Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
This application is a 371 of PCT/CN2023/073828.
The response filed on August 11, 2026 has been entered.
Election/Restrictions
Applicant’s election without traverse of Group I with a species election of (1) SEQ ID NO:1 as the parent nuclease and (2) K169R + R589H (SEQ ID NO:3) as the amino acid modifications made in the parent in the reply filed on August 11, 2026 is acknowledged.
Claims 7-8, 10-11, and 13-22 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on August 11, 2026.
Status of Claims
Claims 1-8, 10-11, and 13-22 are pending.
Claims 7-8, 10-11, and 13-22 are withdrawn.
Claims 1-6 are under examination.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Drawings
The drawings are objected to because Fig. 4-9 are blurry and illegible. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Nucleotide and/or Amino Acid Sequence Disclosures
Summary of Requirements for Patent Applications Filed On Or After July 1, 2022, That Have Sequence Disclosures
37 CFR 1.831(a) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.831(b) must contain a “Sequence Listing XML”, as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.831-1.835. This “Sequence Listing XML” part of the disclosure may be submitted:
1. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter “Legal Framework”) in XML format, together with an incorporation by reference statement of the material in the XML file in a separate paragraph of the specification (an incorporation by reference paragraph) as required by 37 CFR 1.835(a)(2) or 1.835(b)(2) identifying:
a. the name of the XML file
b. the date of creation; and
c. the size of the XML file in bytes; or
2. In accordance with 37 CFR 1.831(a) using the symbols and format requirements of 37 CFR 1.832 through 1.834 on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation by reference statement of the material in the XML format according to 37 CFR 1.52(e)(8) and 37 CFR 1.835(a)(2) or 1.835(b)(2) in a separate paragraph of the specification identifying:
a. the name of the XML file;
b. the date of creation; and
c. the size of the XML file in bytes.
SPECIFIC DEFICIENCIES AND THE REQUIRED RESPONSE TO THIS NOTICE ARE AS FOLLOWS:
Specific deficiency - Sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.831(c), see Fig. 4-8. Sequence identifiers for sequences (i.e., “SEQ ID NO:X” or the like) must appear either in the drawings or in the Brief Description of the Drawings.
Required response – Applicant must provide:
Amended drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required sequence identifiers (i.e., “SEQ ID NO:X” or the like) into the Brief Description of the Drawings, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
Specific deficiency - Sequences appearing in the specification are not identified by sequence identifiers (i.e., “SEQ ID NO:X” or the like) in accordance with 37 CFR 1.831(c), see page 20, 21, 23, 24, and5.
Required response – Applicant must provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3), and 1.125 inserting the required sequence identifiers, consisting of:
• A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
• A copy of the amended specification without markings (clean version); and
• A statement that the substitute specification contains no new matter.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code, for example page 23. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on November 25, 2024 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 2 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 2 recites the phrase “preferably”. The phrase renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim 3 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 3 recites the broad recitation “at least 80%...99%” and “at least 99%”. The limitation “at least 80%” is equivalent to a range of “80%-100%” and the limitation “at least…99%” is equivalent to a range of “99%-100%”. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
Claim 4 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 3 recites the broad recitation “at least 92%...99%” and “at least 99%..100%”. The limitation “at least 92%” is equivalent to a range of “92%-100%” and the limitation “at least…99%” is equivalent to a range of “99%-100%”. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-6 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow.'' In this case, the claims have been broadly interpreted to encompass (A) any polypeptide or any polypeptide having at least 80% or at least 92% sequence identity to SEQ ID NO:1, 2, or 3, wherein said polypeptide has an Arg residue at the position corresponding to 169 of SEQ ID NO:1 or (B) any polypeptide having an Arg residue and His residue at the positions corresponding to 169 and 159, respectively of SEQ ID NO:1, wherein the polypeptide of (A) or (B) has nuclease activity and improved editing activity in yeast. Therefore, the claims are drawn to a genus of polypeptides having unknown structure, except having an Arg and His residues at positions 169 and 589, respectively, of SEQ ID NO:1, but having nuclease activity and improved editing activity. but having nuclease activity and improved editing activity in yeast.
MPEP 2163 I. states that to “satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention.
MPEP 2163. II.A.3.(a) sates that “Possession may be shown in many ways. For example, possession may be shown by describing an actual reduction to practice of the claimed invention. Possession may also be shown by a clear depiction of the invention in detailed drawings or in structural chemical formulas which permit a person skilled in the art to clearly recognize that inventor had possession of the claimed invention. An adequate written description of the invention may be shown by any description of sufficient, relevant, identifying characteristics so long as a person skilled in the art would recognize that the inventor had possession of the claimed invention.
According to MPEP 2163.II.A.3.(a).ii), “Satisfactory disclosure of a ‘representative number’ depends on whether one of skill in the art would recognize that the applicant was in possession of the necessary common attributes or features possessed by the members of the genus in view of the species disclosed. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus…Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are ‘representative of the full variety or scope of the genus,’ or by the establishment of ‘a reasonable structure-function correlation.’"
The recitation of “nuclease” and “improved editing activity in yeast” fails to provide a sufficient description of the genus of the polypeptides as it merely describes the functional features of the genus without providing any definition of the structural features of the species within the genus. The specification does not specifically define any of the species that fall within the genus. The specification does not define any structural features commonly possessed by members of the genus that distinguish them from others. One skilled in the art therefore cannot, as one can do with a fully described genus, visualize or recognize the identity of the members of the genus.
Eubacterium rectale MAD7 nuclease having the amino acid sequence of SEQ ID NO:1 was known in the art. Jones (WO 2021/257716 - form PTO-1449) and Mijts (US 10,604,746 – form PTO-892) both disclose Eubacterium rectale MAD7 nuclease having 100% sequence identity to the MAD7 nuclease of SEQ ID NO:1 of the instant application (see the sequence alignments below). Jones and Mijts disclose some engineered MAD7 nucleases of SEQ ID NO:1. However, the claimed genus of polypeptides having nuclease activity and improved editing activity in yeast were not known in the prior art nor disclosed in the instant specification.
Fransceus (J Ind Microbiol Biotechnol. 2017 May;44(4-5):687-695. – form PTO-892) reviews protein engineering techniques, such as random mutagenesis and recombination, directed evolution and iterative or combinatory saturation “hotspots”. Fransceus states that “a recurring problem, however, is choosing which amino acid positions should be targeted. Answering this question is not an easy feat and requires substantial insight in the relationship between an enzyme’s sequence or structure and its properties.” Sanavia (Computational and Structural Biotechnology Journal, Volume 18, 2020, Pages 1968-1979. – form PTO-892) discloses challenges in the prediction of protein stability in the occurrence of multiple mutations. “Multiple-point mutations are common variations of the protein sequence that may be needed in protein engineering when a single-point mutation is not enough to yield the desired stability change. Dealing with multiple-site variations adds another level of complexity beyond the prediction of the effect of a single variant on protein stability, since it requires the learning of many types of combinatorial effects”.
The specification is limited to description of an engineered MAD 7 nucleases of SEQ ID NO:1 having two point mutations: SEQ ID NO:2 (K169R mutant), SEQ ID NO:3 (K169R + N589Hmutant), SEQ ID NO:11 (K169R + K535R mutant ), SEQ ID NO:12 (K169R + K563R mutant), SEQ ID NO:13 (K169R + T601R mutant ), and SEQ ID NO:14 (K169R +S624R mutant). While MPEP 2163 acknowledges that in certain situations “one species adequately supports a genus,” it also acknowledges that “[f]or inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus.” In view of the widely variant species encompassed by the genus, the examples described above are not enough and does not constitute a representative number of species to describe the whole genus of polypeptides having nuclease activity and improved editing activity in yeast compared to SEQ ID NO:1. Therefore, the specification fails to describe a representative species of the claimed genus.
Further, one of skill in the art could identify polypeptides having at least 80% sequence identity to SEQ ID NO:1 or at least 92% sequence identity to SEQ ID NO:2 or 3. However, there is no teaching regarding which 20% of the amino acids can vary from SEQ ID NO:1 or which 8% of the amino acids can vary from SEQ ID NO:2 or 3 and result in polypeptide having nuclease activity and improved editing activity in yeast compared to SEQ ID NO:1. An important consideration is that structure is not necessarily a reliable indicator of function. In the instant case, there is no disclosure relating similarity of structure to conservation of function. Conservation of structure is not necessarily a surrogate for conservation of function. Since the claimed invention is that of an enzyme, and there is no disclosure of the domains responsible for nuclease activity and improved editing activity in yeast compared to SEQ ID NO:1, the absence of information may be persuasive that those of skill in the art would not take the disclosure as generic.
Given this lack of description of the representative species encompassed by the genus of the claims, the specification fails to sufficiently describe the claimed invention in such full, clear, concise, and exact terms that a skilled artisan would recognize that applicants were in possession of the inventions of claims 1-6.
Claims 1-6 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for an engineered MAD 7 nucleases of SEQ ID NO:1 having two point mutations: SEQ ID NO:2 (K169R mutant), SEQ ID NO:3 (K169R + N589Hmutant), SEQ ID NO:11 (K169R + K535R mutant ), SEQ ID NO:12 (K169R + K563R mutant), SEQ ID NO:13 (K169R + T601R mutant ), and SEQ ID NO:14 (K169R +S624R mutant), does not reasonably provide enablement for any polypeptide having unknown structure, except having an Arg and His residues at positions 169 and 589, respectively of SEQ ID NO:1, but having nuclease activity and improved editing activity in yeast. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.
The breadth of the claims.
MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow.'' In this case, the claims have been broadly interpreted to encompass (A) any polypeptide or any polypeptide having at least 80% or at least 92% sequence identity to SEQ ID NO:1, 2, or 3, wherein said polypeptide has an Arg residue at the position corresponding to 169 of SEQ ID NO:1 or (B) any polypeptide having an Arg residue and His residue at the positions corresponding to 169 and 159, respectively of SEQ ID NO:1, wherein the polypeptide of (A) or (B) has nuclease activity and improved editing activity in yeast. Therefore, the claims are drawn to any polypeptide having unknown structure, except having an Arg and His residues at positions 169 and 589, respectively, of SEQ ID NO:1, but having nuclease activity and improved editing activity in yeast.
The claims are not commensurate with the enablement provided by the disclosure with regard to the extremely large number of polypeptides having nuclease activity and improved editing activity in yeast. In the instant case, the specification is limited to an engineered MAD 7 nucleases of SEQ ID NO:1 having two point mutations: SEQ ID NO:2 (K169R mutant), SEQ ID NO:3 (K169R + N589Hmutant), SEQ ID NO:11 (K169R + K535R mutant ), SEQ ID NO:12 (K169R + K563R mutant), SEQ ID NO:13 (K169R + T601R mutant ), and SEQ ID NO:14 (K169R +S624R mutant).
The quantity of experimentation required to practice the claimed invention based on the teachings of the specification.
While enzyme isolation techniques, recombinant and mutagenesis techniques were known in the art at the time of the invention, e.g. mutagenesis, and it is routine in the art to screen for variants comprising multiple substitutions or multiple modifications as encompassed by the instant claims, the specific amino acid positions within the protein's sequence where amino acid modifications can be made with a reasonable expectation of success in obtaining the desired activity/utility are limited in any protein and the result of such modifications is unpredictable. In addition, one skilled in the art would expect any tolerance to modification for a given protein to diminish with each further and additional modification, e.g. multiple substitutions.
In the absence of: (a) rational and predictable scheme for making any polypeptide or any polypeptide having at least 80% sequence identity to SEQ ID NO:1 or having at least 92% sequence identity to SEQ ID NO:2,3, 11, 12, 14, or 14, wherein the polypeptide has nuclease activity and improved editing activity in yeast, and (b) a correlation between structure and the function of having nuclease activity and improved editing activity in yeast the specification provides insufficient guidance as to which of the essentially infinite possible choices is likely to be successful. One of skill in the art would have to test these infinite possible polynucleotides to determine which polypeptides have nuclease activity and improved editing activity in yeast While enablement is not precluded by the necessity for routine screening, if a large amount of screening is required, as is the case herein, the specification must provide a reasonable amount of guidance which respect to the direction in which the experimentation should proceed so that a reasonable number of species can be selected for testing. In view of the fact that such guidance has not been provided in the instant specification, it would require undue experimentation to enable the full scope of the claims.
The state of prior art, the relative skill of those in the art, and predictability or unpredictability of the art.
Since the amino acid sequence of the mutant determines its structural and functional properties, predictability of which changes can be tolerated in a protein's amino acid sequence and obtain the desired activity requires a knowledge of and guidance with regard to which amino acids in the protein's sequence, if any, are tolerant of modification and which are conserved (i.e. expectedly intolerant to modification), and detailed knowledge of the ways in which the proteins' structure relates to its function. In the instant case, neither the specification or the art provide a correlation between structure and activity such that one of skill in the art can envision the structure of any polypeptides having nuclease activity and improved editing activity in yeast or predict said function of a polypeptide from its primary structure. In addition, the art does not provide any teaching or guidance as to (1) which amino acids (other than those at positions 169 and 589) within any polypeptide or the polypeptide of SEQ ID NO:1 hat can be modified and which ones are conserved such that one of skill in the art can make the recited polypeptides having nuclease activity and improved editing activity in yeast, (2) which segments (except having an Arg and His residues at positions 169 and 589, respectively of SEQ ID NO:1) of any polypeptide or the polypeptide of SEQ ID NO:1 that are essential for polypeptides having nuclease activity and improved editing activity in yeast and (3) the general tolerance of any polypeptide or the polypeptide SEQ ID NO:1 to structural modifications and the extent of such tolerance. The art clearly teaches that changes in a protein's amino acid sequence to obtain the desired activity without any guidance/knowledge as to which amino acids in a protein are required for that activity is highly unpredictable. At the time of the invention there was a high level of unpredictability associated with altering a polypeptide sequence with an expectation that the polypeptide will maintain the desired activity. For example, Studer (Residue mutations and their impact on protein structure and function: detecting beneficial and pathogenic changes. Biochem. J. (2013) 449, 581–594. – form PTO-892) teach that (1) protein engineers are frequently surprised by the range of effects caused by single mutations that they hoped would change only one specific and simple property in enzymes, (2) the often surprising results obtained by experiments where single mutations are made reveal how little is known about the rules of protein stability, and (3) the difficulties in designing de novo stable proteins with specific functions.
Eubacterium rectale MAD7 nuclease having the amino acid sequence of SEQ ID NO:1 was known in the art. Jones (WO 2021/257716 - form PTO-1449) and Mijts (US 10,604,746 – form PTO-892) both disclose Eubacterium rectale MAD7 nuclease having 100% sequence identity to the MAD7 nuclease of SEQ ID NO:1 of the instant application (see the sequence alignments below). Jones and Mijts disclose some engineered MAD7 nucleases of SEQ ID NO:1. However, the claimed genus of polypeptides having nuclease activity and improved editing activity in yeast were not known in the prior art nor disclosed in the instant specification.
Fransceus (J Ind Microbiol Biotechnol. 2017 May;44(4-5):687-695. – form PTO-892) reviews protein engineering techniques, such as random mutagenesis and recombination, directed evolution and iterative or combinatory saturation “hotspots”. Fransceus states that “a recurring problem, however, is choosing which amino acid positions should be targeted. Answering this question is not an easy feat and requires substantial insight in the relationship between an enzyme’s sequence or structure and its properties.” Sanavia (Computational and Structural Biotechnology Journal, Volume 18, 2020, Pages 1968-1979. – form PTO-892) discloses challenges in the prediction of protein stability in the occurrence of multiple mutations. “Multiple-point mutations are common variations of the protein sequence that may be needed in protein engineering when a single-point mutation is not enough to yield the desired stability change. Dealing with multiple-site variations adds another level of complexity beyond the prediction of the effect of a single variant on protein stability, since it requires the learning of many types of combinatorial effects”.
The amount of direction or guidance presented and the existence of working examples.
The specification is limited to an engineered MAD 7 nucleases of SEQ ID NO:1 having two point mutations: SEQ ID NO:2 (K169R mutant), SEQ ID NO:3 (K169R + N589Hmutant), SEQ ID NO:11 (K169R + K535R mutant ), SEQ ID NO:12 (K169R + K563R mutant), SEQ ID NO:13 (K169R + T601R mutant ), and SEQ ID NO:14 (K169R +S624R mutant). However, the speciation fails to provide any information as to (1) specific substrates associated with any polypeptide or any polypeptide having at least 80% sequence identity to SEQ ID NO: 1 or (2) structural elements (other than having an Arg and His residues at positions 169 and 589, respectively of SEQ ID NO:1) required in a polypeptide having nuclease activity and improved editing activity in yeast. No correlation between structure and function of having nuclease activity and improved editing activity in yeas has been presented.
Thus, in view of the overly broad scope of the claims, the lack of guidance and working examples provided in the specification, the high level of unpredictability of the prior art in regard to structural changes and their effect on function and the lack of knowledge about a correlation between structure and function, an undue experimentation would be necessary one having ordinary skill in the art to make and use the claimed invention in a manner reasonably correlated with the scope of the claims. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of polypeptides having the desired biological characteristics recited in the claims are unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988).
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1-6 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Jones (WO 2021/257716 - form PTO-1449).
Jones discloses an Eubacterium rectale MAD7 nuclease having the amino acid sequence of SEQ ID NO:1, which is identical to the Eubacterium rectale MAD7 having the amino acid sequence of SEQ ID NO:1 of the instant application ([0005]-[0006] and see the sequence alignment below).
Regarding claim 1, Jones discloses an hyperactive engineered MAD7 nuclease of SEQ ID NO:1, wherein the engineered MAD7 nuclease has a K169R amino acid substitution ([0010]-[0014] and [0078]).
Regarding claim 2, Jones discloses that the hyperactive engineered MAD7 nuclease of SEQ ID NO:1 further has L535R ([0010]-[0014] and [0078]).
Regarding claim 2, Jones discloses that the hyperactive engineered MAD7 nuclease of SEQ ID NO:1, further has an N589R/K amino acid substitution, which reads on position 589 mutated from asparagine into any other amino acid ([0013] and [0082]).
Regarding claim 3, the hyperactive engineered MAD7 nuclease of Jones has at least 80-99% sequence identity to SEQ ID NO:1 of the instant application since the engineered MAD7 nuclease has one or two amino acid substitutions compared to the unmodified MAD7 nuclease.
Regarding claim 4, the hyperactive engineered MAD7 nuclease of Jones has at least 92-100% sequence identity to SEQ ID NO:2 (MAD7 K169R), SEQ ID NO:3 (K169R+N589H), or SEQ ID NO:11 (MAD7 K169R+K535R) of the instant application since the engineered MAD7 nuclease has one or amino acid substitutions compared to the unmodified MAD7 nuclease.
Regarding claim 5, the hyperactive engineered MAD7 nuclease has increased nuclease activity, which encompasses improved editing activity, compared to the unmodified MAD7 ([0010]). Since the engineered MAD7 nucleases of Jones are identical to the engineered MAD7 of SEQ ID NO:2, 3, or 11 of the instant application, the engineered MAD7 nucleases of Jones have improved editing activity in yeast compared to the unmodified MAD7 nuclease.
Regarding claim 6, Jones discloses a kit comprising the hyperactive engineered MAD7 nuclease and other components, which reads on an enzyme cocktail (abstract and [00122]).
Therefore, the reference of Jones anticipates claims 1-6.
Claim(s) 1-6 is/are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Kobayashi (WO 2023027041- form PTO-892. English Translation of WO 2023027041. Retrieved on October 5, 2026 – form PTO-892).
Regarding claims 1 and 4, Kobayashi discloses an engineered MAD7 nuclease of SEQ ID NO:6, which has K169R amino acid substitution and has 100% sequence identity to the engineered MAD7 nuclease of SEQ ID NO:2 (MAD7 K169R mutant) of the instant application (bottom of page 2, claim 1, and Figures 6 and 8 and see the sequence alignment below).
Regarding claim 2, Kobayashi discloses that the engineered MAD7 nuclease of SEQ ID NO:6 further has K535R amino acid substitution, resulting in K169R+K535R amino acid substitutions (Preparation example 1-8 at page 17).
Regarding claim 3, the engineered MAD7 nuclease of Kobayashi has at least 80-99% sequence identity to SEQ ID NO:1 of the instant application since the engineered MAD7 nuclease has one or two amino acid substitutions compared to the unmodified MAD7 nuclease.
Regarding claim 4, the engineered MAD7 nuclease of Kobayashi has at least 92-100% sequence identity to SEQ ID NO:2 (MAD7 K169R) or SEQ ID NO:11 (MAD7 K169R+K535R) of the instant application since the engineered MAD7 nuclease of Kobayashi consists of K169R or K169+K535R amino acid substitutions.
Regarding claim 5, the engineered MAD7 nuclease of Kobayashi has increased nuclease activity, which encompasses improved editing activity, compared to the unmodified MAD (Test example 1-2 at page 20). Since the engineered MAD7 nucleases of Kobayashi are identical to the engineered MAD7 of SEQ ID NO:2 or 11, the engineered MAD7 nucleases of Kobayashi have improved editing activity in yeast compared to the unmodified MAD7 nuclease.
Regarding claim 6, Kobayashi discloses a composition comprising the engineered MAD7 nuclease and other components, which reads on an enzyme cocktail bottom of page 23).
Therefore, the reference of Kobayashi anticipates claims 1-6.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1-6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Jones (WO 2021/257716 - form PTO-1449) and Mijts (US 10,604,746 – form PTO-892).
Jones discloses an Eubacterium rectale MAD7 nuclease having the amino acid sequence of SEQ ID NO:1, which is identical to the Eubacterium rectale MAD7 having the amino acid sequence of SEQ ID NO:1 of the instant application ([0005]-[0006] and see the sequence alignment below).
Regarding claim 1, Jones discloses an hyperactive engineered MAD7 nuclease of SEQ ID NO:1, wherein the engineered MAD7 nuclease has a K169R amino acid substitution ([0010]-[0014] and [0078]).
Regarding claim 3, the hyperactive engineered MAD7 nuclease of Jones has at least 80-99% sequence identity to SEQ ID NO:1 of the instant application since the engineered MAD7 nuclease has one amino acid substitution compared to the unmodified MAD7 nuclease.
Regarding claim 4, the hyperactive engineered MAD7 nuclease of Jones has at least 92-100% sequence identity to SEQ ID NO:2 (MAD7 K169R), since the engineered MAD7 nuclease has one amino acid substitution compared to the unmodified MAD7 nuclease.
Regarding claim 5, the hyperactive engineered MAD7 nuclease has increased nuclease activity, which encompasses increased editing activity, compared to the unmodified MAD7 ([0010]). Since the engineered MAD7 nuclease of Jones is identical to the engineered MAD7 of SEQ ID NO:2 of the instant application, the engineered MAD7 nuclease of Jones has improved editing activity in yeast compared to the unmodified MAD7 nuclease.
Regarding claim 6, Jones discloses a kit comprising the hyperactive engineered MAD7 nuclease and other components, which reads on an enzyme cocktail (abstract and [00122]).
Jones does not disclose an engineered MAD7 nuclease having K169R+N589H amino acid substitutions.
Mijts discloses an Eubacterium rectale MAD7 nuclease having the amino acid sequence of SEQ ID NO:1, which is identical to the Eubacterium rectale MAD7 having the amino acid sequence of SEQ ID NO:1 of the instant application (Column 1, line 65 through Column 2, line 2 and see the sequence alignment below).
Regarding claim 2, Mijts discloses an engineered MAD7 nuclease of SEQ ID NO:1, wherein the engineered MAD7 nuclease has an N589H amino acid substitution (Example 13).
Regarding claim 4, an engineered MAD7 nuclease comprising both K169R+N589H amino acid substitutions has 100% sequence identity to the engineered MAD7 nuclease having the amino acid sequence of SEQ ID NO:3 (MAD7 K169R+N589H) of the instant application.
Regarding claim 5, the engineered MAD7 nuclease of Mijts has increased genome editing activity in yeast compared to the unmodified MAD7 nuclease (Example 13).
Therefore, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to introduce an N589H amino acid substitution into the hyperactive engineered MAD7 K169R nuclease of Jones. One having ordinary skill in the art would have been motivated to do so in order to further improve genome editing activity. One having ordinary skill in the art would have had a reasonable expectation of success since Jones discloses an engineered MAD7 nuclease having K169R amino acid substitution having increased genome editing activity and Mijts discloses an engineered MAD7 nuclease having N589H amino acid substitution having increased genome editing activity
Therefore, the above references render claims 1-6 prima facie obvious.
Claim(s) 1-6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kobayashi (WO 2023027041- form PTO-892. English Translation of WO 2023027041. Retrieved on October 5, 2026 – form PTO-892) and Mijts (US 10,604,746 – form PTO-892).
Regarding claims 1 and 4, Kobayashi discloses an engineered MAD7 nuclease of SEQ ID NO:6, which has a K169R amino acid substitution and has 100% sequence identity to the engineered MAD7 nuclease of SEQ ID NO:2 (MAD7 K169R mutant) of the instant application (bottom of page 2, claim 1, and Figures 6 and 8 and see the sequence alignment below).
Regarding claim 3, the engineered MAD7 nuclease of Kobayashi has at least 80-99% sequence identity to SEQ ID NO:1 of the instant application since the engineered MAD7 nuclease has one amino acid substitution compared to the unmodified MAD7 nuclease.
Regarding claim 5, the engineered MAD7 nuclease of Kobayashi has increased nuclease activity, which encompasses increased editing activity, compared to the unmodified MAD (Test example 1-2 at page 20). Since the engineered MAD7 nuclease of Kobayashi is identical to the engineered MAD7 of SEQ ID NO:2 of the instant application, the engineered MAD7 nuclease of Kobayashi has improved editing activity in yeast compared to the unmodified MAD7 nuclease.
Regarding claim 6, Kobayashi discloses a composition comprising the engineered MAD7 nuclease and other components, which reads on an enzyme cocktail (bottom of page 23).
Kobayashi does not disclose an engineered MAD7 nuclease having K169R+N589H amino acid substitutions.
Mijts discloses an Eubacterium rectale MAD7 nuclease having the amino acid sequence of SEQ ID NO:1, which is identical to the Eubacterium rectale MAD7 having the amino acid sequence of SEQ ID NO:1 of the instant application (Column 1, line 65 through Column 2, line 2 and see the sequence alignment below).
Regarding claim 2, Mijts discloses an engineered MAD7 nuclease of SEQ ID NO:1, wherein the engineered MAD7 nuclease has an N589H amino acid substitution (Example 13).
Regarding claim 4, an engineered MAD7 nuclease comprising both K169R+N589H has 100% sequence identity to the engineered MAD7 nuclease having the amino acid sequence of SEQ ID NO:3 (MAD7 K169R+N589H) of the instant application.
Regarding claim 5, the engineered MAD7 nuclease of Mijts has increased genome editing activity in yeast compared to the unmodified MAD7 nuclease (Example 13).
Therefore, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to introduce an N589H amino acid substitution into the hyperactive engineered MAD7 K169R nuclease of Kobayashi. One having ordinary skill in the art would have been motivated to do so in order to further improve genome editing activity. One having ordinary skill in the art would have had a reasonable expectation of success since Kobayashi discloses an engineered MAD7 nuclease having K169R amino acid substitution having increased genome editing activity and Mijts discloses an engineered MAD7 nuclease having N589H amino acid substitution having increased genome editing activity in yeast.
Therefore, the above references render claims 1-6 prima facie obvious.
Conclusion
Claims 1-8, 10-11, and 13-22 are pending.
Claims 7-8, 10-11, and 13-22 are withdrawn.
Claims 1-6 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YONG D PAK whose telephone number is (571)272-0935. The examiner can normally be reached M-Th: 5:30 am - 3:30 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert Mondesi can be reached on 408-918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/YONG D PAK/Primary Examiner, Art Unit 1652
Sequence alignment between the MAD7 nuclease of SEQ ID NO:1 of the instant application (“Qy”) and the MAD7 nuclease of SEQ ID NO:1 of Jones (“Db”)
GBKL71722
ID BKL71722 standard; protein; 1263 AA.
XX
AC BKL71722;
XX
DT 16-JUN-2022 (revised)
DT 10-FEB-2022 (first entry)
XX
DE Eubacterium rectale derived MAD7 protein SEQ ID 1.
XX
KW CRISPR-Cas system; MAD7 protein; enzyme engineering; genome editing;
KW recombinant protein.
XX
OS Eubacterium rectale.
XX
CC PN WO2021257716-A2.
XX
CC PD 23-DEC-2021.
XX
CC PF 16-JUN-2021; 2021WO-US037649.
XX
PR 16-JUN-2020; 2020US-0039580P.
XX
CC PA (BIOT-) BIO-TECHNE CORP.
XX
CC PI Jones B, Otto N, Barnes B;
XX
DR WPI; 2021-E80463/009.
DR REFSEQ; WP_055225123.1.
XX
CC PT New modified MAD7 enzyme useful for altering target genomic DNA sequence
CC PT in host cell, preferably mammalian or human cell, comprises mutation in
CC PT catalytic domains and domain chosen from peptidylglycine alpha-amidating
CC PT monooxygenase binding domain, RuvC endonuclease domain, and nuclease
CC PT domain.
XX
CC PS Claim 3; SEQ ID NO 1; 92pp; English.
XX
CC The present invention relates to a modified MAD7 enzyme useful for
CC altering a target genomic DNA sequence in a host cell. The modified MAD7
CC enzyme comprises a mutation in one or more catalytic domains, where the
CC modified MAD7 enzyme possesses a nickase activity, where the enzyme is
CC catalytically inactive. The enzyme comprises a mutation in a domain
CC chosen from a PAM binding domain, a RuvC endonuclease domain, and a
CC nuclease domain, where the enzyme possesses increased nuclease activity
CC compared to a wild-type MAD7 enzyme. The invention further relates to:
CC (1) a fusion protein comprising the modified MAD7 enzyme; (2) a system
CC comprising a modified MAD7 enzyme or a fusion protein, and a nucleic acid
CC molecule comprising a guide RNA sequence; (3) a vector comprising a
CC nucleic acid sequence encoding the modified MAD7 enzyme or the fusion
CC protein, and a nucleic acid molecule comprising a guide RNA sequence; (4)
CC a host cell comprising the system or the vector; and (5) a method for
CC altering a target genomic DNA sequence in a host cell which involves
CC introducing the system or the vector into a host cell comprising a target
CC genomic DNA sequence. The modified MAD7 enzyme is useful for altering a
CC target genomic DNA sequence in a host cell. The modified MAD7 enzyme has
CC increased nuclease activity such as cleavage of target and non-target DNA
CC strands.
CC
CC Revised record issued on 16-JUN-2022 : Addition of DWPI-enhanced title
CC (PT field).
XX
SQ Sequence 1263 AA;
Query Match 100.0%; Score 6618; Length 1263;
Best Local Similarity 100.0%;
Matches 1263; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDD 60
Qy 61 YYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFK 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 YYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFK 120
Qy 121 NMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRANCFSADDI 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 NMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRANCFSADDI 180
Qy 181 SSSSCHRIVNDNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKY 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SSSSCHRIVNDNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKY 240
Qy 241 GEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKF 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 GEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKF 300
Qy 301 ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE 360
Qy 361 TINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETY 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 TINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETY 420
Qy 421 IHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKD 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 IHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKD 480
Qy 481 NNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNN 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 NNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNN 540
Qy 541 AIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 AIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK 600
Qy 601 TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT 660
Qy 661 STYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLH 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 STYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLH 720
Qy 721 TMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGN 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 TMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGN 780
Qy 781 IQIVRKNIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYF 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 IQIVRKNIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYF 840
Qy 841 LHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKS 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 LHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKS 900
Qy 901 FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIA 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIA 960
Qy 961 MEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKL 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 MEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKL 1020
Qy 1021 KNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLF 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 KNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLF 1080
Qy 1081 CFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTD 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 CFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTD 1140
Qy 1141 INWRDGHDLRQDIIDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYD 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 INWRDGHDLRQDIIDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYD 1200
Qy 1201 SAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNK 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 SAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNK 1260
Qy 1261 RYL 1263
|||
Db 1261 RYL 1263
Sequence alignment between the MAD7 nuclease of SEQ ID NO:2 of the instant application (“Qy”) and the MAD7 nuclease of SEQ ID NO:6 of Kobayashi (“Db”)
BMN39907
ID BMN39907 standard; protein; 1263 AA.
XX
AC BMN39907;
XX
DT 13-APR-2023 (first entry)
XX
DE Eubacterium rectale MAD7 (Cas12a) protein, SEQ:6.
XX
KW Cas12a protein; Genome editing; MAD7 protein; cell culture;
KW enzyme engineering.
XX
OS Eubacterium rectale.
XX
CC PN WO2023027041-A1.
XX
CC PD 02-MAR-2023.
XX
CC PF 22-AUG-2022; 2022WO-JP031615.
XX
PR 23-AUG-2021; 2021JP-00135502.
XX
CC PA (GRAG-) GRA & GREEN INC.
XX
CC PI Kobayashi T;
XX
DR WPI; 2023-23746K/028.
DR N-PSDB; BMN39906.
XX
CC PT New protein useful in composition for editing genome, and producing
CC PT genome-edited cells or non-human organisms comprises amino acid sequence
CC PT of MAD7 protein comprising specific mutation.
XX
CC PS Claim 1; SEQ ID NO 6; 59pp; Japanese.
XX
CC The present invention relates to a novel MAD7 protein mutant (amino acid
CC sequence b1), which contains K169R mutation in reference to SEQ ID NO:6
CC (see BMN39907). The invention further discloses: (1) a protein,
CC comprising: (a) 5'-3' exonuclease domain comprising 5'-3' exonuclease
CC amino acid sequence a1 and a nuclear localization signal sequence a2; and
CC (b) a MAD7 domain comprising the amino acid sequence b1 of the MAD7
CC protein and a linking sequence b2 of multiple nuclear localization signal
CC ; (2) a polynucleotide encoding the protein; (3) a cell comprising the
CC polynucleotide; (4) a composition for genome editing, comprising at least
CC one selected from the group consisting of the protein and the
CC polynucleotide; (5) a genome editing method, which involves introducing
CC at least one selected from the group consisting of the protein and the
CC polynucleotide into a cell or non-human organism; and (6) a method for
CC producing a genome-edited cell or non-human organism, which involves
CC introducing at least one selected from the group consisting of the
CC protein and the polynucleotide into the cell or non-human organism. The
CC novel MAD7 protein mutant of the invention is used in a genome-editing
CC technique to produce genome-edited cell or non-human organism, which has
CC higher genome-editing efficiency. Note: The present sequence is used as
CC the parent sequence for the creation of variants (see BMN40003-BMN40035)
CC based on the information given in paragraph [0109], paragraph [0133],
CC figure 4, figure 5 and figure 6 of the specification.
XX
SQ Sequence 1263 AA;
Query Match 100.0%; Score 6618; Length 1263;
Best Local Similarity 100.0%;
Matches 1263; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDD 60
Qy 61 YYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFK 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 YYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFK 120
Qy 121 NMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRANCFSADDI 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 NMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRANCFSADDI 180
Qy 181 SSSSCHRIVNDNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKY 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SSSSCHRIVNDNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKY 240
Qy 241 GEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKF 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 GEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKF 300
Qy 301 ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE 360
Qy 361 TINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETY 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 TINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETY 420
Qy 421 IHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKD 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 IHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKD 480
Qy 481 NNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNN 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 NNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNN 540
Qy 541 AIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 AIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK 600
Qy 601 TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT 660
Qy 661 STYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLH 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 STYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLH 720
Qy 721 TMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGN 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 TMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGN 780
Qy 781 IQIVRKNIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYF 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 IQIVRKNIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYF 840
Qy 841 LHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKS 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 LHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKS 900
Qy 901 FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIA 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIA 960
Qy 961 MEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKL 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 MEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKL 1020
Qy 1021 KNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLF 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 KNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLF 1080
Qy 1081 CFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTD 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 CFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTD 1140
Qy 1141 INWRDGHDLRQDIIDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYD 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 INWRDGHDLRQDIIDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYD 1200
Qy 1201 SAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNK 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 SAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNK 1260
Qy 1261 RYL 1263
|||
Db 1261 RYL 1263
Sequence alignment between the MAD7 nuclease of SEQ ID NO:3 of the instant application (“Qy”) and the MAD7 nuclease of Kobayashi (“Db”)
BMN40034
ID BMN40034 standard; protein; 1263 AA.
XX
AC BMN40034;
XX
DT 13-APR-2023 (first entry)
XX
DE Eubacterium rectale MAD7 (Cas12a) protein mutant (K169R/K535R).
XX
KW Cas12a protein; Genome editing; MAD7 protein; cell culture;
KW enzyme engineering; mutein.
XX
OS Eubacterium rectale.
OS Synthetic.
XX
CC PN WO2023027041-A1.
XX
CC PD 02-MAR-2023.
XX
CC PF 22-AUG-2022; 2022WO-JP031615.
XX
PR 23-AUG-2021; 2021JP-00135502.
XX
CC PA (GRAG-) GRA & GREEN INC.
XX
CC PI Kobayashi T;
XX
DR WPI; 2023-23746K/028.
XX
CC PT New protein useful in composition for editing genome, and producing
CC PT genome-edited cells or non-human organisms comprises amino acid sequence
CC PT of MAD7 protein comprising specific mutation.
XX
CC PS Example; Page; 59pp; Japanese.
XX
CC The present invention relates to a novel MAD7 protein mutant (amino acid
CC sequence b1), which contains K169R mutation in reference to SEQ ID NO:6
CC (see BMN39907). The invention further discloses: (1) a protein,
CC comprising: (a) 5'-3' exonuclease domain comprising 5'-3' exonuclease
CC amino acid sequence a1 and a nuclear localization signal sequence a2; and
CC (b) a MAD7 domain comprising the amino acid sequence b1 of the MAD7
CC protein and a linking sequence b2 of multiple nuclear localization signal
CC ; (2) a polynucleotide encoding the protein; (3) a cell comprising the
CC polynucleotide; (4) a composition for genome editing, comprising at least
CC one selected from the group consisting of the protein and the
CC polynucleotide; (5) a genome editing method, which involves introducing
CC at least one selected from the group consisting of the protein and the
CC polynucleotide into a cell or non-human organism; and (6) a method for
CC producing a genome-edited cell or non-human organism, which involves
CC introducing at least one selected from the group consisting of the
CC protein and the polynucleotide into the cell or non-human organism. The
CC novel MAD7 protein mutant of the invention is used in a genome-editing
CC technique to produce genome-edited cell or non-human organism, which has
CC higher genome-editing efficiency. Note: The present sequence is derived
CC from SEQ ID NO:6 (see BMN39907) based on the information given in figure
CC 6 of the specification.
XX
SQ Sequence 1263 AA;
Query Match 99.9%; Score 6615; Length 1263;
Best Local Similarity 99.9%;
Matches 1262; Conservative 1; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDD 60
Qy 61 YYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFK 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 YYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFK 120
Qy 121 NMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFRNRANCFSADDI 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 NMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFRNRANCFSADDI 180
Qy 181 SSSSCHRIVNDNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKY 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SSSSCHRIVNDNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKY 240
Qy 241 GEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKF 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 GEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKF 300
Qy 301 ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE 360
Qy 361 TINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETY 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 TINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETY 420
Qy 421 IHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKD 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 IHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKD 480
Qy 481 NNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNN 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||:|||||
Db 481 NNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSREYSNN 540
Qy 541 AIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 AIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK 600
Qy 601 TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT 660
Qy 661 STYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLH 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 STYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLH 720
Qy 721 TMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGN 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 TMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGN 780
Qy 781 IQIVRKNIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYF 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 IQIVRKNIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYF 840
Qy 841 LHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKS 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 LHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKS 900
Qy 901 FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIA 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIA 960
Qy 961 MEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKL 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 MEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKL 1020
Qy 1021 KNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLF 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 KNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLF 1080
Qy 1081 CFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTD 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 CFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTD 1140
Qy 1141 INWRDGHDLRQDIIDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYD 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 INWRDGHDLRQDIIDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYD 1200
Qy 1201 SAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNK 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 SAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNK 1260
Qy 1261 RYL 1263
|||
Db 1261 RYL 1263
Sequence alignment between the MAD7 nuclease of SEQ ID NO:1 of the instant application (“Qy”) and the MAD7 nuclease of SEQ ID NO:1 of Mijts (“Db”)
US-16-658-948-1
Filing date in PALM: 2019-10-21
Sequence 1, US/16658948
Patent No. 10604746
GENERAL INFORMATION
APPLICANT: Inscripta, Inc.
TITLE OF INVENTION: ENGINEERED ENZYMES
FILE REFERENCE: INSC029US
CURRENT APPLICATION NUMBER: US/16/658,948
CURRENT FILING DATE: 2019-10-21
PRIOR APPLICATION NUMBER: 62/748,668
PRIOR FILING DATE: 2018-10-22
NUMBER OF SEQ ID NOS: 90
SEQ ID NO 1
LENGTH: 1263
TYPE: PRT
ORGANISM: Eubacterium rectale
Query Match 100.0%; Score 6618; Length 1263;
Best Local Similarity 100.0%;
Matches 1263; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MNNGTNNFQNFIGISSLQKTLRNALIPTETTQQFIVKNGIIKEDELRGENRQILKDIMDD 60
Qy 61 YYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFK 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 YYRGFISETLSSIDDIDWTSLFEKMEIQLKNGDNKDTLIKEQTEYRKAIHKKFANDDRFK 120
Qy 121 NMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRANCFSADDI 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 NMFSAKLISDILPEFVIHNNNYSASEKEEKTQVIKLFSRFATSFKDYFKNRANCFSADDI 180
Qy 181 SSSSCHRIVNDNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKY 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SSSSCHRIVNDNAEIFFSNALVYRRIVKSLSNDDINKISGDMKDSLKEMSLEEIYSYEKY 240
Qy 241 GEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKF 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 GEFITQEGISFYNDICGKVNSFMNLYCQKNKENKNLYKLQKLHKQILCIADTSYEVPYKF 300
Qy 301 ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 ESDEEVYQSVNGFLDNISSKHIVERLRKIGDNYNGYNLDKIYIVSKFYESVSQKTYRDWE 360
Qy 361 TINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETY 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 TINTALEIHYNNILPGNGKSKADKVKKAVKNDLQKSITEINELVSNYKLCSDDNIKAETY 420
Qy 421 IHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKD 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 IHEISHILNNFEAQELKYNPEIHLVESELKASELKNVLDVIMNAFHWCSVFMTEELVDKD 480
Qy 481 NNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNN 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 NNFYAELEEIYDEIYPVISLYNLVRNYVTQKPYSTKKIKLNFGIPTLADGWSKSKEYSNN 540
Qy 541 AIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 AIILMRDNLYYLGIFNAKNKPDKKIIEGNTSENKGDYKKMIYNLLPGPNKMIPKVFLSSK 600
Qy 601 TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 TGVETYKPSAYILEGYKQNKHIKSSKDFDITFCHDLIDYFKNCIAIHPEWKNFGFDFSDT 660
Qy 661 STYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLH 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 STYEDISGFYREVELQGYKIDWTYISEKDIDLLQEKGQLYLFQIYNKDFSKKSTGNDNLH 720
Qy 721 TMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGN 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 TMYLKNLFSEENLKDIVLKLNGEAEIFFRKSSIKNPIIHKKGSILVNRTYEAEEKDQFGN 780
Qy 781 IQIVRKNIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYF 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 IQIVRKNIPENIYQELYKYFNDKSDKELSDEAAKLKNVVGHHEAATNIVKDYRYTYDKYF 840
Qy 841 LHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKS 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 LHMPITINFKANKTGFINDRILQYIAKEKDLHVIGIDRGERNLIYVSVIDTCGNIVEQKS 900
Qy 901 FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIA 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 FNIVNGYDYQIKLKQQEGARQIARKEWKEIGKIKEIKEGYLSLVIHEISKMVIKYNAIIA 960
Qy 961 MEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKL 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 MEDLSYGFKKGRFKVERQVYQKFETMLINKLNYLVFKDISITENGGLLKGYQLTYIPDKL 1020
Qy 1021 KNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLF 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 KNVGHQCGCIFYVPAAYTSKIDPTTGFVNIFKFKDLTVDAKREFIKKFDSIRYDSEKNLF 1080
Qy 1081 CFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTD 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 CFTFDYNNFITQNTVMSKSSWSVYTYGVRIKRRFVNGRFSNESDTIDITKDMEKTLEMTD 1140
Qy 1141 INWRDGHDLRQDIIDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYD 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 INWRDGHDLRQDIIDYEIVQHIFEIFRLTVQMRNSLSELEDRDYDRLISPVLNENNIFYD 1200
Qy 1201 SAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNK 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 SAKAGDALPKDADANGAYCIALKGLYEIKQITENWKEDGKFSRDKLKISNKDWFDFIQNK 1260
Qy 1261 RYL 1263
|||
Db 1261 RYL 1263
