggcttc atcaagcgtc 2760aactcgtcga gaccagacag atcacaaagc acgtcgcaca gattctcgat tctcggatga 2820acaccaagta cgacgagaac gacaagctca tccgagaggt caaggtgatt actctcaagt 2880ccaaactggt ctccgatttc cgaaaggact ttcagttcta caaggtgcga gagatcaaca 2940attaccacca tgcccacgat gcttacctca acgccgtcgt tggcactgcg ctcatcaaga 3000aataccccaa gctcgaaagc gagttcgttt acggcgatta caaggtctac gacgttcgaa 3060agatgattgc caagtccgaa caggagattg gcaaggctac tgccaagtac ttcttttact 3120ccaacatcat gaactttttc aagaccgaga tcaccttggc caacggagag attcgaaaga 3180gaccacttat cgagaccaac ggcgaaactg gagagatcgt gtgggacaag ggtcgagact 3240ttgcaaccgt gcgaaaggtt ctgtcgatgc ctcaggtcaa catcgtcaag aaaaccgagg 3300ttcagactgg cggattctcc aaggagtcga ttctgcccaa gcgaaactcc gacaagctca 3360tcgctcgaaa gaaagactgg gatcccaaga aatacggtgg cttcgattct cctaccgtcg 3420cctattccgt gcttgtcgtt gcgaaggtcg agaagggcaa gtccaaaaag ctcaagtccg 3480tcaaggagct gctcggaatt accatcatgg agcgatcgag cttcgagaag aatcccatcg 3540acttcttgga agccaagggt tacaaggagg tcaagaaaga cctcattatc aagctgccca 3600agtactctct gttcgaactg gagaacggtc gaaagcgtat gctcgcctcc gctggcgagc 3660tgcagaaggg aaacgagctt gccttgcctt cgaagtacgt caactttctc tatctggctt 3720ctcactacga gaagctcaag ggttctcccg aggacaacga acagaagcaa ctcttcgttg 3780agcagcacaa acattacctc gacgagatta tcgagcagat ttccgagttt tcgaagcgag 3840tcatcctggc tgatgccaac ttggacaagg tgctctctgc ctacaacaag catcgggaca 3900aacccattcg agaacaggcg gagaacatca ttcacctgtt tactcttacc aacctgggtg 3960ctcctgcagc tttcaagtac ttcgatacca ctatcgaccg aaagcggtac acatccacca 4020aggaggttct cgatgccacc ctgattcacc agtccatcac tggcctgtac gagacccgaa 4080tcgacctgtc tcagcttggt ggcgactcca gagccgatcc caagaaaaag cgaaaggtct 4140aagcggccgc taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga 4200ttaaatcaga acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg 4260tggtcccacc tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg 4320tggggtctcc ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag 4380tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg 4440acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca 4500ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc 4560ctttttgcgt ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc 4620gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag 4680tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt 4740tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt 4800gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga 4860acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt 4920tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga 4980gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag 5040tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg 5100accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg 5160ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt 5220agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg 5280gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc 5340ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg 5400tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac 5460ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact 5520gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa 5580acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa 5640aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 5700atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 5760gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 5820tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 5880ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 5940ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 6000ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 6060aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 6120cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 6180gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 6240ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 6300cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 6360tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 6420cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg 6480cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tatggtgcac 6540tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 6600cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 6660gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 6720tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa ttcgcgcgcg 6780aaggcgaagc ggcatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc 6840ctatgctact ccgtcaagcc gtcaattgtc tgattcgtta ccaattatga caacttgacg 6900gctacatcat tcactttttc ttcacaaccg gcacggaact cgctcgggct ggccccggtg 6960cattttttaa atacccgcga gaaatagagt tgatcgtcaa aaccaacatt gcgaccgacg 7020gtggcgatag gcatccgggt ggtgctcaaa agcagcttcg cctggctgat acgttggtcc 7080tcgcgccagc ttaagacgct aatccctaac tgctggcgga aaagatgtga cagacgcgac 7140ggcgacaagc aaacatgctg tgcgacgctg gcgatatcaa aattgctgtc tgccaggtga 7200tcgctgatgt actgacaagc ctcgcgtacc cgattatcca tcggtggatg gagcgactcg 7260ttaatcgctt ccatgcgccg cagtaacaat tgctcaagca gatttatcgc cagcagctcc 7320gaatagcgcc cttccccttg cccggcgtta atgatttgcc caaacaggtc gctgaaatgc 7380ggctggtgcg cttcatccgg gcgaaagaac cccgtattgg caaatattga cggccagtta 7440agccattcat gccagtaggc gcgcggacga aagtaaaccc actggtgata ccattcgcga 7500gcctccggat gacgaccgta gtgatgaatc tctcctggcg ggaacagcaa aatatcaccc 7560ggtcggcaaa caaattctcg tccctgattt ttcaccaccc cctgaccgcg aatggtgaga 7620ttgagaatat aacctttcat tcccagcggt cggtcgataa aaaaatcgag ataaccgttg 7680gcctcaatcg gcgttaaacc cgccaccaga tgggcattaa acgagtatcc cggcagcagg 7740ggatcatttt gcgcttcagc catacttttc atactcccgc cattcagaga agaaaccaat 7800tgtccatatt gcatcagaca ttgccgtcac tgcgtctttt actggctctt ctcgctaacc 7860aaaccggtaa ccccgcttat taaaagcatt ctgtaacaaa gcgggaccaa agccatgaca 7920aaaacgcgta acaaaagtgt ctataatcac ggcagaaaag tccacattga ttatttgcac 7980ggcgtcacac tttgctatgc catagcattt ttatccataa gattagcgga tcctacctga 8040cgctttttat cgcaactctc tactgtttct ccatacccgt tttttgggct aacaggagga 8100attaaccatg gggggttctc atcatcatca tcatcatggt atggctagca tgactggtgg 8160acagcaaatg ggtcgggatc tgtacgacga tgacgataag gatccgagct cgagatctgc 8220agctggtacc atatggg 82371188153DNAArtificial sequencepRF243 118catggggcat caccatcacc accattgttt tttcaaagac gaactggaat tcgacaagaa 60atactccatc ggcctggaca ttggaaccaa ctctgtcggc tgggctgtca tcaccgacga 120gtacaaggtg ccctccaaga aattcaaggt cctcggaaac accgatcgac actccatcaa 180gaaaaacctc attggtgccc tgttgttcga ttctggcgag actgccgaag ctaccagact 240caagcgaact gctcggcgac gttacacccg acggaagaac cgaatctgct acctgcagga 300gatcttttcc aacgagatgg ccaaggtgga cgattcgttc tttcatcgac tggaggaatc 360cttcctcgtc gaggaagaca agaaacacga gcgtcatccc atctttggca acattgtgga 420cgaggttgct taccacgaga agtatcctac catctaccac ctgcgaaaga aactcgtcga 480ttccaccgac aaggcggatc tcagacttat ctacctcgct ctggcacaca tgatcaagtt 540tcgaggtcat ttcctcatcg agggcgatct caatcccgac aacagcgatg tggacaagct 600gttcattcag ctcgttcaga cctacaacca gctgttcgag gaaaacccca tcaatgcctc 660cggagtcgat gcaaaggcca tcttgtctgc tcgactctcg aagagcagac gactggagaa 720cctcattgcc caacttcctg gcgagaaaaa gaacggactg tttggcaacc tcattgccct 780ttctcttggt ctcacaccca acttcaagtc caacttcgat ctggcggagg acgccaagct 840ccagctgtcc aaggacacct acgacgatga cctcgacaac ctgcttgcac agattggcga 900tcagtacgcc gacctgtttc tcgctgccaa gaacctttcg gatgctattc tcttgtctga 960cattctgcga gtcaacaccg agatcacaaa ggctcccctt tctgcctcca tgatcaagcg 1020atacgacgag caccatcagg atctcacact gctcaaggct cttgtccgac agcaactgcc 1080cgagaagtac aaggagatct ttttcgatca gtcgaagaac ggctacgctg gatacatcga 1140cggcggagcc tctcaggaag agttctacaa gttcatcaag ccaattctcg agaagatgga 1200cggaaccgag gaactgcttg tcaagctcaa tcgagaggat ctgcttcgga agcaacgaac 1260cttcgacaac ggcagcattc ctcatcagat ccacctcggt gagctgcacg ccattcttcg 1320acgtcaggaa gacttctacc cctttctcaa ggacaaccga gagaagatcg agaagattct 1380tacctttcga atcccctact atgttggtcc tcttgccaga ggaaactctc gatttgcttg 1440gatgactcga aagtccgagg aaaccatcac tccctggaac ttcgaggaag tcgtggacaa 1500gggtgcctct gcacagtcct tcatcgagcg aatgaccaac ttcgacaaga atctgcccaa 1560cgagaaggtt cttcccaagc attcgctgct ctacgagtac tttacagtct acaacgaact 1620caccaaagtc aagtacgtta ccgagggaat gcgaaagcct gccttcttgt ctggcgaaca 1680gaagaaagcc attgtcgatc tcctgttcaa gaccaaccga aaggtcactg ttaagcagct 1740caaggaggac tacttcaaga aaatcgagtg tttcgacagc gtcgagattt ccggagttga 1800ggaccgattc aacgcctctt tgggcaccta tcacgatctg ctcaagatta tcaaggacaa 1860ggattttctc gacaacgagg aaaacgagga cattctggag gacatcgtgc tcactcttac 1920cctgttcgaa gatcgggaga tgatcgagga acgactcaag acatacgctc acctgttcga 1980cgacaaggtc atgaaacaac tcaagcgacg tagatacacc ggctggggaa gactttcgcg 2040aaagctcatc aacggcatca gagacaagca gtccggaaag accattctgg actttctcaa 2100gtccgatggc tttgccaacc gaaacttcat gcagctcatt cacgacgatt ctcttacctt 2160caaggaggac atccagaagg cacaagtgtc cggtcagggc gacagcttgc acgaacatat 2220tgccaacctg gctggttcgc cagccatcaa gaaaggcatt ctccagactg tcaaggttgt 2280cgacgagctg gtgaaggtca tgggacgtca caagcccgag aacattgtga tcgagatggc 2340cagagagaac cagacaactc aaaagggtca gaaaaactcg cgagagcgga tgaagcgaat 2400cgaggaaggc atcaaggagc tgggatccca gattctcaag gagcatcccg tcgagaacac 2460tcaactgcag aacgagaagc tgtatctcta ctatctgcag aatggtcgag acatgtacgt 2520ggatcaggaa ctggacatca atcgtctcag cgactacgat gtggaccaca ttgtccctca 2580atcctttctc aaggacgatt ctatcgacaa caaggtcctt acacgatccg acaagaacag 2640aggcaagtcg gacaacgttc ccagcgaaga ggtggtcaaa aagatgaaga actactggcg 2700acagctgctc aacgccaagc tcattaccca gcgaaagttc gacaatctta ccaaggccga 2760gcgaggcggt ctgtccgagc tcgacaaggc tggcttcatc aagcgtcaac tcgtcgagac 2820cagacagatc acaaagcacg tcgcacagat tctcgattct cggatgaaca ccaagtacga 2880cgagaacgac aagctcatcc gagaggtcaa ggtgattact ctcaagtcca aactggtctc 2940cgatttccga aaggactttc agttctacaa ggtgcgagag atcaacaatt accaccatgc 3000ccacgatgct tacctcaacg ccgtcgttgg cactgcgctc atcaagaaat accccaagct 3060cgaaagcgag ttcgtttacg gcgattacaa ggtctacgac gttcgaaaga tgattgccaa 3120gtccgaacag gagattggca aggctactgc caagtacttc ttttactcca acatcatgaa 3180ctttttcaag accgagatca ccttggccaa cggagagatt cgaaagagac cacttatcga 3240gaccaacggc gaaactggag agatcgtgtg ggacaagggt cgagactttg caaccgtgcg 3300aaaggttctg tcgatgcctc aggtcaacat cgtcaagaaa accgaggttc agactggcgg 3360attctccaag gagtcgattc tgcccaagcg aaactccgac aagctcatcg ctcgaaagaa 3420agactgggat cccaagaaat acggtggctt cgattctcct accgtcgcct attccgtgct 3480tgtcgttgcg aaggtcgaga agggcaagtc caaaaagctc aagtccgtca aggagctgct 3540cggaattacc atcatggagc gatcgagctt cgagaagaat cccatcgact tcttggaagc 3600caagggttac aaggaggtca agaaagacct cattatcaag ctgcccaagt actctctgtt 3660cgaactggag aacggtcgaa agcgtatgct cgcctccgct ggcgagctgc agaagggaaa 3720cgagcttgcc ttgccttcga agtacgtcaa ctttctctat ctggcttctc actacgagaa 3780gctcaagggt tctcccgagg acaacgaaca gaagcaactc ttcgttgagc agcacaaaca 3840ttacctcgac gagattatcg agcagatttc cgagttttcg aagcgagtca tcctggctga 3900tgccaacttg gacaaggtgc tctctgccta caacaagcat cgggacaaac ccattcgaga 3960acaggcggag aacatcattc acctgtttac tcttaccaac ctgggtgctc ctgcagcttt 4020caagtacttc gataccacta tcgaccgaaa gcggtacaca tccaccaagg aggttctcga 4080tgccaccctg attcaccagt ccatcactgg cctgtacgag acccgaatcg acctgtctca 4140gcttggtggc gactccagag ccgatcccaa gaaaaagcga aaggtctaag cggccgctaa 4200gcttggctgt tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg 4260cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga 4320ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca 4380tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg 4440cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg 4500gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat 4560aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc 4620tacaaactct tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa 4680taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc 4740cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa 4800acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa 4860ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg 4920atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa 4980gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc 5040acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc 5100atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta 5160accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag 5220ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca 5280acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata 5340gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc 5400tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca 5460ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca 5520actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg 5580taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa 5640tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt 5700gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat 5760cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg 5820gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga 5880gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac 5940tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt 6000ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag 6060cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc 6120gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag 6180gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca 6240gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt 6300cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc 6360tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc 6420cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc 6480cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat 6540tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct cagtacaatc 6600tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca 6660tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 6720cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 6780caccgtcatc accgaaacgc gcgaggcagc agatcaattc gcgcgcgaag gcgaagcggc 6840atgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta tgctactccg 6900tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct acatcattca 6960ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat tttttaaata 7020cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg gcgataggca 7080tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg cgccagctta 7140agacgctaat ccctaactgc

tggcggaaaa gatgtgacag acgcgacggc gacaagcaaa 7200catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg ctgatgtact 7260gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta atcgcttcca 7320tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa tagcgccctt 7380ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc tggtgcgctt 7440catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc cattcatgcc 7500agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc tccggatgac 7560gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt cggcaaacaa 7620attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg agaatataac 7680ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc tcaatcggcg 7740ttaaacccgc caccagatgg gcattaaacg agtatcccgg cagcagggga tcattttgcg 7800cttcagccat acttttcata ctcccgccat tcagagaaga aaccaattgt ccatattgca 7860tcagacattg ccgtcactgc gtcttttact ggctcttctc gctaaccaaa ccggtaaccc 7920cgcttattaa aagcattctg taacaaagcg ggaccaaagc catgacaaaa acgcgtaaca 7980aaagtgtcta taatcacggc agaaaagtcc acattgatta tttgcacggc gtcacacttt 8040gctatgccat agcattttta tccataagat tagcggatcc tacctgacgc tttttatcgc 8100aactctctac tgtttctcca tacccgtttt ttgggctaac aggaggaatt aac 81531198204DNAArtificial sequencepRF238 119catggggcat catcatcacc atcacggcgc cctgttctta ggccagctgg gcgccgcggg 60atccacgatg ggtgcgccga agaaaaagcg caaagttgaa ttcgacaaga aatactccat 120cggcctggac attggaacca actctgtcgg ctgggctgtc atcaccgacg agtacaaggt 180gccctccaag aaattcaagg tcctcggaaa caccgatcga cactccatca agaaaaacct 240cattggtgcc ctgttgttcg attctggcga gactgccgaa gctaccagac tcaagcgaac 300tgctcggcga cgttacaccc gacggaagaa ccgaatctgc tacctgcagg agatcttttc 360caacgagatg gccaaggtgg acgattcgtt ctttcatcga ctggaggaat ccttcctcgt 420cgaggaagac aagaaacacg agcgtcatcc catctttggc aacattgtgg acgaggttgc 480ttaccacgag aagtatccta ccatctacca cctgcgaaag aaactcgtcg attccaccga 540caaggcggat ctcagactta tctacctcgc tctggcacac atgatcaagt ttcgaggtca 600tttcctcatc gagggcgatc tcaatcccga caacagcgat gtggacaagc tgttcattca 660gctcgttcag acctacaacc agctgttcga ggaaaacccc atcaatgcct ccggagtcga 720tgcaaaggcc atcttgtctg ctcgactctc gaagagcaga cgactggaga acctcattgc 780ccaacttcct ggcgagaaaa agaacggact gtttggcaac ctcattgccc tttctcttgg 840tctcacaccc aacttcaagt ccaacttcga tctggcggag gacgccaagc tccagctgtc 900caaggacacc tacgacgatg acctcgacaa cctgcttgca cagattggcg atcagtacgc 960cgacctgttt ctcgctgcca agaacctttc ggatgctatt ctcttgtctg acattctgcg 1020agtcaacacc gagatcacaa aggctcccct ttctgcctcc atgatcaagc gatacgacga 1080gcaccatcag gatctcacac tgctcaaggc tcttgtccga cagcaactgc ccgagaagta 1140caaggagatc tttttcgatc agtcgaagaa cggctacgct ggatacatcg acggcggagc 1200ctctcaggaa gagttctaca agttcatcaa gccaattctc gagaagatgg acggaaccga 1260ggaactgctt gtcaagctca atcgagagga tctgcttcgg aagcaacgaa ccttcgacaa 1320cggcagcatt cctcatcaga tccacctcgg tgagctgcac gccattcttc gacgtcagga 1380agacttctac ccctttctca aggacaaccg agagaagatc gagaagattc ttacctttcg 1440aatcccctac tatgttggtc ctcttgccag aggaaactct cgatttgctt ggatgactcg 1500aaagtccgag gaaaccatca ctccctggaa cttcgaggaa gtcgtggaca agggtgcctc 1560tgcacagtcc ttcatcgagc gaatgaccaa cttcgacaag aatctgccca acgagaaggt 1620tcttcccaag cattcgctgc tctacgagta ctttacagtc tacaacgaac tcaccaaagt 1680caagtacgtt accgagggaa tgcgaaagcc tgccttcttg tctggcgaac agaagaaagc 1740cattgtcgat ctcctgttca agaccaaccg aaaggtcact gttaagcagc tcaaggagga 1800ctacttcaag aaaatcgagt gtttcgacag cgtcgagatt tccggagttg aggaccgatt 1860caacgcctct ttgggcacct atcacgatct gctcaagatt atcaaggaca aggattttct 1920cgacaacgag gaaaacgagg acattctgga ggacatcgtg ctcactctta ccctgttcga 1980agatcgggag atgatcgagg aacgactcaa gacatacgct cacctgttcg acgacaaggt 2040catgaaacaa ctcaagcgac gtagatacac cggctgggga agactttcgc gaaagctcat 2100caacggcatc agagacaagc agtccggaaa gaccattctg gactttctca agtccgatgg 2160ctttgccaac cgaaacttca tgcagctcat tcacgacgat tctcttacct tcaaggagga 2220catccagaag gcacaagtgt ccggtcaggg cgacagcttg cacgaacata ttgccaacct 2280ggctggttcg ccagccatca agaaaggcat tctccagact gtcaaggttg tcgacgagct 2340ggtgaaggtc atgggacgtc acaagcccga gaacattgtg atcgagatgg ccagagagaa 2400ccagacaact caaaagggtc agaaaaactc gcgagagcgg atgaagcgaa tcgaggaagg 2460catcaaggag ctgggatccc agattctcaa ggagcatccc gtcgagaaca ctcaactgca 2520gaacgagaag ctgtatctct actatctgca gaatggtcga gacatgtacg tggatcagga 2580actggacatc aatcgtctca gcgactacga tgtggaccac attgtccctc aatcctttct 2640caaggacgat tctatcgaca acaaggtcct tacacgatcc gacaagaaca gaggcaagtc 2700ggacaacgtt cccagcgaag aggtggtcaa aaagatgaag aactactggc gacagctgct 2760caacgccaag ctcattaccc agcgaaagtt cgacaatctt accaaggccg agcgaggcgg 2820tctgtccgag ctcgacaagg ctggcttcat caagcgtcaa ctcgtcgaga ccagacagat 2880cacaaagcac gtcgcacaga ttctcgattc tcggatgaac accaagtacg acgagaacga 2940caagctcatc cgagaggtca aggtgattac tctcaagtcc aaactggtct ccgatttccg 3000aaaggacttt cagttctaca aggtgcgaga gatcaacaat taccaccatg cccacgatgc 3060ttacctcaac gccgtcgttg gcactgcgct catcaagaaa taccccaagc tcgaaagcga 3120gttcgtttac ggcgattaca aggtctacga cgttcgaaag atgattgcca agtccgaaca 3180ggagattggc aaggctactg ccaagtactt cttttactcc aacatcatga actttttcaa 3240gaccgagatc accttggcca acggagagat tcgaaagaga ccacttatcg agaccaacgg 3300cgaaactgga gagatcgtgt gggacaaggg tcgagacttt gcaaccgtgc gaaaggttct 3360gtcgatgcct caggtcaaca tcgtcaagaa aaccgaggtt cagactggcg gattctccaa 3420ggagtcgatt ctgcccaagc gaaactccga caagctcatc gctcgaaaga aagactggga 3480tcccaagaaa tacggtggct tcgattctcc taccgtcgcc tattccgtgc ttgtcgttgc 3540gaaggtcgag aagggcaagt ccaaaaagct caagtccgtc aaggagctgc tcggaattac 3600catcatggag cgatcgagct tcgagaagaa tcccatcgac ttcttggaag ccaagggtta 3660caaggaggtc aagaaagacc tcattatcaa gctgcccaag tactctctgt tcgaactgga 3720gaacggtcga aagcgtatgc tcgcctccgc tggcgagctg cagaagggaa acgagcttgc 3780cttgccttcg aagtacgtca actttctcta tctggcttct cactacgaga agctcaaggg 3840ttctcccgag gacaacgaac agaagcaact cttcgttgag cagcacaaac attacctcga 3900cgagattatc gagcagattt ccgagttttc gaagcgagtc atcctggctg atgccaactt 3960ggacaaggtg ctctctgcct acaacaagca tcgggacaaa cccattcgag aacaggcgga 4020gaacatcatt cacctgttta ctcttaccaa cctgggtgct cctgcagctt tcaagtactt 4080cgataccact atcgaccgaa agcggtacac atccaccaag gaggttctcg atgccaccct 4140gattcaccag tccatcactg gcctgtacga gacccgaatc gacctgtctc agcttggtgg 4200cgactccaga gccgatccca agaaaaagcg aaaggtctaa gcggccgcta agcttggctg 4260ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac gcagaagcgg 4320tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg accccatgcc 4380gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc atgcgagagt 4440agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt 4500ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg ggagcggatt 4560tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca taaactgcca 4620ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt ctacaaactc 4680ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 4740taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 4800cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 4860aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 4920aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 4980tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc 5040ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 5100catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 5160aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 5220ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 5280gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 5340aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 5400gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 5460gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 5520gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 5580gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 5640gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 5700atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 5760ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 5820ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 5880ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 5940ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 6000ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 6060tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 6120tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 6180tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 6240tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 6300gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 6360tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 6420ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 6480gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 6540gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt 6600acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat 6660gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc atggctgcgc 6720cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 6780cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 6840caccgaaacg cgcgaggcag cagatcaatt cgcgcgcgaa ggcgaagcgg catgcataat 6900gtgcctgtca aatggacgaa gcagggattc tgcaaaccct atgctactcc gtcaagccgt 6960caattgtctg attcgttacc aattatgaca acttgacggc tacatcattc actttttctt 7020cacaaccggc acggaactcg ctcgggctgg ccccggtgca ttttttaaat acccgcgaga 7080aatagagttg atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc atccgggtgg 7140tgctcaaaag cagcttcgcc tggctgatac gttggtcctc gcgccagctt aagacgctaa 7200tccctaactg ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa acatgctgtg 7260cgacgctggc gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac tgacaagcct 7320cgcgtacccg attatccatc ggtggatgga gcgactcgtt aatcgcttcc atgcgccgca 7380gtaacaattg ctcaagcaga tttatcgcca gcagctccga atagcgccct tccccttgcc 7440cggcgttaat gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct tcatccgggc 7500gaaagaaccc cgtattggca aatattgacg gccagttaag ccattcatgc cagtaggcgc 7560gcggacgaaa gtaaacccac tggtgatacc attcgcgagc ctccggatga cgaccgtagt 7620gatgaatctc tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca aattctcgtc 7680cctgattttt caccaccccc tgaccgcgaa tggtgagatt gagaatataa cctttcattc 7740ccagcggtcg gtcgataaaa aaatcgagat aaccgttggc ctcaatcggc gttaaacccg 7800ccaccagatg ggcattaaac gagtatcccg gcagcagggg atcattttgc gcttcagcca 7860tacttttcat actcccgcca ttcagagaag aaaccaattg tccatattgc atcagacatt 7920gccgtcactg cgtcttttac tggctcttct cgctaaccaa accggtaacc ccgcttatta 7980aaagcattct gtaacaaagc gggaccaaag ccatgacaaa aacgcgtaac aaaagtgtct 8040ataatcacgg cagaaaagtc cacattgatt atttgcacgg cgtcacactt tgctatgcca 8100tagcattttt atccataaga ttagcggatc ctacctgacg ctttttatcg caactctcta 8160ctgtttctcc atacccgttt tttgggctaa caggaggaat taac 82041201149DNAEscherichia colimisc_feature(1)..(1149)galK gene 120atgagtctga aagaaaaaac acaatctctg tttgccaacg catttggcta ccctgccact 60cacaccattc aggcgcctgg ccgcgtgaat ttgattggtg aacacaccga ctacaacgac 120ggtttcgttc tgccctgcgc gattgattat caaaccgtga tcagttgtgc accacgcgat 180gaccgtaaag ttcgcgtgat ggcagccgat tatgaaaatc agctcgacga gttttccctc 240gatgcgccca ttgtcgcaca tgaaaactat caatgggcta actacgttcg tggcgtggtg 300aaacatctgc aactgcgtaa caacagcttc ggcggcgtgg acatggtgat cagcggcaat 360gtgccgcagg gtgccgggtt aagttcttcc gcttcactgg aagtcgcggt cggaaccgta 420ttgcagcagc tttatcatct gccgctggac ggcgcacaaa tcgcgcttaa cggtcaggaa 480gcagaaaacc agtttgtagg ctgtaactgc gggatcatgg atcagctaat ttccgcgctc 540ggcaagaaag atcatgcctt gctgatcgat tgccgctcac tggggaccaa agcagtttcc 600atgcccaaag gtgtggctgt cgtcatcatc aacagtaact tcaaacgtac cctggttggc 660agcgaataca acacccgtcg tgaacagtgc gaaaccggtg cgcgtttctt ccagcagcca 720gccctgcgtg atgtcaccat tgaagagttc aacgctgttg cgcatgaact ggacccgatc 780gtggcaaaac gcgtgcgtca tatactgact gaaaacgccc gcaccgttga agctgccagc 840gcgctggagc aaggcgacct gaaacgtatg ggcgagttga tggcggagtc tcatgcctct 900atgcgcgatg atttcgaaat caccgtgccg caaattgaca ctctggtaga aatcgtcaaa 960gctgtgattg gcgacaaagg tggcgtacgc atgaccggcg gcggatttgg cggctgtatc 1020gtcgcgctga tcccggaaga gctggtgcct gccgtacagc aagctgtcgc tgaacaatat 1080gaagcaaaaa caggtattaa agagactttt tacgtttgta aaccatcaca aggagcagga 1140cagtgctga 11491211017DNAEscherichia coli 121atgagagttc tggttaccgg tggtagcggt tacattggaa gtcatacctg tgtgcaatta 60ctgcaaaacg gtcatgatgt catcattctt gataacctct gtaacagtaa gcgcagcgta 120ctgcctgtta tcgagcgttt aggcggcaaa catccaacgt ttgttgaagg cgatattcgt 180aacgaagcgt tgatgaccga gatcctgcac gatcacgcta tcgacaccgt gatccacttc 240gccgggctga aagccgtggg cgaatcggta caaaaaccgc tggaatatta cgacaacaat 300gtcaacggca ctctgcgcct gattagcgcc atgcgcgccg ctaacgtcaa aaactttatt 360tttagctcct ccgccaccgt ttatggcgat cagcccaaaa ttccatacgt tgaaagcttc 420ccgaccggca caccgcaaag cccttacggc aaaagcaagc tgatggtgga acagatcctc 480accgatctgc aaaaagccca gccggactgg agcattgccc tgctgcgcta cttcaacccg 540gttggcgcgc atccgtcggg cgatatgggc gaagatccgc aaggcattcc gaataacctg 600atgccataca tcgcccaggt tgctgtaggc cgtcgcgact cgctggcgat ttttggtaac 660gattatccga ccgaagatgg tactggcgta cgcgattaca tccacgtaat ggatctggcg 720gacggtcacg tcgtggcgat ggaaaaactg gcgaacaagc caggcgtaca catctacaac 780ctcggcgctg gcgtaggcaa cagcgtgctg gacgtggtta atgccttcag caaagcctgc 840ggcaaaccgg ttaattatca ttttgcaccg cgtcgcgagg gcgaccttcc ggcctactgg 900gcggacgcca gcaaagccga ccgtgaactg aactggcgcg taacgcgcac actcgatgaa 960atggcgcagg acacctggca ctggcagtca cgccatccac agggatatcc cgattaa 10171221047DNAEscherichia coli 122atgacgcaat ttaatcccgt tgatcatcca catcgccgct acaacccgct caccgggcaa 60tggattctgg tttcaccgca ccgcgctaag cgcccctggc agggggcgca ggaaacgcca 120gccaaacagg tgttacctgc gcacgatcca gattgcttcc tctgcgcagg taatgtgcgg 180gtgacaggcg ataaaaaccc cgattacacc gggacttacg ttttcactaa tgactttgcg 240gctttgatgt ctgacacgcc agatgcgcca gaaagtcacg atccgctgat gcgttgccag 300agcgcgcgcg gcaccagccg ggtgatctgc ttttcaccgg atcacagtaa aacgctgcca 360gagctcagcg ttgcagcatt gacggaaatc gtcaaaacct ggcaggagca aaccgcagaa 420ctggggaaaa cgtacccatg ggtgcaggtt tttgaaaaca aaggcgcggc gatgggctgc 480tctaacccgc atccgcacgg tcagatttgg gcaaatagct tcctgcctaa cgaagctgag 540cgcgaagacc gcctgcaaaa agaatatttt gccgaacaga aatcaccaat gctggtggat 600tatgttcagc gcgagctggc agacggtagc cgtaccgttg tcgaaaccga acactggtta 660gccgtcgtgc cttactgggc tgcctggccg ttcgaaacgc tactgctgcc caaagcccac 720gttttacgga tcaccgattt gaccgacgcc cagcgcagcg atctggcgct ggcgttgaaa 780aagctgacca gtcgttatga caacctcttc cagtgctcct tcccctactc tatgggctgg 840cacggcgcgc catttaatgg cgaagagaat caacactggc agctgcacgc gcacttttat 900ccgcctctgc tgcgctccgc caccgtacgt aaatttatgg ttggttatga aatgctggca 960gagacccagc gagacctgac cgcagaacag gcagcagagc gtttgcgcgc agtcagcgat 1020atccattttc gcgaatccgg agtgtaa 104712376RNAArtificial sequenceCER domainl 123guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc cguuaucaac uugaaaaagu 60ggcaccgagu cggugc 7612476DNAArtificial sequenceCER encoding DNA PCR 124gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60ggcaccgagt cggtgc 7612511714DNAArtificial sequencepRF291 125cgataaaaaa caaaaaaaaa agcaccgact cggtgccact ttttcaagtt gataacggac 60tagccttatt ttaacttgct atttctagct ctaaaacgca ggtgtaaaaa taaaaaggcc 120tgcgattacc agcaggcctg ttattaacct aagccttagg acgcttcacg ccatacttgg 180aacgagcctg cttacggtct ttaacgccgg agcagtcaag cgcaccacgt acggtgtggt 240aacgaacacc cgggaggtct ttaacacgac cgccacggat caggatcacg gagtgctcct 300gcaggttgtg accttcacca ccgatgtagg aagtcacttc gaaaccgtta gtcagacgaa 360cacggcatac tttacgcagc gcggagttcg gttttttagg agtggtagta tatacacgag 420tacatacgcc acgtttttgc gggcatgctt ccagcgcagg cacgttgctt ttcgcaactt 480tgcgagcacg tggtttgcgt accagctggt taactgttgc cattaaatag ctcctggttt 540tagcttttgc ttcgtaaaca cgtaataaaa cgtcctcaca caatatgagg acgccgaatt 600tagggcgatg ccgaaaaggt gtcaagaaat atacaacgat cccgccatca cctgcgtccc 660attcgccatg ccgaagcatg ttgcccagcc ggcgccagcg aggaggctgg gaccatgccg 720gccattattt tgcgttaagt ttctaatcat cacgaaatta tctatcaaaa ataactaggt 780cccaccgaga ttcgaactcg ggaccttaag atttgcaatc tcacgcgcta ccgctgtgcc 840ataggaccga agttaaaatt tggccaaaga aggacctggg caccctggac tgtgggttag 900ggtaatattc cttatggaga caatgggcta gggtaaatta cctaaaatgg gtcgataaag 960aggggtgttc ccagttggga agtgtaattg aagacggggt caaaaaagaa aatcaaaaaa 1020aatttaatta agtcatacac aagtcagctt tcttcgagcc tcatataagt ataagtagtt 1080caacgtatta gcactgtacc cagcatctcc gtatcgagaa acacaacaac atgccccatt 1140ggacagatca tgcggataca caggttgtgc agtatcatac atactcgatc agacaggtcg 1200tctgaccatc atacaagctg aacaagcgct ccatacttgc acgctctcta tatacacagt 1260taaattacat atccatagtc taacctctaa cagttaatct tctggtaagc ctcccagcca 1320gccttctggt atcgcttggc ctcctcaata ggatctcggt tctggccgta cagacctcgg 1380ccgacaatta tgatatccgt tccggtagac atgacatcct caacagttcg gtactgctgt 1440ccgagagcgt ctcccttgtc gtcaagaccc accccggggg tcagaataag ccagtcctca 1500gagtcgccct taggtcggtt ctgggcaatg aagccaacca caaactcggg gtcggatcgg 1560gcaagctcaa tggtctgctt ggagtactcg ccagtggcca gagagccctt gcaagacagc 1620tcggccagca tgagcagacc tctggccagc ttctcgttgg gagaggggac taggaactcc 1680ttgtactggg agttctcgta gtcagagacg tcctccttct tctgttcaga gacagtttcc 1740tcggcaccag ctcgcaggcc agcaatgatt ccggttccgg gtacaccgtg ggcgttggtg 1800atatcggacc actcggcgat tcggtgacac cggtactggt gcttgacagt gttgccaata 1860tctgcgaact ttctgtcctc gaacaggaag aaaccgtgct taagagcaag ttccttgagg 1920gggagcacag tgccggcgta ggtgaagtcg tcaatgatgt cgatatgggt tttgatcatg 1980cacacataag gtccgacctt atcggcaagc tcaatgagct ccttggtggt ggtaacatcc

2040agagaagcac acaggttggt tttcttggct gccacgagct tgagcactcg agcggcaaag 2100gcggacttgt ggacgttagc tcgagcttcg taggagggca ttttggtggt gaagaggaga 2160ctgaaataaa tttagtctgc agaacttttt atcggaacct tatctggggc agtgaagtat 2220atgttatggt aatagttacg agttagttga acttatagat agactggact atacggctat 2280cggtccaaat tagaaagaac gtcaatggct ctctgggcgt cgcctttgcc gacaaaaatg 2340tgatcatgat gaaagccagc aatgacgttg cagctgatat tgttgtcggc caaccgcgcc 2400gaaaacgcag ctgtcagacc cacagcctcc aacgaagaat gtatcgtcaa agtgatccaa 2460gcacactcat agttggagtc gtactccaaa ggcggcaatg acgagtcaga cagatactcg 2520tcgacgttta aaccatcatc taagggcctc aaaactacct cggaactgct gcgctgatct 2580ggacaccaca gaggttccga gcactttagg ttgcaccaaa tgtcccacca ggtgcaggca 2640gaaaacgctg gaacagcgtg tacagtttgt cttaacaaaa agtgagggcg ctgaggtcga 2700gcagggtggt gtgacttgtt atagccttta gagctgcgaa agcgcgtatg gatttggctc 2760atcaggccag attgagggtc tgtggacaca tgtcatgtta gtgtacttca atcgccccct 2820ggatatagcc ccgacaatag gccgtggcct catttttttg ccttccgcac atttccattg 2880ctcggtaccc acaccttgct tctcctgcac ttgccaacct taatactggt ttacattgac 2940caacatctta caagcggggg gcttgtctag ggtatatata aacagtggct ctcccaatcg 3000gttgccagtc tcttttttcc tttctttccc cacagattcg aaatctaaac tacacatcac 3060accatggaca agaaatactc catcggcctg gacattggaa ccaactctgt cggctgggct 3120gtcatcaccg acgagtacaa ggtgccctcc aagaaattca aggtcctcgg aaacaccgat 3180cgacactcca tcaagaaaaa cctcattggt gccctgttgt tcgattctgg cgagactgcc 3240gaagctacca gactcaagcg aactgctcgg cgacgttaca cccgacggaa gaaccgaatc 3300tgctacctgc aggagatctt ttccaacgag atggccaagg tggacgattc gttctttcat 3360cgactggagg aatccttcct cgtcgaggaa gacaagaaac acgagcgtca tcccatcttt 3420ggcaacattg tggacgaggt tgcttaccac gagaagtatc ctaccatcta ccatctccga 3480aagaaactcg tcgattccac cgacaaggcg gatctcagac ttatctacct cgctctggca 3540cacatgatca agtttcgagg tcatttcctc atcgagggcg atctcaatcc cgacaacagc 3600gatgtggaca agctgttcat tcagctcgtt cagacctaca accagctgtt cgaggaaaac 3660cccatcaatg cctccggagt cgatgcaaag gccatcttgt ctgctcgact ctcgaagagc 3720agacgactgg agaacctcat tgcccaactt cctggcgaga aaaagaacgg actgtttggc 3780aacctcattg ccctttctct tggtctcaca cccaacttca agtccaactt cgatctggcg 3840gaggacgcca agctccagct gtccaaggac acctacgacg atgacctcga caacctgctt 3900gcacagattg gcgatcagta cgccgacctg tttctcgctg ccaagaacct ttcggatgct 3960attctcttgt ctgacattct gcgagtcaac accgagatca caaaggctcc cctttctgcc 4020tccatgatca agcgatacga cgagcaccat caggatctca cactgctcaa ggctcttgtc 4080cgacagcaac tgcccgagaa gtacaaggag atctttttcg atcagtcgaa gaacggctac 4140gctggataca tcgacggcgg agcctctcag gaagagttct acaagttcat caagccaatt 4200ctcgagaaga tggacggaac cgaggaactg cttgtcaagc tcaatcgaga ggatctgctt 4260cggaagcaac gaaccttcga caacggcagc attcctcatc agatccacct cggtgagctg 4320cacgccattc ttcgacgtca ggaagacttc tacccctttc tcaaggacaa ccgagagaag 4380atcgagaaga ttcttacctt tcgaatcccc tactatgttg gtcctcttgc cagaggaaac 4440tctcgatttg cttggatgac tcgaaagtcc gaggaaacca tcactccctg gaacttcgag 4500gaagtcgtgg acaagggtgc ctctgcacag tccttcatcg agcgaatgac caacttcgac 4560aagaatctgc ccaacgagaa ggttcttccc aagcattcgc tgctctacga gtactttaca 4620gtctacaacg aactcaccaa agtcaagtac gttaccgagg gaatgcgaaa gcctgccttc 4680ttgtctggcg aacagaagaa agccattgtc gatctcctgt tcaagaccaa ccgaaaggtc 4740actgttaagc agctcaagga ggactacttc aagaaaatcg agtgtttcga cagcgtcgag 4800atttccggag ttgaggaccg attcaacgcc tctttgggca cctatcacga tctgctcaag 4860attatcaagg acaaggattt tctcgacaac gaggaaaacg aggacattct ggaggacatc 4920gtgctcactc ttaccctgtt cgaagatcgg gagatgatcg aggaacgact caagacatac 4980gctcacctgt tcgacgacaa ggtcatgaaa caactcaagc gacgtagata caccggctgg 5040ggaagacttt cgcgaaagct catcaacggc atcagagaca agcagtccgg aaagaccatt 5100ctggactttc tcaagtccga tggctttgcc aaccgaaact tcatgcagct cattcacgac 5160gattctctta ccttcaagga ggacatccag aaggcacaag tgtccggtca gggcgacagc 5220ttgcacgaac atattgccaa cctggctggt tcgccagcca tcaagaaagg cattctccag 5280actgtcaagg ttgtcgacga gctggtgaag gtcatgggac gtcacaagcc cgagaacatt 5340gtgatcgaga tggccagaga gaaccagaca actcaaaagg gtcagaaaaa ctcgcgagag 5400cggatgaagc gaatcgagga aggcatcaag gagctgggat cccagattct caaggagcat 5460cccgtcgaga acactcaact gcagaacgag aagctgtatc tctactatct gcagaatggt 5520cgagacatgt acgtggatca ggaactggac atcaatcgtc tcagcgacta cgatgtggac 5580cacattgtcc ctcaatcctt tctcaaggac gattctatcg acaacaaggt ccttacacga 5640tccgacaaga acagaggcaa gtcggacaac gttcccagcg aagaggtggt caaaaagatg 5700aagaactact ggcgacagct gctcaacgcc aagctcatta cccagcgaaa gttcgacaat 5760cttaccaagg ccgagcgagg cggtctgtcc gagctcgaca aggctggctt catcaagcgt 5820caactcgtcg agaccagaca gatcacaaag cacgtcgcac agattctcga ttctcggatg 5880aacaccaagt acgacgagaa cgacaagctc atccgagagg tcaaggtgat tactctcaag 5940tccaaactgg tctccgattt ccgaaaggac tttcagttct acaaggtgcg agagatcaac 6000aattaccacc atgcccacga tgcttacctc aacgccgtcg ttggcactgc gctcatcaag 6060aaatacccca agctcgaaag cgagttcgtt tacggcgatt acaaggtcta cgacgttcga 6120aagatgattg ccaagtccga acaggagatt ggcaaggcta ctgccaagta cttcttttac 6180tccaacatca tgaacttttt caagaccgag atcaccttgg ccaacggaga gattcgaaag 6240agaccactta tcgagaccaa cggcgaaact ggagagatcg tgtgggacaa gggtcgagac 6300tttgcaaccg tgcgaaaggt tctgtcgatg cctcaggtca acatcgtcaa gaaaaccgag 6360gttcagactg gcggattctc caaggagtcg attctgccca agcgaaactc cgacaagctc 6420atcgctcgaa agaaagactg ggatcccaag aaatacggtg gcttcgattc tcctaccgtc 6480gcctattccg tgcttgtcgt tgcgaaggtc gagaagggca agtccaaaaa gctcaagtcc 6540gtcaaggagc tgctcggaat taccatcatg gagcgatcga gcttcgagaa gaatcccatc 6600gacttcttgg aagccaaggg ttacaaggag gtcaagaaag acctcattat caagctgccc 6660aagtactctc tgttcgaact ggagaacggt cgaaagcgta tgctcgcctc cgctggcgag 6720ctgcagaagg gaaacgagct tgccttgcct tcgaagtacg tcaactttct ctatctggct 6780tctcactacg agaagctcaa gggttctccc gaggacaacg aacagaagca actcttcgtt 6840gagcagcaca aacattacct cgacgagatt atcgagcaga tttccgagtt ttcgaagcga 6900gtcatcctgg ctgatgccaa cttggacaag gtgctctctg cctacaacaa gcatcgggac 6960aaacccattc gagaacaggc ggagaacatc attcacctgt ttactcttac caacctgggt 7020gctcctgcag ctttcaagta cttcgatacc actatcgacc gaaagcggta cacatccacc 7080aaggaggttc tcgatgccac cctgattcac cagtccatca ctggcctgta cgagacccga 7140atcgacctgt ctcagcttgg tggcgactcc agagccgatc ccaagaaaaa gcgaaaggtc 7200taagcggccg caagtgtgga tggggaagtg agtgcccggt tctgtgtgca caattggcaa 7260tccaagatgg atggattcaa cacagggata tagcgagcta cgtggtggtg cgaggatata 7320gcaacggata tttatgtttg acacttgaga atgtacgata caagcactgt ccaagtacaa 7380tactaaacat actgtacata ctcatactcg tacccgggca acggtttcac ttgagtgcag 7440tggctagtgc tcttactcgt acagtgtgca atactgcgta tcatagtctt tgatgtatat 7500cgtattcatt catgttagtt gcgtacgagc cggaagcata aagtgtaaag cctggggtgc 7560ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 7620aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 7680tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 7740gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 7800cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 7860gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 7920aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 7980ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 8040cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 8100ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 8160cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 8220agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 8280gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 8340gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 8400tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 8460agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 8520agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 8580atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 8640cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 8700actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 8760aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 8820cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 8880ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 8940cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 9000ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 9060cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 9120ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 9180tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 9240ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 9300aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 9360gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 9420gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 9480ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 9540catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 9600atttccccga aaagtgccac ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 9660ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 9720tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 9780gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 9840gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt 9900ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat 9960ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa 10020tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc 10080cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 10140ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg 10200ttttcccagt cacgacgttg taaaacgacg gccagtgaat tgtaatacga ctcactatag 10260ggcgaattgg gtaccgggcc ccccctcgag gtcgatggtg tcgataagct tgatatcgaa 10320ttcatgtcac acaaaccgat cttcgcctca aggaaaccta attctacatc cgagagactg 10380ccgagatcca gtctacactg attaattttc gggccaataa tttaaaaaaa tcgtgttata 10440taatattata tgtattatat atatacatca tgatgatact gacagtcatg tcccattgct 10500aaatagacag actccatctg ccgcctccaa ctgatgttct caatatttaa ggggtcatct 10560cgcattgttt aataataaac agactccatc taccgcctcc aaatgatgtt ctcaaaatat 10620attgtatgaa cttattttta ttacttagta ttattagaca acttacttgc tttatgaaaa 10680acacttccta tttaggaaac aatttataat ggcagttcgt tcatttaaca atttatgtag 10740aataaatgtt ataaatgcgt atgggaaatc ttaaatatgg atagcataaa tgatatctgc 10800attgcctaat tcgaaatcaa cagcaacgaa aaaaatccct tgtacaacat aaatagtcat 10860cgagaaatat caactatcaa agaacagcta ttcacacgtt actattgaga ttattattgg 10920acgagaatca cacactcaac tgtctttctc tcttctagaa atacaggtac aagtatgtac 10980tattctcatt gttcatactt ctagtcattt catcccacat attccttgga tttctctcca 11040atgaatgaca ttctatcttg caaattcaac aattataata agatatacca aagtagcggt 11100atagtggcaa tcaaaaagct tctctggtgt gcttctcgta tttattttta ttctaatgat 11160ccattaaagg tatatattta tttcttgtta tataatcctt ttgtttatta catgggctgg 11220atacataaag gtattttgat ttaatttttt gcttaaattc aatcccccct cgttcagtgt 11280caactgtaat ggtaggaaat taccatactt ttgaagaagc aaaaaaaatg aaagaaaaaa 11340aaaatcgtat ttccaggtta gacgttccgc agaatctaga atgcggtatg cggtacattg 11400ttcttcgaac gtaaaagttg cgctccctga gatattgtac atttttgctt ttacaagtac 11460aagtacatcg tacaactatg tactactgtt gatgcatcca caacagtttg ttttgttttt 11520ttttgttttt tttttttcta atgattcatt accgctatgt atacctactt gtacttgtag 11580taagccgggt tattggcgtt caattaatca tagacttatg aatctgcacg gtgtgcgctg 11640cgagttactt ttagcttatg catgctactt gggtgtaata ttgggatctg ttcggaaatc 11700aacggatgct caat 1171412620DNAArtificial sequenceCER forward 126gttttagagc tagaaatagc 2012720DNAArtificial sequenceuniveral reverse 127gcaccgactc ggtgccactt 2012818DNAArtificial sequenceuniversal forward T7 primer 128taatacgact cactatag 1812934DNAArtificial sequencegalK2-1 forward primer 129taatacgact cactatagat cagcggcaat gtgc 3413034DNAArtificial sequencegalK2-1 reverse primer 130ttctagctct aaaactgcgg cacattgccg ctga 34131114DNAArtificial SequencegalK2-1 sgRNA in vitro transcription template 131taatacgact cactatagat cagcggcaat gtgccgcagt tttagagcta gaaatagcaa 60gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgc 11413218DNAT7 phage 132taatacgact cactatag 1813320DNAEscherichia coli 133atcagcggca atgtgccgca 2013423DNAEscherichia coli 134atcagcggca atgtgccgca ggg 2313596RNAArtificial sequencegalK2-1 sgRNA 135aucagcggca augugccgca guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugc 96136262PRTArtificial sequencehis-MPG1-dsREDexpress 136Met Gly His His His His His His Gly Ala Leu Phe Leu Gly Gln Leu1 5 10 15Gly Ala Ala Gly Ser Thr Met Gly Ala Pro Lys Lys Lys Arg Lys Val 20 25 30Glu Phe Gly Gly Gly Gly Ala Ser Ser Glu Asp Val Ile Lys Glu Phe 35 40 45Met Arg Phe Lys Val Arg Met Glu Gly Ser Val Asn Gly His Glu Phe 50 55 60Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr65 70 75 80Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp 85 90 95Ile Leu Ser Pro Gln Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His 100 105 110Pro Ala Asp Ile Pro Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe 115 120 125Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val 130 135 140Thr Gln Asp Ser Ser Leu Gln Asp Gly Ser Phe Ile Tyr Lys Val Lys145 150 155 160Phe Ile Gly Val Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys 165 170 175Thr Met Gly Trp Glu Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly 180 185 190Val Leu Lys Gly Glu Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly 195 200 205His Tyr Leu Val Glu Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val 210 215 220Gln Leu Pro Gly Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser225 230 235 240His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly 245 250 255Arg His His Leu Phe Leu 260137256PRTArtificial sequencepVEC-dsREDexpress 137Met Gly His His His His His His Leu Leu Ile Ile Leu Arg Arg Arg1 5 10 15Ile Arg Lys Gln Ala His Ala His Ser Lys Glu Phe Gly Gly Gly Gly 20 25 30Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe Lys Val Arg 35 40 45Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly 50 55 60Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr65 70 75 80Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe 85 90 95Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro Asp 100 105 110Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met 115 120 125Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu 130 135 140Gln Asp Gly Ser Phe Ile Tyr Lys Val Lys Phe Ile Gly Val Asn Phe145 150 155 160Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala 165 170 175Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu Ile 180 185 190His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu Phe 195 200 205Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr Tyr 210 215 220Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr225 230 235 240Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His His Leu Phe Leu 245 250 255138245PRTArtificial sequenceCFFKDEL-dsREDexpress 138Met Gly His His His His His His Cys Phe Phe Lys Asp Glu Leu Glu1 5 10 15Phe Gly Gly Gly Gly Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met 20 25 30Arg Phe Lys Val Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu 35 40 45Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala 50 55 60Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile65 70 75 80Leu Ser Pro Gln Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro 85 90 95Ala Asp Ile Pro Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys 100 105 110Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr 115 120 125Gln Asp Ser Ser Leu Gln Asp Gly Ser Phe Ile Tyr Lys Val Lys Phe 130 135 140Ile Gly Val Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys

Thr145 150 155 160Met Gly Trp Glu Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val 165 170 175Leu Lys Gly Glu Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly His 180 185 190Tyr Leu Val Glu Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln 195 200 205Leu Pro Gly Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His 210 215 220Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg225 230 235 240His His Leu Phe Leu 245139257PRTArtificial SequenceTLM-dsREDexpress 139Met Gly His His His His His His Pro Leu Ser Ser Ile Phe Ser Arg1 5 10 15Ile Gly Asp Pro Pro Lys Lys Lys Arg Lys Val Glu Phe Gly Gly Gly 20 25 30Gly Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe Lys Val 35 40 45Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu 50 55 60Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val65 70 75 80Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln 85 90 95Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp Ile Pro 100 105 110Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val 115 120 125Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser 130 135 140Leu Gln Asp Gly Ser Phe Ile Tyr Lys Val Lys Phe Ile Gly Val Asn145 150 155 160Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu 165 170 175Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys Gly Glu 180 185 190Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu Val Glu 195 200 205Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro Gly Tyr 210 215 220Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr225 230 235 240Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His His Leu Phe 245 250 255Leu140292PRTArtificial sequenceZebra-dsREDexpress 140Met Gly His His His His His His Glu Cys Asp Ser Glu Leu Glu Ile1 5 10 15Lys Arg Tyr Lys Arg Val Arg Val Ala Ser Arg Lys Cys Arg Ala Lys 20 25 30Phe Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser 35 40 45Ser Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Gln Met Cys Glu Phe 50 55 60Gly Gly Gly Gly Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg65 70 75 80Phe Lys Val Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile 85 90 95Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys 100 105 110Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu 115 120 125Ser Pro Gln Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala 130 135 140Asp Ile Pro Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp145 150 155 160Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln 165 170 175Asp Ser Ser Leu Gln Asp Gly Ser Phe Ile Tyr Lys Val Lys Phe Ile 180 185 190Gly Val Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met 195 200 205Gly Trp Glu Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu 210 215 220Lys Gly Glu Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr225 230 235 240Leu Val Glu Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu 245 250 255Pro Gly Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn 260 265 270Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His 275 280 285His Leu Phe Leu 290141259PRTArtificial sequencepep1-dsREDexpress 141Met Gly His His His His His His Lys Glu Thr Trp Trp Glu Thr Trp1 5 10 15Trp Thr Glu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val Glu Phe Gly 20 25 30Gly Gly Gly Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe 35 40 45Lys Val Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu 50 55 60Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu65 70 75 80Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser 85 90 95Pro Gln Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp 100 105 110Ile Pro Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu 115 120 125Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp 130 135 140Ser Ser Leu Gln Asp Gly Ser Phe Ile Tyr Lys Val Lys Phe Ile Gly145 150 155 160Val Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly 165 170 175Trp Glu Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys 180 185 190Gly Glu Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu 195 200 205Val Glu Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro 210 215 220Gly Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn Glu225 230 235 240Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His His 245 250 255Leu Phe Leu142259PRTArtificial sequencetp10-dsREDexpress 142Met Gly His His His His His His Ala Gly Tyr Leu Leu Gly Lys Ile1 5 10 15Asn Leu Lys Ala Cys Ala Ala Cys Ala Lys Lys Ile Leu Glu Phe Gly 20 25 30Gly Gly Gly Ala Ser Ser Glu Asp Val Ile Lys Glu Phe Met Arg Phe 35 40 45Lys Val Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu 50 55 60Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr Ala Lys Leu65 70 75 80Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser 85 90 95Pro Gln Phe Gln Tyr Gly Ser Lys Val Tyr Val Lys His Pro Ala Asp 100 105 110Ile Pro Asp Tyr Lys Lys Leu Ser Phe Pro Glu Gly Phe Lys Trp Glu 115 120 125Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val Thr Gln Asp 130 135 140Ser Ser Leu Gln Asp Gly Ser Phe Ile Tyr Lys Val Lys Phe Ile Gly145 150 155 160Val Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly 165 170 175Trp Glu Ala Ser Thr Glu Arg Leu Tyr Pro Arg Asp Gly Val Leu Lys 180 185 190Gly Glu Ile His Lys Ala Leu Lys Leu Lys Asp Gly Gly His Tyr Leu 195 200 205Val Glu Phe Lys Ser Ile Tyr Met Ala Lys Lys Pro Val Gln Leu Pro 210 215 220Gly Tyr Tyr Tyr Val Asp Ser Lys Leu Asp Ile Thr Ser His Asn Glu225 230 235 240Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly Arg His His 245 250 255Leu Phe Leu1431442PRTArtificial sequenceZebra-Cas9 143Met Gly His His His His His His Glu Cys Asp Ser Glu Leu Glu Ile1 5 10 15Lys Arg Tyr Lys Arg Val Arg Val Ala Ser Arg Lys Cys Arg Ala Lys 20 25 30Phe Lys Gln Leu Leu Gln His Tyr Arg Glu Val Ala Ala Ala Lys Ser 35 40 45Ser Glu Asn Asp Arg Leu Arg Leu Leu Leu Lys Gln Met Cys Glu Phe 50 55 60Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly65 70 75 80Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys 85 90 95Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly 100 105 110Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys 115 120 125Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr 130 135 140Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe145 150 155 160Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His 165 170 175Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His 180 185 190Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser 195 200 205Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met 210 215 220Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp225 230 235 240Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn 245 250 255Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys 260 265 270Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu 275 280 285Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu 290 295 300Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp305 310 315 320Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp 325 330 335Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu 340 345 350Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile 355 360 365Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met 370 375 380Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala385 390 395 400Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp 405 410 415Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln 420 425 430Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly 435 440 445Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys 450 455 460Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly465 470 475 480Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu 485 490 495Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro 500 505 510Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met 515 520 525Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val 530 535 540Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn545 550 555 560Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu 565 570 575Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr 580 585 590Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys 595 600 605Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val 610 615 620Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser625 630 635 640Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr 645 650 655Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn 660 665 670Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu 675 680 685Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His 690 695 700Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr705 710 715 720Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys 725 730 735Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala 740 745 750Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys 755 760 765Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His 770 775 780Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile785 790 795 800Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg 805 810 815His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr 820 825 830Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu 835 840 845Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val 850 855 860Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln865 870 875 880Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu 885 890 895Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp 900 905 910Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly 915 920 925Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn 930 935 940Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe945 950 955 960Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys 965 970 975Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys 980 985 990His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu 995 1000 1005Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 1010 1015 1020Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val 1025 1030 1035Arg Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn 1040 1045 1050Ala Val Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu 1055 1060 1065Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys 1070 1075 1080Met Ile Ala Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys 1085 1090 1095Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile 1100 1105 1110Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr 1115 1120 1125Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe 1130 1135 1140Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val 1145 1150 1155Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile 1160 1165 1170Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp 1175 1180 1185Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala 1190 1195 1200Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys 1205 1210 1215Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu 1220 1225 1230Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys 1235 1240 1245Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys 1250 1255 1260Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala 1265 1270 1275Ser Ala Gly Glu

Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser 1280 1285 1290Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu 1295 1300 1305Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu 1310 1315 1320Gln His Lys His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu 1325 1330 1335Phe Ser Lys Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val 1340 1345 1350Leu Ser Ala Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln 1355 1360 1365Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala 1370 1375 1380Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg 1385 1390 1395Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln 1400 1405 1410Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu 1415 1420 1425Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val 1430 1435 14401441406PRTArtificial sequencepVEC-Cas9 144Met Gly His His His His His His Leu Leu Ile Ile Leu Arg Arg Arg1 5 10 15Ile Arg Lys Gln Ala His Ala His Ser Lys Glu Phe Asp Lys Lys Tyr 20 25 30Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile 35 40 45Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn 50 55 60Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe65 70 75 80Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg 85 90 95Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile 100 105 110Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu 115 120 125Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His Pro 130 135 140Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro145 150 155 160Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala 165 170 175Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg 180 185 190Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val 195 200 205Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu 210 215 220Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser225 230 235 240Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu 245 250 255Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser 260 265 270Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp 275 280 285Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn 290 295 300Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala305 310 315 320Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn 325 330 335Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr 340 345 350Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln 355 360 365Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn 370 375 380Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr385 390 395 400Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu 405 410 415Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe 420 425 430Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His Ala 435 440 445Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg 450 455 460Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly465 470 475 480Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser 485 490 495Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly 500 505 510Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn 515 520 525Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr 530 535 540Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly545 550 555 560Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val 565 570 575Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys 580 585 590Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser 595 600 605Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu 610 615 620Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu625 630 635 640Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg 645 650 655Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp 660 665 670Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg 675 680 685Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys 690 695 700Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe705 710 715 720Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln 725 730 735Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile Ala 740 745 750Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val 755 760 765Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro Glu 770 775 780Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly785 790 795 800Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys 805 810 815Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln 820 825 830Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp 835 840 845Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp 850 855 860Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp865 870 875 880Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn 885 890 895Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln 900 905 910Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr 915 920 925Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile 930 935 940Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln945 950 955 960Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu 965 970 975Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp 980 985 990Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr 995 1000 1005His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala 1010 1015 1020Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly 1025 1030 1035Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu 1040 1045 1050Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn 1055 1060 1065Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu 1070 1075 1080Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu 1085 1090 1095Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val 1100 1105 1110Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln 1115 1120 1125Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser 1130 1135 1140Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr 1145 1150 1155Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val 1160 1165 1170Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys 1175 1180 1185Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys 1190 1195 1200Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys 1205 1210 1215Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu 1220 1225 1230Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln 1235 1240 1245Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu 1250 1255 1260Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp 1265 1270 1275Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu 1280 1285 1290Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile 1295 1300 1305Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys 1310 1315 1320His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His 1325 1330 1335Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr 1340 1345 1350Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu 1355 1360 1365Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr 1370 1375 1380Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala 1385 1390 1395Asp Pro Lys Lys Lys Arg Lys Val 1400 1405

Claims

1. A composition comprising at least one protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein said protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, and wherein said RGEN protein-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a microbial cell.

2. The composition of claim 1, wherein the protein component of the RGEN is associated with at least one RNA component that comprises a sequence complementary to a target site sequence on a chromosome or episome in the microbial cell, wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence.

3. The composition of claim 2, wherein the RNA component comprises a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) operably linked to a trans-activating CRISPR RNA (tracrRNA).

4. The composition of claim 2, wherein the RGEN can cleave one or both DNA strands at the target site sequence.

5. The composition of claim 1, wherein the RGEN comprises a CRISPR-associated (Cas) protein-9 (Cas9) amino acid sequence.

6. The composition of claim 1, wherein the RGEN protein component and CPP are covalently linked.

7. The composition of claim 1, wherein the RGEN protein component and CPP are non-covalently linked.

8. The composition of claim 1, wherein the CPP is cationic or amphipathic.

9. The composition of claim 1, wherein the CPP comprises: (i) a CPP from an Epstein-Barr virus Zebra trans-activator protein, (ii) a CPP having 6 or more contiguous arginine residues, (iii) a transportan-10 (TP10) CPP, or (iv) a CPP from a vascular endothelium cadherin protein.

10. The composition of claim 1, wherein said RGEN protein-CPP complex can traverse a cell wall and cell membrane of a microbial cell.

11. A cell comprising the composition according to claim 1.

12. A method of delivering a protein component of an RNA-guided endonuclease (RGEN) into a microbial cell, said method comprising: contacting the microbial cell with a composition comprising the protein component of the RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein said protein component and CPP are covalently, or non-covalently, linked to each other in an RGEN protein-CPP complex, wherein said RGEN protein-CPP complex traverses (i) a cell membrane, or (ii) a cell wall and cell membrane, of the cell, thereby entering the microbial cell.

13. The method of claim 12, wherein: (i) the composition further comprises at least one RNA component that is associated with the protein component of the RGEN; or (ii) the microbial cell comprises the RNA component, wherein the RNA component associates with the protein component of the RGEN after the RGEN protein-CPP complex enters the microbial cell; wherein the RNA component comprises a sequence complementary to a target site sequence on a chromosome or episome in the cell, wherein the RGEN can bind to the target site sequence, and optionally cleave one or both DNA strands at the target site sequence.

14. The method of claim 13, wherein the RGEN can cleave one or both DNA strands at the target site sequence.

15. The method of claim 14, wherein the microbial cell further comprises a donor polynucleotide comprising at least one sequence homologous to a sequence at or near the target site sequence, and wherein the donor polynucleotide integrates at or near the target site sequence by homologous recombination.

16. A polynucleotide sequence comprising a nucleotide sequence encoding an RGEN protein-CPP fusion protein that comprises a protein component of an RNA-guided endonuclease (RGEN) and at least one cell-penetrating peptide (CPP), wherein optionally, said nucleotide sequence is operably linked to a promoter sequence.

17. A method of producing an RGEN protein-CPP fusion protein comprising: (a) providing the polynucleotide sequence of claim 16; (b) expressing the RGEN protein-CPP fusion protein from the polynucleotide sequence, thereby producing the RGEN protein-CPP fusion protein, wherein said expressing is optionally performed in a cell; and (c) optionally, isolating the RGEN protein-CPP fusion protein produced in step (b).

18. A composition comprising at least one protein component of a guide polynucleotide/Cas endonuclease complex and at least one cell-penetrating peptide (CPP), wherein said protein component and CPP are covalently, or non-covalently, linked to each other in a guide polynucleotide/Cas endonuclease-CPP complex, and wherein said guide polynucleotide/Cas endonuclease-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of a microbial cell.

19. A method for modifying a target site in the genome of a cell, the method comprising providing a guide polynucleotide, a cell-penetrating peptide (CPP) and a Cas endonuclease to the cell, wherein said guide polynucleotide, Cas endonuclease and CPP are covalently, or non-covalently, linked to each other in a guide polynucleotide/Cas endonuclease-CPP complex, and wherein said guide polynucleotide/Cas endonuclease-CPP complex can traverse (i) a cell membrane, or (ii) a cell wall and cell membrane, of the microbial cell

Images