DETAILED CORRESPONDENCE
Application Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
2. Applicants’ amendment to the claims filed on 07/26/2023 is acknowledged. This listing of claims replaces all prior listings of claims in the application.
3. Claims 1-28 are pending.
Priority
4. Acknowledgement is made of applicant’s claim for foreign priority under 35 U.S.C. 119(a)-(d) to European Patent Application No. 20201096.3, filing date 10/08/2021. The certified copy has been filed in the present application, filed on 03/28/2023.
Information Disclosure Statement
5. The IDSs filed on 07/26/2023, 08/01/2023, and 07/21/2025 have been considered by the examiner and copies of the Form PTO/SB/08 are attached to the office action.
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 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 p. 24 of the specification as filed.
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.
Specific deficiency - Sequences appearing in the drawings are not identified by sequence identifiers in accordance with 37 CFR 1.831(c). 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. See Figure 2.
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.
Claim Rejections - 35 USC § 112(b)
6. 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.
7. Claims 2 and 4 are 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.
Regarding claim 2, the recitation of “the pMB1 origin of replication comprising a nucleotide sequence with at least 90% sequence identity to the nucleotide sequence of SEQ ID NO” is indefinite because it is unclear which SEQ ID NO the claim is referring to. It appears that the sequence of “16” was inadvertently removed from the claim by the amendment filed on 07/26/2023; however, such removal renders the claim indefinite. It is suggested that applicants clarify the meaning of the claim. In the interest of compact prosecution, claim 2 will be interpreted as “at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 16”.
Regarding claim 4, there is insufficient antecedent basis for the limitation “the AmpR promoter” in the claim. It is suggested that applicants clarify the meaning of the claim.
Claim Rejections - 35 USC § 103
8. 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.
9. Claim(s) 1-3, 5-10, 12-18, and 21-28 is/are rejected under 35 U.S.C. 103 as being unpatentable over Bhambure et al. (WO 2018/211529 A1; examiner cited) in view of Brass et al. (WO 2006/061174 A1; cited on IDS filed on 07/26/2023), Luirink et al. (WO 2012/041899 A1; cited on IDS filed on 07/26/2023), and Farmer et al. (WO 2013/142033 A1; examiner cited), as evidenced by Crowe et al. (WO 2016/156465 A1; examiner cited).
10. With respect to claim 1, Bhambure et al. teach a DNA construct for expressing rannibzumab in a bacterial host cell [see Abstract; p. 3, bottom; p. 5], wherein said DNA construct comprises a nucleotide sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 3 and a nucleotide sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 [see alignment attached as APPENDIX A], at least one nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding an antibiotic resistance marker, such as kanamycin [see p. 2; p. 8] under control of a promoter [see Figure 3].
With respect to claim 2, Bhambure et al. teach a DNA construct for expressing rannibzumab in a bacterial host cell [see Abstract; p. 3, bottom; p. 5], wherein said DNA construct comprises a nucleotide sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 3 and a nucleotide sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 [see alignment attached as APPENDIX A], at least one nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding an antibiotic resistance marker, such as kanamycin [see p. 2; p. 8] under control of a promoter [see Figure 3].
With respect to claims 6 and 7, Bhambure et al. teach the DNA construct wherein the nucleotide sequence encoding the light chain is 100% identical to SQ ID NO: 5 and the nucleotide sequence encoding the heavy chain is 100% identical to SEQ ID NO: 6 [see alignment attached as APPENDIX B].
With respect to claim 12, Bhambure et al. teach an expression vector comprising the DNA construct [see p. 4].
With respect to claim 13, Bhambure et al. teach a bacterial host cell comprising the DNA construct [see p. 4].
With respect to claim 17, Bhambure et al. teach a method of producing ranibizumab comprising the step of inducing expression of ranibizumab [see p. 4].
With respect to claim 18, Bhambure et al. teach the method further comprising a step of recovering the ranibizumab from the bacterial host cell [see p. 4].
With respect to claim 22, Bhambure et al. teach a bacterial host cell comprising the expression vector [see p. 4].
With respect to claim 23, Bhambure et al. teach the bacterial host cell wherein said bacterial host cell is an E. coli cell [see p. 4].
With respect to claim 27, Bhambure et al. teach the method further comprising one or more steps of purifying the recovered ranbizumab [see p. 22, bottom to top of p. 23].
With respect to claim 28, Bhambure et al. teach the method wherein said one or more steps of purifying the recovered ranibizumab comprise one or more chromatography steps [see p. 22, bottom to top of p. 23].
However, Bhambure et al. does not teach the DNA construct of claim 1, wherein said DNA construct further comprising nucleotide sequences encoding an rhaBAD promoter, an RhaR transcription activator, an RhaS transcription activator, an rrnB T1 terminator, an rrnB T2 terminator, and a pMB1 origin of replication; the construct of claim 3, wherein the rrnBT2 teminator comprises the nucleotide sequence of SEQ ID NO: 15; the construct of claim 5, wherein the nucleotide sequence encoding ranibizumab is operably linked to the rhaBAD promoter; the construct of claim 8, wherein the nucleotide encoding the signal peptide is operably linked in the direction to both of nucleotide sequences; the DNA construct of claim 9, wherein the signal peptide is PelB; the DNA construct of claim 10, wherein the nucleotide sequence encoding the PelB signal peptide comprises a sequence with at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 7; the host cell of claim 14, wherein said host cell either comprises (i) a chromosome which comprises a mutation in the nucleotide sequence of the rhaB gene which renders RhaB inactive, or a chromosome in which the nucleotide encoding RhaB is deleted; the host cell of claim 15, wherein said host cell is an Escherichia coli W3110 cell; the host cell of claim 16, wherein said host cell is an Escherichia coli W3110 cell comprising a chromosome which comprises a sequence with at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 2; the method of claim 17, exposing the bacterial host cell to rhamnose; the DNA construct of claim 21, wherein the nucleotide sequence encoding the rrnB T2 terminator comprises the nucleotide sequence of SEQ ID NO: 15; the host cell of claim 24, wherein said E. coli cell is an E. coli K-12 cell; the host cell of claim 25, wherein the host cell comprises a chromosome which comprises a frame shift mutation in the nucleotide sequence encoding RhaB; the host cell of claim 26, wherein the chromosome of the host cell further comprises a nucleotide sequence encoding RhaT.
Brass et al. teach expression constructs for the heterologous expression of nucleic acids in prokaryotic systems comprising nucleotide sequences encoding signal peptides operably linked in the direction of transcription of the nucleotide encoding the heterologous protein, an rhaBAD promoter inducible by rhamnose [see p. 2], RhaR and RhaS transcription activators [see p. 4], antibiotic resistance marker, promoter operably linked to the nucleotide sequence encoding the resistance marker [see p. 20], and an rrnB terminator or rrnB T1 terminator [see p. 15, bottom to top of p. 16; p. 25, bottom].
With respect to claim 5, Brass eta l. teach the construct wherein the nucleotide sequence encoding the heterologous protein is operably linked to the rhaBAD promoter [see p. 2].
With respect to claim 8, Brass et al. teach the construct, wherein the nucleotide sequence encoding the signal peptide is operably linked in the direction of transcription of the heterologous polypeptide [see p. 18].
With respect to claims 9 and 10, Brass et al. teach the DNA construct wherein the signal peptide is PelB [see p. 4]. Crowe et al. is cited to demonstrate that the PelB signal sequence is 100% identical to SEQ ID NO: 7 [see alignment attached as APPENDIX C].
With respect to claim 14, Brass et al. teach the host cell comprising a mutation or deletion of the rhaB gene in the chromosome [see p. 21, top].
With respect to claim 15, Brass et al. teach the host cell wherein said host cell is an E. coli W3110 cell [see p. 21, top].
With respect to claim 16, Brass et al. teach the host cell wherein said host cell is an E. coli W3110 cell [see p. 21, top]. Although Brass et al. does not explicitly teach that the chromosome comprises a sequence that is at least 90% identical to SEQ ID NO: 2, it is the examiner’s position given that Brass et al. teach identical host cell, that the E. coli W3110 cell of Brass et al. would share a chromosome having at least 90% sequence identity to SEQ ID NO: 2. Since the Office does not have the facilities for examining and comparing applicants’ host cell with the host cell of the prior art, the burden is on the applicant to show a novel or unobvious difference between the claimed product and the product of the prior art (i.e., that the host cell of the prior art does not possess the same material structural and functional characteristics of the claimed host cell). See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and In re Fitzgerald et al., 205 USPQ 594.
With respect to claim 17, Brass et al. teach a method of producing a polypeptide of interest comprising exposing the bacterial host cell to rhamnose [see p. 22].
With respect to claim 24, Brass et al. teach the host cell wherein said host cell is an E. coli TG1, W3110, DH1, and XL-1 Blue cell [see p. 21, top]. These are derivatives or strains of E. coli K-12.
With respect to claim 25, Brass et al. teach the host cell comprising a mutation or deletion of the rhaB gene in the chromosome, through transposon mutagenesis, which can institute a frameshift mutation [see p. 21, top].
With respect to claim 26, Brass et al. teach the host cell wherein said host cell is an E. coli W3110 cell comprising a rhaBAD operon [see p. 2; p. 21, top]. It is the examiner’s position that given that Brass et al. teach identical strains and constructs of the claimed host cells, that the RhaT chromosome would be intact in the strains of Brass et al. Since the Office does not have the facilities for examining and comparing applicants’ host cell with the host cell of the prior art, the burden is on the applicant to show a novel or unobvious difference between the claimed product and the product of the prior art (i.e., that the host cell of the prior art does not possess the same material structural and functional characteristics of the claimed host cell). See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and In re Fitzgerald et al., 205 USPQ 594.
Liurink et al. teach expression constructs encoding fusion proteins for secretion of one or more polypeptides that comprise a pMB1 origin of replication [see Abstract; paragraph 00171; Figure 9].
Farmer et al. teach constructs for genetically engineering host cells for expression of a polypeptide of interest that include rrnB terminators such as rrnB-T1 and rrnB-T2, wherein said rrnB-T2 terminator sequence comprises a sequence that is 100% identical to SEQ ID NO: 15 [see Abstract; paragraphs 0092, 0108-0112; alignment attached as APPENDIX D].
Before the effective filing date of the claimed invention, it would have been obvious for one of ordinary skill in the art to combine the teachings of Bhambure et al., Brass et al., Liurink et al. and Farmer et al. to develop a DNA construct for expression of ranibizumab because Bhambure et al. teach the difficulty of expressing ranibizumab. Brass et al. teach DNA constructs based on the rhaBAD operon operably linked to a signal peptide are useful in the expression of hard to express proteins by secretion of the protein of interest from the bacterial host cell. Liurink et al. teach that vectors comprising a pMB1 origin of replication are useful for the expression and secretion of fusion proteins. Farmer et al. teach that rrnB terminators such as rrnB-T1 and rrnB-T2 are useful terminators for genetically engineering host cells for expression polypeptides of interest. One of ordinary skill in the art would have had a reasonable expectation of success and a reasonable level of predictability to combine the teachings of Bhambure et al., Brass et al., Liurink et al. and Farmer et al. because Brass et al. acknowledges constructs based on the rhaBAD operon operably linked to a signal peptide are useful in the expression of hard to express proteins by secretion of the protein of interest from the bacterial host cell. Liurink et al. acknowledges that vectors comprising a pMB1 origin of replication are useful for the expression and secretion of fusion proteins, and Farmer et al. acknowledges that rrnB terminators such as rrnB-T1 and rrnB-T2 are useful terminators for genetically engineering host cells for expression polypeptides of interest. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
11. Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Bhambure et al. (WO 2018/211529 A1; examiner cited) in view of Brass et al. (WO 2006/061174 A1; cited on IDS filed on 07/26/2023), Luirink et al. (WO 2012/041899 A1; cited on IDS filed on 07/26/2023), and Farmer et al. (WO 2013/142033 A1; examiner cited), as evidenced by Crowe et al. (WO 2016/156465 A1; examiner cited) as applied to claims 1-3, 5-10, 12-18, and 21-28 above, and further in view of Horton et al. (WO 2020/254876 A1, priority to 06/18/2019; examiner cited), Sun et al. (WO 2018/122794; examiner cited), Sorokin et al. (EP 3260542, 2017; examiner cited), Hajitou et al. (WO 2017/077275 A1; examiner cited), and Kebeler et al. (US Patent Application Publication 2006/0014291 A1; examiner cited).
12. The relevant teachings of Bhambure et al., Brass et al., Liurink et al. and Farmer et al. as applied to claims 1-3, 5-10, 12-18, and 21-28 are set forth above.
With respect to claim 4, Bhambure et al. teach a DNA construct for expressing rannibzumab in a bacterial host cell [see Abstract; p. 3, bottom; p. 5], wherein said DNA construct comprises a nucleotide sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 3 and a nucleotide sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 [see alignment attached as APPENDIX A], at least one nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding an antibiotic resistance marker, such as kanamycin [see p. 2; p. 8] under control of a promoter [see Figure 3]. Bhambure et al. teach the construct wherein the rhaBAD promoter comprises a nucleotide sequence with at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 8 [see alignment attached as APPENDX A].
With respect to claim 4, Brass et al. teach expression constructs for the heterologous expression of nucleic acids in prokaryotic systems comprising nucleotide sequences encoding signal peptides operably linked in the direction of transcription of the nucleotide encoding the heterologous protein, an rhaBAD promoter inducible by rhamnose [see p. 2], RhaR and RhaS transcription activators [see p. 4], antibiotic resistance marker, promoter operably linked to the nucleotide sequence encoding the resistance marker [see p. 20], and an rrnB terminator or rrnB T1 terminator [see p. 15, bottom to top of p. 16; p. 25, bottom].
With respect to claim 4, Liurink et al. teach expression constructs encoding fusion proteins for secretion of one or more polypeptides that comprise a pMB1 origin of replication [see Abstract; paragraph 00171; Figure 9].
With respect to claim 4, Farmer et al. teach constructs for genetically engineering host cells for expression of a polypeptide of interest that include rrnB terminators such as rrnB-T1 and rrnB-T2, wherein said rrnB-T2 terminator sequence comprises a sequence that is 100% identical to SEQ ID NO: 15 [see Abstract; paragraphs 0092, 0108-0112; alignment attached as APPENDIX D].
However, the combination of Bhambure et al., Brass et al., Liurink et al. and Farmer et al. do not teach the DNA construct of claim 4, wherein the RhaR and RhaS transcription activator comprises a sequence with at least 90% sequence identity to SEQ ID NO:O 9 and 11, the antibiotic resistance marker is a kanamycin resistance marker comprising a sequence with at least 90% sequence identity to SEQ ID NO: 12, the AmpR promoter comprises a sequence with at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 13, the rrnB T1 teminator comprises a sequence with at least 90% sequence identity to SEQ ID NO: 14, and the pMB1 origin of replication comprises a sequence with at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 16.
Horton et al. teach expression constructs comprising a kanamycin resistance gene under control of a AmpR promoter that shares 100% identity with SEQ ID NO: 13 [see Abstract; p. 31; alignment attached as APPENDIX E].
Sun et al. teach genetic constructs for the expression of polypeptides in host cells using vectors expressing kanamycin resistance wherein said gene for kanamycin resistance is 100% identical to the nucleotide sequence of SEQ ID NO: 12 [see Abstract; alignment attached as APPENDIX F].
Sorokin et al. teach recombinant constructs for expression of polypeptides comprising rrnB terminator sequences such as rrnB T1 having 100% sequence identity to SEQ ID NO: 14 [see Abstract; paragraphs 0010-0011; alignment attached as APPENDIX G].
Hajitou et al. teach recombinant vector constructs comprising pMB1 origin of replication sharing 100% sequence identity to SEQ ID NO: 16 [see Abstract; p. 12; alignment attached as APPENDIX H].
Kebeler et al. teach nucleic acid constructs comprising an L-rhamnose inducible promoter and RhaR and RhaS transcriptional activators sharing at least 90% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 11 useful for the expression of heterologous proteins [see paragraphs 0001-0012; alignment attached as APPENDIX I].
Before the effective filing date of the claimed invention, it would have been obvious for one of ordinary skill in the art to combine the teachings of Bhambure et al., Brass et al., Liurink et al., Farmer et al., Horton et al., Sun et al., Sorokin et al., Hajitou et al., and Kebeler et al. according to the teachings of Horton et al., Sun et al., Sorokin et al., Hajitou et al., and Kebeler et al. because Horton et al. teach that AmpR can be used as a promoter linked to a kanamycin resistance gene in expression vectors. Sun et al. teach genetic constructs for the expression of polypeptides in host cells using vectors expressing kanamycin resistance wherein said gene for kanamycin resistance is 100% identical to the nucleotide sequence of SEQ ID NO: 12. Sorokin et al. teach recombinant constructs for expression of polypeptides comprising rrnB terminator sequences such as rrnB T1 having 100% sequence identity to SEQ ID NO: 14. Hajitou et al. teach recombinant vector constructs comprising pMB1 origin of replication sharing 100% sequence identity to SEQ ID NO: 16. Kebeler et al. teach nucleic acid constructs comprising an L-rhamnose inducible promoter and RhaR and RhaS transcriptional activators sharing at least 90% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 11 useful for the expression of heterologous proteins. One of ordinary skill in the art would have had a reasonable expectation of success and a reasonable level of predictability to combine the teachings of Bhambure et al., Brass et al., Liurink et al., Farmer et al., Horton et al., Sun et al., Sorokin et al., Hajitou et al., and Kebeler et al. because Horton et al. acknowledges that AmpR promoters can be used to drive the expression of a kanamycin resistance gene. Sun et al. acknowledges that genetic constructs for the expression of polypeptides in host cells using vectors expressing kanamycin resistance wherein said gene for kanamycin resistance is 100% identical to the nucleotide sequence of SEQ ID NO: 12, and Sorokin et al. acknowledges recombinant constructs for expression of polypeptides comprising rrnB terminator sequences such as rrnB T1 having 100% sequence identity to SEQ IDN O: 14. Hajitou et al. acknowledges recombinant vector constructs comprising pMB1 origin of replication sharing 100% sequence identity to SEQ ID NO: 16, and Kebeler et al. acknowledges nucleic acid constructs comprising an L-rhamnose inducible promoter and RhaR and RhaS transcriptional activators sharing at least 90% sequence identity to SEQ ID NO: 9 and SEQ ID NO: 11 useful for the expression of heterologous proteins. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
13. Claims 19-20 are rejected under 35 U.S.C. 103 as being unpatentable over Bhambure et al. (WO 2018/211529 A1; examiner cited) in view of Brass et al. (WO 2006/061174 A1; cited on IDS filed on 07/26/2023), Luirink et al. (WO 2012/041899 A1; cited on IDS filed on 07/26/2023), and Farmer et al. (WO 2013/142033 A1; examiner cited), as evidenced by Crowe et al. (WO 2016/156465 A1; examiner cited) as applied to claims 1-3, 5-10, 12-18, and 21-28 above, and further in view of Horton et al. (WO 2020/254876 A1, priority to 06/18/2019; examiner cited).
14. The relevant teachings of Bhambure et al., Brass et al., Liurink et al. and Farmer et al. as applied to claims 1-3, 5-10, 12-18, and 21-28 are set forth above.
With respect to claims 19-20, Bhambure et al. teach a DNA construct for expressing rannibzumab in a bacterial host cell [see Abstract; p. 3, bottom; p. 5], wherein said DNA construct comprises a nucleotide sequence encoding a light chain comprising the amino acid sequence of SEQ ID NO: 3 and a nucleotide sequence encoding a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 [see alignment attached as APPENDIX A], at least one nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding an antibiotic resistance marker, such as kanamycin [see p. 2; p. 8] under control of a promoter [see Figure 3].
With respect to claims 19-20, Brass et al. teach expression constructs for the heterologous expression of nucleic acids in prokaryotic systems comprising nucleotide sequences encoding signal peptides operably linked in the direction of transcription of the nucleotide encoding the heterologous protein, an rhaBAD promoter inducible by rhamnose [see p. 2], RhaR and RhaS transcription activators [see p. 4], antibiotic resistance marker, promoter operably linked to the nucleotide sequence encoding the resistance marker [see p. 20], and an rrnB terminator or rrnB T1 terminator [see p. 15, bottom to top of p. 16; p. 25, bottom].
However, the combination of Bhambure et al., Brass et al., Liurink et al. and Farmer et al. do not teach the DNA construct of claims 19 and 20, wherein the nucleotide sequence encoding the kanamycin resistance marker comprises a nucleotide sequence with at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 12 and/or the nucleotide sequence encoding the AmpR promoter comprises a nucleotide sequence with at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 13.
Horton et al. teach expression constructs comprising a kanamycin resistance gene under control of a AmpR promoter that shares 100% identity with SEQ ID NO: 13 [see Abstract; p. 31; alignment attached as APPENDIX E].
Before the effective filing date of the claimed invention, it would have been obvious for one of ordinary skill in the art to combine the teachings of Bhambure et al., Brass et al., Liurink et al., Farmer et al., and Horton et al. according to the teachings of Horton et al. to include a kanamycin resistance marker operably linked to a AmpR promoter because the combination of Bhambure et al., Brass et al., Liurink et al., and Farmer et al. teach a DNA construct comprising a kanamycin resistant marker for the expression of ranibizumab. Horton et al. teach that AmpR can be used as a promoter linked to a kanamycin resistance gene in expression vectors. One of ordinary skill in the art would have had a reasonable expectation of success and a reasonable level of predictability to combine the teachings of Bhambure et al., Brass et al., Liurink et al., Farmer et al., and Horton et al. because Horton et al. acknowledges that AmpR promoters can be used to drive the expression of a kanamycin resistance gene. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Claim Objections
15. Claim 11 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Conclusion
16. Status of the claims:
Claims 1-28 are pending.
Claims 1-10 and 12-28 are rejected.
Claim 11 is objected to.
No claims are in condition for an allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to PAUL J HOLLAND whose telephone number is (571)270-3537. The examiner can normally be reached Monday to Friday from 8AM to 5PM.
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, Manjunath Rao can be reached at 571-272-0939. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/PAUL J HOLLAND/Primary Examiner, Art Unit 1656
APPENDIX A
Bhambure et al. with SEQ ID NO: 3
Query Match 100.0%; Score 1114; Length 214;
Best Local Similarity 100.0%;
Matches 214; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPS 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPS 60
Qy 61 RFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPP 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 RFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPP 120
Qy 121 SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT 180
Qy 181 LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 214
||||||||||||||||||||||||||||||||||
Db 181 LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 214
Bhambure et al. with SEQ ID NO: 4
Query Match 100.0%; Score 1241; Length 231;
Best Local Similarity 100.0%;
Matches 231; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTY 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTY 60
Qy 61 AADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVT 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVT 120
Qy 121 VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL 180
Qy 181 QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL 231
|||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL 231
APPENDIX B
Bhambure et al. with SEQ ID NO: 5
Query Match 100.0%; Score 645; Length 669;
Best Local Similarity 100.0%;
Matches 645; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 GATATTCAGCTGACCCAGAGCCCGAGCAGCCTGAGCGCAAGCGTTGGTGATCGTGTTACC 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 11 GATATTCAGCTGACCCAGAGCCCGAGCAGCCTGAGCGCAAGCGTTGGTGATCGTGTTACC 70
Qy 61 ATTACCTGTAGCGCAAGCCAGGATATTAGCAATTATCTGAATTGGTATCAGCAGAAACCG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 71 ATTACCTGTAGCGCAAGCCAGGATATTAGCAATTATCTGAATTGGTATCAGCAGAAACCG 130
Qy 121 GGTAAAGCACCGAAAGTGCTGATCTATTTTACCAGCAGCCTGCATAGCGGTGTTCCGAGC 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 131 GGTAAAGCACCGAAAGTGCTGATCTATTTTACCAGCAGCCTGCATAGCGGTGTTCCGAGC 190
Qy 181 CGTTTTAGCGGTAGCGGTAGTGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 191 CGTTTTAGCGGTAGCGGTAGTGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCG 250
Qy 241 GAAGATTTTGCAACCTATTATTGTCAGCAGTATAGCACCGTTCCGTGGACCTTTGGTCAG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 251 GAAGATTTTGCAACCTATTATTGTCAGCAGTATAGCACCGTTCCGTGGACCTTTGGTCAG 310
Qy 301 GGCACCAAAGTTGAAATTAAACGTACCGTTGCAGCACCGAGCGTTTTTATCTTTCCGCCT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 311 GGCACCAAAGTTGAAATTAAACGTACCGTTGCAGCACCGAGCGTTTTTATCTTTCCGCCT 370
Qy 361 AGTGATGAACAGCTGAAAAGCGGCACCGCAAGCGTTGTTTGTCTGCTGAATAACTTTTAT 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 371 AGTGATGAACAGCTGAAAAGCGGCACCGCAAGCGTTGTTTGTCTGCTGAATAACTTTTAT 430
Qy 421 CCGCGTGAAGCAAAAGTTCAGTGGAAAGTTGATAATGCACTGCAGAGCGGTAATAGCCAA 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 431 CCGCGTGAAGCAAAAGTTCAGTGGAAAGTTGATAATGCACTGCAGAGCGGTAATAGCCAA 490
Qy 481 GAAAGCGTTACCGAACAGGATAGCAAAGATAGCACCTATAGCCTGAGCAGCACCCTGACC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 491 GAAAGCGTTACCGAACAGGATAGCAAAGATAGCACCTATAGCCTGAGCAGCACCCTGACC 550
Qy 541 CTGAGCAAAGCAGATTATGAAAAACACAAAGTGTATGCCTGCGAAGTTACCCATCAGGGT 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 551 CTGAGCAAAGCAGATTATGAAAAACACAAAGTGTATGCCTGCGAAGTTACCCATCAGGGT 610
Qy 601 CTGAGCAGTCCGGTTACCAAAAGTTTTAATCGTGGTGAATGCTAA 645
|||||||||||||||||||||||||||||||||||||||||||||
Db 611 CTGAGCAGTCCGGTTACCAAAAGTTTTAATCGTGGTGAATGCTAA 655
Bhambure et al. with SEQ ID NO: 6
Query Match 100.0%; Score 696; Length 721;
Best Local Similarity 100.0%;
Matches 696; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 GAAGTTCAGCTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGGTAGCCTGCGTCTG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 12 GAAGTTCAGCTGGTTGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGGTAGCCTGCGTCTG 71
Qy 61 AGCTGTGCAGCAAGCGGTTATGATTTTACCCATTATGGTATGAATTGGGTTCGTCAGGCA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 72 AGCTGTGCAGCAAGCGGTTATGATTTTACCCATTATGGTATGAATTGGGTTCGTCAGGCA 131
Qy 121 CCGGGTAAAGGTCTGGAATGGGTTGGTTGGATTAATACCTATACCGGTGAACCGACCTAT 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 132 CCGGGTAAAGGTCTGGAATGGGTTGGTTGGATTAATACCTATACCGGTGAACCGACCTAT 191
Qy 181 GCAGCAGATTTTAAACGTCGTTTTACCTTTAGCCTGGATACCAGCAAAAGCACCGCATAT 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 192 GCAGCAGATTTTAAACGTCGTTTTACCTTTAGCCTGGATACCAGCAAAAGCACCGCATAT 251
Qy 241 CTGCAGATGAATAGCCTGCGTGCAGAGGATACCGCAGTGTATTATTGTGCAAAATATCCG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 252 CTGCAGATGAATAGCCTGCGTGCAGAGGATACCGCAGTGTATTATTGTGCAAAATATCCG 311
Qy 301 TATTATTACGGCACCAGCCATTGGTATTTCGATGTTTGGGGTCAGGGCACCCTGGTTACC 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 312 TATTATTACGGCACCAGCCATTGGTATTTCGATGTTTGGGGTCAGGGCACCCTGGTTACC 371
Qy 361 GTTAGCAGCGCAAGCACCAAAGGTCCGAGCGTTTTTCCGCTGGCACCGAGCAGCAAAAGT 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 372 GTTAGCAGCGCAAGCACCAAAGGTCCGAGCGTTTTTCCGCTGGCACCGAGCAGCAAAAGT 431
Qy 421 ACCAGCGGTGGCACCGCAGCACTGGGTTGTCTGGTTAAAGATTATTTTCCGGAACCGGTT 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 432 ACCAGCGGTGGCACCGCAGCACTGGGTTGTCTGGTTAAAGATTATTTTCCGGAACCGGTT 491
Qy 481 ACCGTGAGCTGGAATAGCGGTGCACTGACCAGCGGTGTTCATACCTTTCCGGCAGTTCTG 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 492 ACCGTGAGCTGGAATAGCGGTGCACTGACCAGCGGTGTTCATACCTTTCCGGCAGTTCTG 551
Qy 541 CAGAGCAGCGGTCTGTATAGCCTGAGCAGCGTTGTTACCGTTCCGAGCAGCAGCCTGGGC 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 552 CAGAGCAGCGGTCTGTATAGCCTGAGCAGCGTTGTTACCGTTCCGAGCAGCAGCCTGGGC 611
Qy 601 ACCCAGACCTATATTTGTAATGTTAATCATAAACCGAGCAATACCAAAGTGGATAAAAAA 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 612 ACCCAGACCTATATTTGTAATGTTAATCATAAACCGAGCAATACCAAAGTGGATAAAAAA 671
Qy 661 GTGGAACCGAAAAGCTGCGATAAAACCCATCTGTAA 696
||||||||||||||||||||||||||||||||||||
Db 672 GTGGAACCGAAAAGCTGCGATAAAACCCATCTGTAA 707
APPENDIX C
Crowe et al. with SEQ ID NO: 7
Query Match 100.0%; Score 66; Length 501;
Best Local Similarity 100.0%;
Matches 66; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 ATGAAATATCTGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTGCA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 ATGAAATATCTGCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTGCA 60
Qy 61 ATGGCA 66
||||||
Db 61 ATGGCA 66
APPENDIX D
Farmer et al. with SEQ ID NO: 15
Query Match 100.0%; Score 28; Length 28;
Best Local Similarity 100.0%;
Matches 28; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 AGAAGGCCATCCTGACGGATGGCCTTTT 28
||||||||||||||||||||||||||||
Db 1 AGAAGGCCATCCTGACGGATGGCCTTTT 28
APPENDIX E
Horton et al. with SEQ ID NO: 13
Query Match 100.0%; Score 99; DB 1; Length 105;
Best Local Similarity 100.0%;
Matches 99; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 ACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 7 ACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAA 66
Qy 61 CCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT 99
|||||||||||||||||||||||||||||||||||||||
Db 67 CCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGT 105
APPENDIX F
Sun et al. with SEQ ID NO: 12
Query Match 100.0%; Score 816; Length 875;
Best Local Similarity 100.0%;
Matches 816; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 ATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGAT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 51 ATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGAT 110
Qy 61 GCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 111 GCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATC 170
Qy 121 TATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGC 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 171 TATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGC 230
Qy 181 GTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCT 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 231 GTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCT 290
Qy 241 CTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 291 CTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCG 350
Qy 301 ATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATT 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 351 ATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATT 410
Qy 361 GTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCT 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 411 GTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCT 470
Qy 421 TTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTG 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 471 TTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTG 530
Qy 481 GTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAA 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 531 GTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAA 590
Qy 541 GAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 591 GAAATGCATAAACTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCA 650
Qy 601 CTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTC 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 651 CTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTC 710
Qy 661 GGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCT 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 711 GGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCT 770
Qy 721 CCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAA 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 771 CCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAA 830
Qy 781 TTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA 816
||||||||||||||||||||||||||||||||||||
Db 831 TTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAA 866
APPENDIX G
Sorokin et al. with SEQ ID NO: 14
Query Match 100.0%; Score 87; Length 99;
Best Local Similarity 100.0%;
Matches 87; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 7 CAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCG 66
Qy 61 GTGAACGCTCTCCTGAGTAGGACAAAT 87
|||||||||||||||||||||||||||
Db 67 GTGAACGCTCTCCTGAGTAGGACAAAT 93
APPENDIX H
Hajitou et al. with SEQ ID NO: 16
Query Match 100.0%; Score 589; Length 589;
Best Local Similarity 100.0%;
Matches 589; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTG 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 589 TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTG 530
Qy 61 GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 529 GCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG 470
Qy 121 CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 469 CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAG 410
Qy 181 CGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 409 CGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTC 350
Qy 241 CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 349 CAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA 290
Qy 301 CTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 289 CTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGG 230
Qy 361 TAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCC 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 229 TAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCC 170
Qy 421 TAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTAC 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 169 TAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTAC 110
Qy 481 CTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 109 CTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGG 50
Qy 541 TTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA 589
|||||||||||||||||||||||||||||||||||||||||||||||||
Db 49 TTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA 1
APPENDIX I
Kebler et al. with SEQ ID NO: 9
Query Match 99.7%; Score 932.8; Length 939;
Best Local Similarity 99.8%;
Matches 934; Conservative 0; Mismatches 2; Indels 0; Gaps 0;
Qy 1 ATGGCTTTCTGCAATAACGCGAATCTTCTCAACGTATTTGTACGCCATATTGCGAATAAT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 ATGGCTTTCTGCAATAACGCGAATCTTCTCAACGTATTTGTACGCCATATTGCGAATAAT 60
Qy 61 CAACTTCGTTCTCTGGCCGAGGTAGCCACGGTGGCGCATCAGTTAAAACTTCTCAAAGAT 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 CAACTTCGTTCTCTGGCCGAGGTAGCCACGGTGGCGCATCAGTTAAAACTTCTCAAAGAT 120
Qy 121 GATTTTTTTGCCAGCGACCAGCAGGCAGTCGCTGTGGCTGACCGTTATCCGCAAGATGTC 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 GATTTTTTTGCCAGCGACCAGCAGGCAGTCGCTGTGGCTGACCGTTATCCGCAAGATGTC 180
Qy 181 TTTGCTGAACATACACATGATTTTTGTGAGCTGGTGATTGTCTGGCGCGGTAATGGCCTG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 TTTGCTGAACATACACATGATTTTTGTGAGCTGGTGATTGTCTGGCGCGGTAATGGCCTG 240
Qy 241 CATGTACTCAACGATCGCCCTTATCGCATTACCCGTGGCGATCTCTTTTACATTCATGCT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 CATGTACTCAACGATCGCCCTTATCGCATTACCCGTGGCGATCTCTTTTACATTCATGCT 300
Qy 301 GATGATAAACACTCCTACGCTTCCGTTAACGATCTGGTTTTGCAGAATATTATTTATTGC 360
|| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 GACGATAAACACTCCTACGCTTCCGTTAACGATCTGGTTTTGCAGAATATTATTTATTGC 360
Qy 361 CCGGAGCGTCTGAAGCTGAATCTTGACTGGCAGGGGGCGATTCCGGGATTTAACGCCAGC 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 CCGGAGCGTCTGAAGCTGAATCTTGACTGGCAGGGGGCGATTCCGGGATTTAACGCCAGC 420
Qy 421 GCAGGGCAACCACACTGGCGCTTAGGTAGCATGGGGATGGCGCAGGCGCGGCAGGTTATT 480
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 GCAGGGCAACCACACTGGCGCTTAGGTAGCATGGGGATGGCGCAGGCGCGGCAGGTTATC 480
Qy 481 GGTCAGCTTGAGCATGAAAGTAGTCAGCATGTGCCGTTTGCTAACGAAATGGCTGAGTTG 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 GGTCAGCTTGAGCATGAAAGTAGTCAGCATGTGCCGTTTGCTAACGAAATGGCTGAGTTG 540
Qy 541 CTGTTCGGGCAGTTGGTGATGTTGCTGAATCGCCATCGTTACACCAGTGATTCGTTGCCG 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 CTGTTCGGGCAGTTGGTGATGTTGCTGAATCGCCATCGTTACACCAGTGATTCGTTGCCG 600
Qy 601 CCAACATCCAGCGAAACGTTGCTGGATAAGCTGATTACCCGGCTGGCGGCTAGCCTGAAA 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 CCAACATCCAGCGAAACGTTGCTGGATAAGCTGATTACCCGGCTGGCGGCTAGCCTGAAA 660
Qy 661 AGTCCCTTTGCGCTGGATAAATTTTGTGATGAGGCATCGTGCAGTGAGCGCGTTTTGCGT 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 AGTCCCTTTGCGCTGGATAAATTTTGTGATGAGGCATCGTGCAGTGAGCGCGTTTTGCGT 720
Qy 721 CAGCAATTTCGCCAGCAGACTGGAATGACCATCAATCAATATCTGCGACAGGTCAGAGTG 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 CAGCAATTTCGCCAGCAGACTGGAATGACCATCAATCAATATCTGCGACAGGTCAGAGTG 780
Qy 781 TGTCATGCGCAATATCTTCTCCAGCATAGCCGCCTGTTAATCAGTGATATTTCGACCGAA 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 TGTCATGCGCAATATCTTCTCCAGCATAGCCGCCTGTTAATCAGTGATATTTCGACCGAA 840
Qy 841 TGTGGCTTTGAAGATAGTAACTATTTTTCGGTGGTGTTTACCCGGGAAACCGGGATGACG 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 TGTGGCTTTGAAGATAGTAACTATTTTTCGGTGGTGTTTACCCGGGAAACCGGGATGACG 900
Qy 901 CCCAGCCAGTGGCGTCATCTCAATTCGCAGAAAGAT 936
||||||||||||||||||||||||||||||||||||
Db 901 CCCAGCCAGTGGCGTCATCTCAATTCGCAGAAAGAT 936
Kebler et al. with SEQ ID NO: 11
Query Match 100.0%; Score 837; Length 837;
Best Local Similarity 100.0%;
Matches 837; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 ATGACCGTATTACATAGTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 ATGACCGTATTACATAGTGTGGATTTTTTTCCGTCTGGTAACGCGTCCGTGGCGATAGAA 60
Qy 61 CCCCGGCTCCCGCAGGCGGATTTTCCTGAACATCATCATGATTTTCATGAAATTGTGATT 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 CCCCGGCTCCCGCAGGCGGATTTTCCTGAACATCATCATGATTTTCATGAAATTGTGATT 120
Qy 121 GTCGAACATGGCACGGGTATTCATGTGTTTAATGGGCAGCCCTATACCATCACCGGTGGC 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 GTCGAACATGGCACGGGTATTCATGTGTTTAATGGGCAGCCCTATACCATCACCGGTGGC 180
Qy 181 ACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATGAACATACCGATAATCTGTGT 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 ACGGTCTGTTTCGTACGCGATCATGATCGGCATCTGTATGAACATACCGATAATCTGTGT 240
Qy 241 CTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCCGGGCTGAATCAG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 CTGACCAATGTGCTGTATCGCTCGCCGGATCGATTTCAGTTTCTCGCCGGGCTGAATCAG 300
Qy 301 TTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAGCGTA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 TTGCTGCCACAAGAGCTGGATGGGCAGTATCCGTCTCACTGGCGCGTTAACCACAGCGTA 360
Qy 361 TTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 TTGCAGCAGGTGCGACAGCTGGTTGCACAGATGGAACAGCAGGAAGGGGAAAATGATTTA 420
Qy 421 CCCTCGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGC 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 CCCTCGACCGCCAGTCGCGAGATCTTGTTTATGCAATTACTGCTCTTGCTGCGTAAAAGC 480
Qy 481 AGTTTGCAGGAGAACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTG 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 AGTTTGCAGGAGAACCTGGAAAACAGCGCATCACGTCTCAACTTGCTTCTGGCCTGGCTG 540
Qy 541 GAGGACCATTTTGCCGATGAGGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 GAGGACCATTTTGCCGATGAGGTGAATTGGGATGCCGTGGCGGATCAATTTTCTCTTTCA 600
Qy 601 CTGCGTACGCTACATCGGCAGCTTAAGCAGCAAACGGGACTGACGCCTCAGCGATACCTG 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 CTGCGTACGCTACATCGGCAGCTTAAGCAGCAAACGGGACTGACGCCTCAGCGATACCTG 660
Qy 661 AACCGCCTGCGACTGATGAAAGCCCGACATCTGCTACGCCACAGCGAGGCCAGCGTTACT 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 AACCGCCTGCGACTGATGAAAGCCCGACATCTGCTACGCCACAGCGAGGCCAGCGTTACT 720
Qy 721 GACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTCGACGCTTTTTCGCCGA 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 GACATCGCCTATCGCTGTGGATTCAGCGACAGTAACCACTTTTCGACGCTTTTTCGCCGA 780
Qy 781 GAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGCAATAA 837
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 GAGTTTAACTGGTCACCGCGTGATATTCGCCAGGGACGGGATGGCTTTCTGCAATAA 837