DETAILED ACTION
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 .
This action is in response to the amendment filed 03/13/2026, in which claims 1-3 and 5-7 were amended, claims 11-13 were previously presented, claims 4, 8-10, 16 and 17 were canceled and claims 21-26 were newly added. Claims 1-3, 5-7, 11-13 and 18-26 are currently pending.
Applicant’s arguments have been thoroughly reviewed, but are not persuasive for the
reasons that follow. Any rejection and objections not reiterated in this action have been
withdrawn. This action is FINAL.
Claim Rejections - 35 USC § 103
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 (i.e., changing from AIA to pre-AIA ) 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 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.
Claims 1-3, 5-7, 11-13 and 18-26 are rejected under 35 U.S.C. 103 as being unpatentable by Sarrion-Perdigones et al (Plant Physiol. 2013 Jul;162(3):1618-31) in view of Weber et al (PLoS One. 2011 Feb 18;6(2): e16765, Pgs. 1-11) and Zhu et al (J. Mol. Biol (2004) 337, Pgs. 573-583).
Regarding claim 1, Sarrion-Perdigones teaches GB cassettes comprise a LacZ selection cassette flanked by four type IIS restriction sites (BsaI and BsmBI) positioned in inverse orientation (Page 1623, Figure 4 Description). Sarrion-Perdigones teaches BsaI cleavage sequences are boxed in red, BsmBI cleavage sequences are boxed in orange, and sites where both enzymes can digest are boxed in green (Page 1623, Figure 4 Description). Sarrion-Perdigones teaches standardized domestication of GB parts where a cassette comprising two BsmBI restriction sites are flanking a DNA part that is used for allelic exchange with a vector comprising LacZ selection marker flanked by BsmBI, BsaI and BtgZI wherein the exchange results in the DNA part being exchanged within the vector for the LacZ selection marker and the BsmBI restriction site being excised (Page 1622, Figure 3). Sarrion-Perdigones teaches using type IIS restriction enzymes to generate four-nucleotide sticky ends flanking each DNA piece, which can be subsequently joined together efficiently by T4 ligase (Page 1619, Column 1). Sarrion-Perdigones teaches that the DNA part of figure 3 is a luciferase selectable marker (Page 1626, Figure 5).
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Sarrion-Perdigones does not teach the R2 and R2’ restriction enzymes are different in the second vector some from the R1 and R1’ restriction enzymes of the first vector.
Zhu teaches BsmAI can be used in cloning methods as an alternative to BsmBI, because both are Type IIS restriction enzymes that share similar properties, including their recognition sequences and cleavage mechanisms (Page 573, Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the BsmBI restriction enzyme in the second vector as 2 and 2’ restriction enzyme sites in Sarrion-Perdigones for BsmAI as taught by because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Zhu teaches BsmAI can be used in cloning methods as an alternative to BsmBI.
One would have been motivated to make such a modification in order to receive the expected benefit of a type IIs restriction enzyme capable of the necessary function for the cloning as taught by both Sarrion-Perdigones and Zhu.
In view further, Sarrion-Perdigones does not teach that the first vector contains a R2 and R2’ restriction enzyme.
Weber teaches recognition sites are placed to the far 5’ and 3’ end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs to be ligated seamlessly; and since type IIS restriction sites can be designed to create different overhangs, which are referred to as fusion sites from here on, directional assembly of multiple fragments is feasible (Page 2, Column 1). Weber teaches that on both sides of the lacZa fragment two different type IIS recognition sequences - here BsaI and BpiI - are positioned in inverse orientation relative to each other, but creating the identical fusion site which allows cloning of the DNA fragment of interest efficiently via BpiI - removing the BpiI recognition sites and lacZa in the process - but provides the possibility to release the cloned fragment with BsaI creating the identical fusion sites it was cloned in (Page 2, Column 2). Weber teaches cloning after level 2i-1 requires the use of two type 11S enzymes, such as Bpil-Bsal or Bpil-Esp3I (Page 7, Figure 6 Description). Weber teaches using the described set of level 1 destination vectors, level 2 destination vectors and end linkers, and the indefinitely repeatable cloning strategy provided, as many transcription units can be added to a construct as desired by a user, using as many cycles of cloning as required (Page 4, Column 1 bridging Column 2 and Page 7, Figure 6 Description).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Sarrion-Perdigones to include the r2 and R2’ restriction enzymes within the first vector as taught by Weber because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Weber teaches MoClo cloning using levels to remove and add additional segments of DNA fragments including the restriction enzymes.
One would have been motivated to make such a modification in order to receive the expected benefit of successful cloning after level 2i-1 as taught by Weber.
Regarding claim 2, Sarrion-Perdigones teaches one microliter of the reaction was
transformed into E. coli DH5a electrocompetent cells, and positive clones were
selected in solid medium (Page 1629, Column 2).
Regarding claim 3, Sarrion-Perdigones does not teach after step dl, a step of treating the third vector with the third restriction enzyme and the fourth restriction enzyme to remove the structure: 5'-R2-M1-R2'-3', thereby generating a fifth vector containing the structure: 5'-R1-D(i)1-D(ii)1-R1'-3'.
Weber teaches recognition sites are placed to the far 5’ and 3’ end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs to be ligated seamlessly. Since type IIS restriction sites can be designed to create different overhangs, which are referred to as fusion sites from here on, directional assembly of multiple fragments is feasible (Page 2, Column 1). Weber teaches that on both sides of the lacZa fragment two different type IIS recognition sequences - here BsaI and BpiI - are positioned in inverse orientation relative to each other, but creating the identical fusion site which allows cloning of the DNA fragment of interest efficiently via BpiI - removing the BpiI recognition sites and lacZa in the process - but provides the possibility to release the cloned fragment with BsaI creating the identical fusion sites it was cloned in (Page 2, Column 2). Weber teaches cloning after level 2i-1 requires the use of two type 11S enzymes, such as Bpil-Bsal or Bpil-Esp3I (Page 7, Figure 6 Description). Weber teaches using the described set of level 1 destination vectors, level 2 destination vectors and end linkers, and the indefinitely repeatable cloning strategy provided, as many transcription units can be added to a construct as desired by a user, using as many cycles of cloning as required (Page 4, Column 1 bridging Column 2 and Page 7, Figure 6 Description).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Sarrion-Perdigones to include the r2 and R2’ restriction enzymes within the first vector as taught by Weber because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Weber teaches MoClo cloning using levels to remove and add additional segments of DNA fragments including the restriction enzymes.
One would have been motivated to make such a modification in order to receive the expected benefit of successful cloning after level 2i-1 as taught by Weber.
Regarding claim 5, Sarrion-Perdigones teaches GB cassettes comprise a LacZ selection cassette flanked by four type IIS restriction sites (BsaI and BsmBI) positioned in inverse orientation (Page 1623, Figure 4 Description). Sarrion-Perdigones teaches BsaI cleavage sequences are boxed in red, BsmBI cleavage sequences are boxed in orange, and sites where both enzymes can digest are boxed in green (Page 1623, Figure 4 Description). Sarrion-Perdigones teaches standardized domestication of GB parts where a cassette comprising two BsmBI restriction sites are flanking a DNA part that is used for allelic exchange with a vector comprising LacZ selection marker flanked by BsmBI, BsaI and BtgZI wherein the exchange results in the DNA part being exchanged within the vector for the LacZ selection marker and the BsmBI restriction site being excised (Page 1622, Figure 3). Sarrion-Perdigones teaches using type IIS restriction enzymes to generate four-nucleotide sticky ends flanking each DNA piece, which can be subsequently joined together efficiently by T4 ligase (Page 1619, Column 1). Sarrion-Perdigones teaches that the DNA part of figure 3 is a luciferase selectable marker (Page 1626, Figure 5).
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Sarrion-Perdigones does not teach the R2 and R2’ restriction enzymes are different in the second vector some from the R1 and R1’ restriction enzymes of the first vector.
Zhu teaches BsmAI can be used in cloning methods as an alternative to BsmBI, because both are Type IIS restriction enzymes that share similar properties, including their recognition sequences and cleavage mechanisms (Page 573, Abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the BsmBI restriction enzyme in the second vector as 2 and 2’ restriction enzyme sites in Sarrion-Perdigones for BsmAI as taught by because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Zhu teaches BsmAI can be used in cloning methods as an alternative to BsmBI.
One would have been motivated to make such a modification in order to receive the expected benefit of a type IIs restriction enzyme capable of the necessary function for the cloning as taught by both Sarrion-Perdigones and Zhu.
In view further, Sarrion-Perdigones does not teach that the first vector contains a R2 and R2’ restriction enzyme.
Weber teaches recognition sites are placed to the far 5’ and 3’ end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs to be ligated seamlessly. Since type IIS restriction sites can be designed to create different overhangs, which are referred to as fusion sites from here on, directional assembly of multiple fragments is feasible (Page 2, Column 1). Weber teaches that on both sides of the lacZa fragment two different type IIS recognition sequences - here BsaI and BpiI - are positioned in inverse orientation relative to each other, but creating the identical fusion site which allows cloning of the DNA fragment of interest efficiently via BpiI - removing the BpiI recognition sites and lacZa in the process - but provides the possibility to release the cloned fragment with BsaI creating the identical fusion sites it was cloned in (Page 2, Column 2). Weber teaches cloning after level 2i-1 requires the use of two type 11S enzymes, such as Bpil-Bsal or Bpil-Esp3I (Page 7, Figure 6 Description). Weber teaches using the described set of level 1 destination vectors, level 2 destination vectors and end linkers, and the indefinitely repeatable cloning strategy provided, as many transcription units can be added to a construct as desired by a user, using as many cycles of cloning as required (Page 4, Column 1 bridging Column 2 and Page 7, Figure 6 Description).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Sarrion-Perdigones to include the r2 and R2’ restriction enzymes within the first vector as taught by Weber because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Weber teaches MoClo cloning using levels to remove and add additional segments of DNA fragments including the restriction enzymes.
One would have been motivated to make such a modification in order to receive the expected benefit of successful cloning after level 2i-1 as taught by Weber.
Regarding claim 6, Sarrion-Perdigones teaches one microliter of the reaction was
transformed into E. coli DH5a electrocompetent cells, and positive clones were
selected in solid medium (Page 1629, Column 2).
Regarding claim 7, Sarrion-Perdigones does not teach that after step d2, a step of treating the fourth vector with the third restriction enzyme and the fourth restriction enzyme to remove the structure: 5'-R2-M2-R2'-3', thereby generating a sixth vector containing the structure: 5'-R1-D(iii)1-D(iv)1-R1'-3'.
Weber teaches recognition sites are placed to the far 5’ and 3’ end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs to be ligated seamlessly. Since type IIS restriction sites can be designed to create different overhangs, which are referred to as fusion sites from here on, directional assembly of multiple fragments is feasible (Page 2, Column 1). Weber teaches that on both sides of the lacZa fragment two different type IIS recognition sequences - here BsaI and BpiI - are positioned in inverse orientation relative to each other, but creating the identical fusion site which allows cloning of the DNA fragment of interest efficiently via BpiI - removing the BpiI recognition sites and lacZa in the process - but provides the possibility to release the cloned fragment with BsaI creating the identical fusion sites it was cloned in (Page 2, Column 2). Weber teaches cloning after level 2i-1 requires the use of two type 11S enzymes, such as Bpil-Bsal or Bpil-Esp3I (Page 7, Figure 6 Description). Weber teaches using the described set of level 1 destination vectors, level 2 destination vectors and end linkers, and the indefinitely repeatable cloning strategy provided, as many transcription units can be added to a construct as desired by a user, using as many cycles of cloning as required (Page 4, Column 1 bridging Column 2 and Page 7, Figure 6 Description).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Sarrion-Perdigones to include the r2 and R2’ restriction enzymes within the first vector as taught by Weber because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Weber teaches MoClo cloning using levels to remove and add additional segments of DNA fragments including the restriction enzymes.
One would have been motivated to make such a modification in order to receive the expected benefit of successful cloning after level 2i-1 as taught by Weber.
Regarding claims 11, 18 and 20, Sarrion-Perdigones teaches GB cassettes comprise a LacZ selection cassette flanked by four type IIS restriction sites (BsaI and BsmBI) positioned in inverse orientation (Page 1623, Figure 4 Description). Sarrion-Perdigones teaches BsaI cleavage sequences are boxed in red, BsmBI cleavage sequences are boxed in orange, and sites where both enzymes can digest are boxed in green (Page 1623, Figure 4 Description). Sarrion-Perdigones teaches standardized domestication of GB parts where a cassette comprising two BsmBI restriction sites are flanking a DNA part that is used for allelic exchange with a vector comprising LacZ selection marker flanked by BsmBI, BsaI and BtgZI wherein the exchange results in the DNA part being exchanged within the vector for the LacZ selection marker and the BsmBI restriction site being excised (Page 1622, Figure 3). Sarrion-Perdigones teaches using type IIS restriction enzymes to generate four-nucleotide sticky ends flanking each DNA piece, which can be subsequently joined together efficiently by T4 ligase (Page 1619, Column 1).
Sarrion-Perdigones does not teach the specific cleavage of each individual restriction site.
Weber teaches recognition sites are placed to the far 5’ and 3’ end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs to be ligated seamlessly. Since type IIS restriction sites can be designed to create different overhangs, which are referred to as fusion sites from here on, directional assembly of multiple fragments is feasible (Page 2, Column 1). Weber teaches that on both sides of the lacZa fragment two different type IIS recognition sequences - here BsaI and BpiI - are positioned in inverse orientation relative to each other, but creating the identical fusion site which allows cloning of the DNA fragment of interest efficiently via BpiI - removing the BpiI recognition sites and lacZa in the process - but provides the possibility to release the cloned fragment with BsaI creating the identical fusion sites it was cloned in (Page 2, Column 2). Weber teaches cloning after level 2i-1 requires the use of two type 11S enzymes, such as Bpil-Bsal or Bpil-Esp3I (Page 7, Figure 6 Description). Weber teaches using the described set of level 1 destination vectors, level 2 destination vectors and end linkers, and the indefinitely repeatable cloning strategy provided, as many transcription units can be added to a construct as desired by a user, using as many cycles of cloning as required (Page 4, Column 1 bridging Column 2 and Page 7, Figure 6 Description).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Sarrion-Perdigones to include the r2 and R2’ restriction enzymes within the first vector as taught by Weber because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Weber teaches MoClo cloning using levels to remove and add additional segments of DNA fragments including the restriction enzymes.
One would have been motivated to make such a modification in order to receive the expected benefit of successful cloning after level 2i-1 as taught by Weber.
Regarding claims 12, 13 and 19, Sarrion-Perdigones teaches the plasmids differ in the resistance marker that is associated with each level (kanamycin for level α and spectinomycin for level Ω, allowing counterselection) (Page 1622, Column 1). Sarrion-Perdigones teaches GB cassettes comprise a LacZ selection cassette flanked by four type IIS restriction sites (BsaI and BsmBI) positioned in inverse orientation (Page 1623, Figure 4 Description). Sarrion-Perdigones teaches BsaI cleavage sequences are boxed in red, BsmBI cleavage sequences are boxed in orange, and sites where both enzymes can digest are boxed in green (Page 1623, Figure 4 Description). Sarrion-Perdigones teaches standardized domestication of GB parts where a cassette comprising two BsmBI restriction sites are flanking a DNA part that is used for allelic exchange with a vector comprising LacZ selection marker flanked by BsmBI, BsaI and BtgZI wherein the exchange results in the DNA part being exchanged within the vector for the LacZ selection marker and the BsmBI restriction site being excised (Page 1622, Figure 3). Sarrion-Perdigones teaches using type IIS restriction enzymes to generate four-nucleotide sticky ends flanking each DNA piece, which can be subsequently joined together efficiently by T4 ligase (Page 1619, Column 1).
Sarrion-Perdigones does not teach that the first vector contains a R2 and R2’ restriction enzyme.
Weber teaches the MoClo cloning principal that can be repeated indefinitely where every cloning step relies on three elements that are different from one level to the next including an antibiotic selectable marker, type IIS enzymes and a color selectable marker as well as cloning after level 2i-1 requires the simultaneous use of two type IIs enzymes such as BpiI/BasI or BpiI/Esp3I (Page 7, Figure 6). Weber teaches that the system used comprises a cRed, LacZ, Amp resistance, Km resistance and/or a Spec resistance selectable marker to permit an effective counter-selection against the different level module plasmids and selection for the correctly assembled level constructs (Page 5, Column 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Sarrion-Perdigones to include the multiple selection markers and replacement of selection markers as taught by Weber because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Weber teaches MoClo cloning using levels to remove and replace selectable markers for determination of what level the cloning process is at.
One would have been motivated to make such a modification in order to receive the expected benefit of permitting an effective counter-selection against the different level module plasmids and selection for the correctly assembled level constructs as taught by Weber.
Regarding claims 21 and 22, Sarrion-Perdigones teaches GB cassettes comprise a LacZ selection cassette flanked by four type IIS restriction sites (BsaI and BsmBI) positioned in inverse orientation (Page 1623, Figure 4 Description). Sarrion-Perdigones teaches BsaI cleavage sequences are boxed in red, BsmBI cleavage sequences are boxed in orange, and sites where both enzymes can digest are boxed in green (Page 1623, Figure 4 Description). Sarrion-Perdigones teaches standardized domestication of GB parts where a cassette comprising two BsmBI restriction sites are flanking a DNA part that is used for allelic exchange with a vector comprising LacZ selection marker flanked by BsmBI, BsaI and BtgZI wherein the exchange results in the DNA part being exchanged within the vector for the LacZ selection marker and the BsmBI restriction site being excised (Page 1622, Figure 3). Sarrion-Perdigones teaches using type IIS restriction enzymes to generate four-nucleotide sticky ends flanking each DNA piece, which can be subsequently joined together efficiently by T4 ligase (Page 1619, Column 1).
Sarrion-Perdigones does not teach the multiple steps of the level process that results in a multitude of vectors to be used at different levels resulting in the desired DNA sequence.
Weber teaches the MoClo cloning principal that can be repeated indefinitely where every cloning step relies on three elements that are different from one level to the next including an antibiotic selectable marker, type IIS enzymes and a color selectable marker as well as cloning after level 2i-1 requires the simultaneous use of two type IIs enzymes such as BpiI/BasI or BpiI/Esp3I (Page 7, Figure 6). Weber teaches that compatible sets of sequenced level 0 modules (for example promoter, 59 untranslated region, signal peptide, CDS and terminator) are then assembled into a level 1 destination vector with a second Golden Gate reaction using the enzyme BsaI, leading to creation of a level 1 module, which contains a eukaryotic transcription unit (TU1) and a series of 7 level 1 destination vectors was designed in which the BpiI restriction sites generate two fusion sites with new specificities for each plasmid (Page 2, Column 2 and Page 5, Fig. 3). Weber teaches that the desired multigene level 2 constructs are then assembled with BpiI from the chosen level 1 modules, a matching end-linker and a level 2 destination vector (Page 3, Column 2 and Page 5, Figure 3). Weber teaches recognition sites are placed to the far 5’ and 3’ end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs to be ligated seamlessly. Since type IIS restriction sites can be designed to create different overhangs, which are referred to as fusion sites from here on, directional assembly of multiple fragments is feasible (Page 2, Column 1). Weber teaches that on both sides of the lacZa fragment two different type IIS recognition sequences - here BsaI and BpiI - are positioned in inverse orientation relative to each other, but creating the identical fusion site which allows cloning of the DNA fragment of interest efficiently via BpiI - removing the BpiI recognition sites and lacZa in the process - but provides the possibility to release the cloned fragment with BsaI creating the identical fusion sites it was cloned in (Page 2, Column 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Sarrion-Perdigones to include the multi-level process of the MoClo cloning process for easy DNA sequence manipulation as taught by Weber because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Weber teaches MoClo cloning using a level system to continue adding and removing through various levels.
One would have been motivated to make such a modification in order to receive the expected benefit of addition or removal of a gene or aDNA fragment with ligation to the remaining sequence by the MoClo cloning process as taught by Weber.
Regarding claims 23-26, Sarrion-Perdigones teaches that the GoldenBraid destination vectors (pDGBs) are binary vectors that function as recipients of new assemblies wherein each pDGB contains a GBcassette (the selection LacZ gene flanked by two restriction/recognition sites corresponding to two different type IIS enzymes) (Page 1621, Column 2).
Sarrion-Perdigones does not specifically teach using the third vector generated in step dl as the first vector in step al and repeating steps al to dl for an additional n cycles (1+ n cycles in total) to generate a third' vector containing the structure (3'):(3') 5'-R1-D(i)1+n-R2-M1-R2'-D(ii)1+n-R1'-3' wherein D(i)1+~ represents a DNA fragment containing the structure obtained at cycle 1+ n: 5'-D(iii)-D(i)n-3'; D(ii)1+~ represents a DNA fragment containing the structure obtained at cycle 1+ n: 5'-D(ii)n-D(iv)-3'; n represents a natural number; between the cycles, D(iii) of the second vector may be the same or different from each other; and between the cycles, D(iv) of the second vector may be the same or different from each other.
Weber teaches the MoClo cloning principal that can be repeated indefinitely where every cloning step relies on three elements that are different from one level to the next including an antibiotic selectable marker, type IIS enzymes and a color selectable marker as well as cloning after level 2i-1 requires the simultaneous use of two type IIs enzymes such as BpiI/BasI or BpiI/Esp3I (Page 7, Figure 6). Weber teaches that the system used comprises a cRed, LacZ, Amp resistance, Km resistance and/or a Spec resistance selectable marker to permit an effective counter-selection against the different level module plasmids and selection for the correctly assembled level constructs (Page 5, Column 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Sarrion-Perdigones to include multiple iterations or repetitions of the process as taught by Weber because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Weber teaches MoClo cloning using a level system to continue adding and removing through various levels.
One would have been motivated to make such a modification in order to receive the expected benefit of successful addition and removal of genes or DNA fragments as well as selection for the correctly assembled level constructs as taught by Weber.
Response to Arguments - Claim Rejections - 35 USC § 103
The previous rejection of claims 1-13 and 16-20 under 35 U.S.C. 103 has been maintained and re-written to address the amendments filed on 03/13/2026.
Applicant’s arguments have been fully considered and are not found to be persuasive.
Applicant argues the cited references do not describe or suggest the use of two different selective markers (M1, M2). Applicant continues to argue that Sarrion-Perdigones and Weber only teach one selectable marker, LacZ. Applicant argues that Sarrion-Perdigones teaches that the reaction of the donor vector containing the DNA PART and the destination vector containing LacZ results in the insertion of the ligated DNA PART at the LacZ site wherein the LacZ is removed as a result of the ligation, and instead the ligated DNA PART is inserted. Applicant argues the cited references do not describe or suggest two vectors carrying two different selective makers (M1. M2). Applicant continues to argue that, for example, to ligate two DNA fragments A and B, three vectors are required: donor vector 1 carrying DNA fragment A, donor vector 2 carrying DNA fragment B and an empty destination vector carrying LacZ.
However, Sarrion-Perdigones teaches that the DNA part of figure 3 is a luciferase selectable marker (Page 1626, Figure 5). Therefore, the two markers would be LacZ and a luciferase selectable marker. Sarrion-Perdigones teaches standardized domestication of GB parts where a cassette comprising two BsmBI restriction sites are flanking a DNA part that is used for allelic exchange with a vector comprising LacZ selection marker flanked by BsmBI, BsaI and BtgZI wherein the exchange results in the DNA part being exchanged within the vector for the LacZ selection marker and the BsmBI restriction site being excised (Page 1622, Figure 3). Sarrion-Perdigones teaches using type IIS restriction enzymes to generate four-nucleotide sticky ends flanking each DNA piece, which can be subsequently joined together efficiently by T4 ligase (Page 1619, Column 1). Therefore, Sarrion-Perdigones teaches two vectors carrying two different selective makers wherein when ligation of the two DNA fragments occurs the LacZ is replaced with the luciferase (DNA part) as shown in figure 3.
Applicant argues that in the methods described in the cited references, DNA fragments are ligated between the restriction enzyme sites instead of in the selective marker gene. Applicant continues to argue that thus, the methods disclosed in the cited references are based on a completely different concept from the presently claimed methods and are not interchangeable.
However, Applicant does not provide how the cited reference differs from the claimed methods due to the claim limitations not including specifically where the DNA fragment ligation being within the selective marker gene rather than the restriction enzyme sites.
Applicant argues the methods described in Sarrion-Perdigones and Weber require assembling multiple fragments and then ligating them together to link a large number of fragments, with the above-mentioned LacZ selection marker gene in between. Applicant continues to argue that figure 1 of Weber, the TU(P2/U2/SP2/CDS2/T2) created at level 1 from each module at level 0 are linked at positions 1 to 7 at level 2, with flanking overhangs (1234, 5678) inserted between each position, as shown in Fig. 3 of D1. Applicant argues that since the DNA fragments are ligated in a specific assembly order, it can only be used for a specific assembly.
However, in response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Weber is not relied on for the specific choices of modules outline in Figure 1 but instead Weber teaches recognition sites are placed to the far 5’ and 3’ end of any DNA fragment in inverse orientation, they are removed in the cleavage process, allowing two DNA fragments flanked by compatible sequence overhangs to be ligated seamlessly; and since type IIS restriction sites can be designed to create different overhangs, which are referred to as fusion sites from here on, directional assembly of multiple fragments is feasible (Page 2, Column 1). Weber teaches that on both sides of the lacZa fragment two different type IIS recognition sequences - here BsaI and BpiI - are positioned in inverse orientation relative to each other, but creating the identical fusion site which allows cloning of the DNA fragment of interest efficiently via BpiI - removing the BpiI recognition sites and lacZa in the process - but provides the possibility to release the cloned fragment with BsaI creating the identical fusion sites it was cloned in (Page 2, Column 2). Weber teaches cloning after level 2i-1 requires the use of two type 11S enzymes, such as Bpil-Bsal or Bpil-Esp3I (Page 7, Figure 6 Description). Weber teaches using the described set of level 1 destination vectors, level 2 destination vectors and end linkers, and the indefinitely repeatable cloning strategy provided, as many transcription units can be added to a construct as desired by a user, using as many cycles of cloning as required (Page 4, Column 1 bridging Column 2 and Page 7, Figure 6 Description).
Weber is also used to teach the limitations in combination with Sarrion-Perdigones and not alone. Therefore, the argument is not persuasive due to not also acknowledging the limitations in combination with Sarrion-Perdigones.
Applicant argues Zhu does not disclose or suggest making R2 and R2' restriction enzyme sites different from R1 and R1' restriction enzyme sites as recited in the present claims.
The Office acknowledges that Sarrion-Perdigones does not teach the R2 and R2’ restriction enzymes are different in the second vector some from the R1 and R1’ restriction enzymes of the first vector. However, Zhu teaches BsmAI can be used in cloning methods as an alternative to BsmBI, because both are Type IIS restriction enzymes that share similar properties, including their recognition sequences and cleavage mechanisms (Page 573, Abstract). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the BsmBI restriction enzyme in the second vector as 2 and 2’ restriction enzyme sites in Sarrion-Perdigones for BsmAI as taught by because Sarrion-Perdigones teaches it is within the ordinary skill in the art to use Goldenbraid to complete multiple ligase reactions in a one-step process for allele exchange and Zhu teaches BsmAI can be used in cloning methods as an alternative to BsmBI. One would have been motivated to make such a modification in order to receive the expected benefit of a type IIs restriction enzyme capable of the necessary function for the cloning as taught by both Sarrion-Perdigones and Zhu.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/ALEXANDRA ROSE LIPPOLIS/Examiner, Art Unit 1637
/CELINE X QIAN/Primary Examiner, Art Unit 1637
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