Toehold assisted DNA assembly (TADA) as a novel method of DNA assembly

Abstract

Disclosed herein include methods, compositions, and kits suitable for use in polynucleotide assembly. Methods, compositions, systems, and kits provided herein can employ a strategy which implements a DNA strand displacement reaction. The method can comprise individually "masking" the exposed complementary sequences of DNA fragments with masking oligonucleotides. In the absence of the desired match, the masking oligonucleotides can stay on and block pairing with sub-optimal ends. The assembly reaction can be initiated by matching toeholds through an isothermal strand displacement reaction (4-way strand exchange).

Description

CIT 8984-PCT / 30KJ-810009-WO PATENT TOEHOLD ASSISTED DNA ASSEMBLY (TADA) AS A NOVEL METHOD OF DNA ASSEMBLY RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 63 / 777,905, filed March 26, 2025, the content of this related application is incorporated herein by reference in its entirety for all purposes.BACKGROUNDField

[0002] The present disclosure relates generally relates to the field of polynucleotide assembly.Description of the Related Art

[0003] DNA encodes the information required for biological systems to carry out a broad range of functions. The understanding of this relationship has sparked inquiries across vast fields of biology and biological engineering as investigators read, edit, and write the genetic information of organisms. Great advancements have been made toward these pursuits, from revolutions in DNA reading through long read sequencing and the ability to generate terabytes of data from a single run, to the breakthroughs in DNA editing with the major advancements in CRISPR / Cas technologies over the last decade. However, writing DNA, as the ability to construct DNA of any length, complexity, or diversity, lags behind since DNA oligo synthesis can only reach short lengths and DNA assembly of oligos and short DNA fragments is fundamentally limited. While the need for affordable, large, and complex synthetic DNA has grown exponentially, advancements in DNA construction have not sufficiently improved to meet the scale and efficiency which is required for the age of synthetic genomes, biomaterials, massively multiplexed machine-learning Protein Language Models, and directed protein evolution. Current assembly methods share the same intrinsic limitation. Current state-of-the-arts in DNA assembly, in particular Golden Gate, Gibson, and Polymerase Chain Assembly, all rely on “strand annealing” between two single stranded overlapping sequences to direct assembly between fragments. However, due to fundamental physical and chemical limitations, exposed single-stranded sequences can participate in undesired partial annealing with incomplete complementation, leading to mis-assembled final products. This intrinsically limits the number of pieces, and therefore DNA sizes, that can be assembled with high accuracy, yield, throughput, and sequence complexity. There is a need for compositions, methods, systems, and kits for polynucleotide assembly.SUMMARY

[0004] Disclosed herein include compositions. The composition can comprise: n fragments. In some embodiments, the first fragment comprises a first fragment toehold and a first complementary region. In some embodiments, each (z)th fragment comprises a first fragment toehold, a first complementary region, a second complementary region, and a second fragment toehold, wherein 1 < z < n. In some embodiments, each (z)th fragment comprises a first polynucleotide strand and a second polynucleotide strand. In some embodiments, the first polynucleotide strand comprises a 3’ overhang. In some embodiments, the second polynucleotide strand comprises a 3’ overhang. In some embodiments, the 3’ overhang of the first polynucleotide strand comprises the first fragment toehold and the first complementary region. In some embodiments, the 3’ overhang of the second polynucleotide strand comprises the second fragment toehold and the second complementary region. In some embodiments, for each (z)th fragment: the first complementary region of the (z)th fragment is complementary to the second complementary region of the (z+l)th fragment. In some embodiments, for each (z)th fragment: the first fragment toehold of the (z)th fragment is complementary to the second fragment toehold of the (z+l)th fragment. In some embodiments, for each (z)th fragment: the second complementary region of the (z)th fragment is complementary to the first complementary region of the (z-l)th fragment. In some embodiments, for each (z)th fragment: the second fragment toehold of the (z)th fragment is complementary to the first fragment toehold of the (z-l)th fragment. In some embodiments, the (zz)th fragment comprises a second fragment toehold and a second complementary region.

[0005] The composition can comprise: n-1 pairs of masking oligonucleotides, wherein n is an integer greater than 2. In some embodiments, each pair of masking oligonucleotides comprises an upstream masking oligonucleotide and a downstream masking oligonucleotide. In some embodiments, the upstream masking oligonucleotide comprises a masking region and a masking toehold. In some embodiments, the downstream masking oligonucleotide comprises a masking region and a masking toehold. In some embodiments, for each (< / )th pair of masking oligonucleotides, wherein q is a positive integer less than zz: the masking region of the (z / )th upstream masking oligonucleotide is complementary to the first complementary region of the (z / )th fragment; the masking region of the (z / )th downstream masking oligonucleotide is complementary to the second complementary region of the (z / +l)th fragment; and the masking toehold of the (z / )th upstream masking oligonucleotide is complementary to the masking toehold of the (z / )th downstream masking oligonucleotide.

[0006] The composition can comprise: n masked fragments. In some embodiments, the first masked fragment comprises the first fragment hybridized to an upstream maskingoligonucleotide via the first complementary region of the first fragment. In some embodiments, the (zz)th masked fragment comprises the (zz)th fragment hybridized to a downstream masking oligonucleotide via the second complementary region of the (with fragment. In some embodiments, the (z)th masked fragment comprises a (z)th fragment hybridized to: (i) an upstream masking oligonucleotide via the first complementary region of the (z)th fragment; and (ii) a downstream masking oligonucleotide via the second complementary region of the (z)th fragment.

[0007] In some embodiments, the first fragment and / or the (zz)th fragment is singlestranded. In some embodiments, the first fragment and / or the (zz)th fragment comprises a first polynucleotide strand and a second polynucleotide strand. In some embodiments, the first fragment comprises a first terminal region, optionally a 5’ terminal region. In some embodiments, the (zz)th fragment comprises a second terminal region, optionally a 3’ terminal region.

[0008] Disclosed herein include compositions. The composition can comprise: n fragments. In some embodiments, each fragment comprises a first fragment toehold, a first complementary region, a second complementary region, and a second fragment toehold. In some embodiments, each fragment comprises a first polynucleotide strand and a second polynucleotide strand. In some embodiments, the first polynucleotide strand comprises a 3’ overhang. In some embodiments, the second polynucleotide strand comprises a 3’ overhang. In some embodiments, the 3’ overhang of the first polynucleotide strand comprises the first fragment toehold and the first complementary region. In some embodiments, the 3’ overhang of the second polynucleotide strand comprises the second fragment toehold and the second complementary region. In some embodiments, for each (z)th fragment, wherein 1 < z < n the first complementary region of the (z)th fragment is complementary to the second complementary region of the (z+ 1 )th fragment; the first fragment toehold of the (z)th fragment is complementary to the second fragment toehold of the (z+ l)th fragment; the second complementary region of the (z)th fragment is complementary to the first complementary region of the (z-l)th fragment; and the second fragment toehold of the (z)th fragment is complementary to the first fragment toehold of the (z-l)th fragment. In some embodiments, the second complementary region of the first fragment is complementary to the first complementary region of the (zz)th fragment. In some embodiments, the second fragment toehold of the first fragment is complementary to the first fragment toehold of the (zz)th fragment.

[0009] The composition can comprise: n pairs of masking oligonucleotides, wherein n is an integer greater than 2. In some embodiments, each pair of masking oligonucleotides comprises an upstream masking oligonucleotide and a downstream masking oligonucleotide. Insome embodiments, the upstream masking oligonucleotide comprises a masking region and a masking toehold. In some embodiments, the downstream masking oligonucleotide comprises a masking region and a masking toehold. In some embodiments, for each (c / )th pair of masking oligonucleotides, wherein q is a positive integer less than n the masking region of the (c / )th upstream masking oligonucleotide is complementary to the first complementary region of the (c / )th fragment; the masking region of the (k / )th downstream masking oligonucleotide is complementary to the second complementary region of the (c / +l)th fragment; and the masking toehold of the (c / )th upstream masking oligonucleotide is complementary to the masking toehold of the (c / )th downstream masking oligonucleotide. In some embodiments, the masking region of the ( / / )th upstream masking oligonucleotide is complementary to the first complementary region of the («)th fragment. In some embodiments, the masking region of the («)th downstream masking oligonucleotide is complementary to the second complementary region of the first fragment. In some embodiments, the masking toehold of the («)th upstream masking oligonucleotide is complementary to the masking toehold of the («)th downstream masking oligonucleotide.

[0010] The composition can comprise: n masked fragments, wherein a masked fragment comprises a fragment of the n fragments hybridized to: (i) an upstream masking oligonucleotide via the first complementary region of the fragment; and (ii) a downstream masking oligonucleotide via the second complementary region of the fragment.

[0011] Disclosed herein include compositions. The composition can comprise: a first fragment and a second fragment. In some embodiments, each of the first fragment and the second fragment comprises a first polynucleotide strand and a second polynucleotide strand. In some embodiments, the first fragment comprises a first fragment toehold and a first complementary region. In some embodiments, the first polynucleotide strand of the first fragment comprises a 3’ overhang, and wherein said 3’ overhang of the first polynucleotide strand comprises the first fragment toehold and the first complementary region. In some embodiments, the second fragment comprises a second complementary region and a second fragment toehold. In some embodiments, the second polynucleotide strand of the second fragment comprises a 3’ overhang, and wherein said 3’ overhang of the second polynucleotide strand comprises the second fragment toehold and the second complementary region. In some embodiments, the first complementary region of the first fragment is complementary to the second complementary region of the second fragment. In some embodiments, the first fragment toehold of the first fragment is complementary to the second fragment toehold of the second fragment. The composition can comprise: an upstream masking oligonucleotide and a downstream masking oligonucleotide. In some embodiments, the upstream maskingoligonucleotide comprises a masking region and a masking toehold. In some embodiments, the downstream masking oligonucleotide comprises a masking region and a masking toehold. In some embodiments, the masking region of the upstream masking oligonucleotide is complementary to the first complementary region of the first fragment. In some embodiments, the masking region of the downstream masking oligonucleotide is complementary to the second complementary region of the second fragment. In some embodiments, the masking toehold of the upstream masking oligonucleotide is complementary to the masking toehold of the downstream masking oligonucleotide.

[0012] The composition can comprise: a first masked fragment and a second masked fragment. In some embodiments, the first masked fragment comprises the first fragment hybridized to the upstream masking oligonucleotide via the first complementary region of the first fragment. In some embodiments, the second masked fragment comprises the second fragment hybridized to the downstream masking oligonucleotide via the second complementary region of the second fragment. In some embodiments, the upstream masking oligonucleotide and the downstream masking oligonucleotide are single-stranded. In some embodiments, the masking region of the upstream masking oligonucleotide is 5’ of the masking toehold. In some embodiments, the masking region of the downstream masking oligonucleotide is 3’ of the masking toehold.

[0013] In some embodiments, less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, or a number or a range between any two of these values, of fragments in the composition are unmasked fragments, wherein an unmasked fragment comprises: (i) a first complementary region not hybridized to an upstream masking oligonucleotide; and / or (ii) a second complementary region not hybridized to a downstream masking oligonucleotide. In some embodiments, the masking region of each upstream masking oligonucleotide is not complementary to the masking region of any other upstream masking oligonucleotide. In some embodiments, the masking region of each downstream masking oligonucleotide is not complementary to the masking region of any other downstream masking oligonucleotide. In some embodiments, the masking region of the (ii’)th upstream masking oligonucleotide is complementary to the masking region of the (ii’)th downstream masking oligonucleotide, wherein w is an integer from 1 to n. In some embodiments, the masking region of the ( )th upstream masking oligonucleotide is not complementary to the masking region of the (g)th downstream masking oligonucleotide, wherein f^g, optionally each of f and g is independently an integer from 1 to n or from 1 to n- . In some embodiments, each pair of masking oligonucleotides is optimized for maximum mutual specificity within a pair and absolute exclusivity across different pairs.

[0014] In some embodiments, said complementarity is or comprises: at least 80%, 85%, 90%, 95%, 99%, 100%, or a number or a range between any two of these values, complementarity; less than five, four, three, two, or one, base pair mismatches; reverse complementarity; canonical Watson-Crick base pairing; wobble base pairing, optionally G-U wobble; and / or DNA nanotechnology interactions, optionally Hoogsteen base pairing, G-quadruplex(es), DNA origami, aptamer-ligand interactions, or any combination thereof. In some embodiments, one or more of the n fragments comprise an internal segment. In some embodiments, the internal segment does not comprise any of the first fragment toehold, the second fragment toehold, the first complementary region, and the second complementary region. In some embodiments, the internal segment is 5’ of the first fragment toehold and the first complementary region. In some embodiments, the internal segment is 3’ of the second fragment toehold and the second complementary region. In some embodiments, the internal segment is double-stranded. In some embodiments, the first polynucleotide strand and / or second polynucleotide strand of one or more of the n fragments comprises a 5’ phosphate.

[0015] In some embodiments, the first fragment, the (z)th fragment, the ( / / )th fragment, one or more of the n fragments, the first fragment toehold, the second fragment toehold, the first complementary region, the second complementary region, the internal segment, one or more upstream masking oligonucleotides, one or more downstream masking oligonucleotides, the masking region, the masking toehold, the first polynucleotide strand, the second polynucleotide strand, and / or the terminal region: (a) is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 1-5, 1-10, 10-100, 10-250, 25-50, 25-100, 25-250, 50-100, 50-200, 50-250, 75-100, 75-200, 75-250, 100-150, 100-200, 100-250, 150-200, 150-250, 200-250, or a number or a range between any two of these values, nucleotides in length; (b) comprises a GC content of about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 20%-50%, 20%-75%, 20%-100%, 30%-60%, 30%-75%, 30%-100%, 40%-60%, 40%-75%, 40%-100%, 50%-75%, 50%-100%, 60%-75%, 60%-100%, 75%-100%, or a number or a range between any two of these values; (c) comprises a melting temperature (Tm) of about 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C,56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 35°C-55°C, 35°C-75°C, 35°C-100°C, 45°C-55°C, 45°C-75°C, 45°C-100°C, 55°C-75°C, 55°C-100°C, 65°C-75°C, 65°C-100°C, 75°C-100°C, or a number or a range between any two of these values; (d) comprises DNA; (e) comprises RNA; and / or (f) comprises one or more nucleic acid analogs, optionally selected from the group consisting of RNA, 2’-O-methyl RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino, phosphorodiamidate morpholino oligomer (PMO), HNA, FANA, TNA, ANA, GNA, CeNA, UNA, L-DNA, or any combination thereof.

[0016] In some embodiments, the fragments, upstream masking oligonucleotides, downstream masking oligonucleotides, the first polynucleotide strand, and / or the second polynucleotide strand: comprise or are derived from synthetic oligonucleotides; and / or comprise or are derived from rolling circle amplification products, restriction enzyme digestion products, reverse transcription products, CRISPR-excised products, PCR amplification products, templateindependent polymerase products, recombinase-generated products, phage-derived products, or any combination thereof. In some embodiments, n is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 10-25, 10-50, 10-75, 10-100, 10-500, 10-1000, 25-50, 25-75, 25-100, 25-500, 25-1000, 50-75, 50-100, 50-500, 50-1000, 75-100, 75-500, 75-1000, 100-500, 100-1000, 500-1000, or a number or a range between any two of these values. In some embodiments, upon incubation in a reaction mixture, the n masked fragments are capable of joining together via at least one four-way junction (4WJ) intermediate to generate an intermediate product. In some embodiments, a ligase is capable of ligating nicks on the first polynucleotide strands and the second polynucleotide strands of said intermediate product to generate an assembled product. In some embodiments, a ligase and / or a chemical coupling agent is capable of forming a covalent linkage between adjacent first polynucleotide strands and between adjacent second polynucleotide strands of said intermediate product to generate an assembled product. In some embodiments, the covalent linkage is formed by a click ligation between complementary reactive handles on adjacent first polynucleotide strands and / or second polynucleotide strands, optionally copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), strain promoted azidealkyne cycloaddition (SPAAC), or inverse electron demand Diels-Alder (iEDDA) reaction between a trans cyclooctene and a tetrazine oxime formation, hydrazone formation, Michael addition, disulfide formation, carbodiimide-mediated coupling, native chemical ligation, or any combination thereof. In some embodiments, the first polynucleotide strands and / or secondpolynucleotide strands comprise synthetic modifications and / or modified synthetic nucleotides, optionally selected a 5’ alkyne, a 3’ azide, a trans-cyclooctene, a tetrazine, a 5’ amine, an aldehyde, an aminooxy group, a thiol, a maleimide, or a phosphorothioate, or any combination thereof. In some embodiments, the chemical coupling agent comprises a click chemistry reagent, a copper(I) source, a copper(I)-stabilizing ligand, a strain-promoted cycloaddition reagent, a tetrazine, an EDC or other carbodiimide, an aniline or p-phenylenediamine catalyst, or any combination thereof.

[0017] In some embodiments, the assembled product or a product thereof comprises a final synthetic sequence, wherein the final synthetic sequence comprises the scarless assembly of the n fragments. In some embodiments, the final synthetic sequence is at least about 500 bases, 750 bases, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50 kb, 75 kb, 100 kb, 250 kb, 500 kb, 750 kb, 1MB, or a number or a range between any two of these values, in length. In some embodiments, the first fragment, the (z)th fragment, the (zz)th fragment, one or more of the n fragments, the first fragment toehold, the second fragment toehold, the first complementary region, the second complementary region, and / or the internal segment: (a) comprises an elevated GC content of at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values; (b) comprises a reduced GC content of less about 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 40%-30%, 40%-20%, 40%-10%, 40%-5%, 40%-l%, 30%-20%, 30%-10%, 30%-5%, 30%-l%, 20%-10%, 20%-5%, 20%-l%, 10%-5%, 10%-l%, 5%-l%, or a number or a range between any two of these values; (c) comprises two or more repeats, optionally tandem repeats, optionally at least 4 nt in length, optionally occurring at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a number or a range between any two of these values, times within the final synthetic sequence; and / or (d) comprises two or more mononucleotide stretches, optionally at least 4 nt in length, optionally occurring at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a number or a range between any two of these values, times within the final synthetic sequence.

[0018] In some embodiments, for each (z)th fragment, the first fragment toehold of the (z)th fragment is not complementary to the second fragment toehold of any (Zr)th fragment, wherein k is an integer not equal to (z+1). In some embodiments, for at least one (z)th fragment, the first fragment toehold of the (z)th fragment is complementary to the second fragment toehold of one or more (A)th fragments, wherein & is an integer not equal to (z+1).

[0019] In some embodiments, the first fragment: is an invariant fragment, wherein all instances of the invariant first fragment in the composition are identical; or is a variant fragment, wherein two or more instances of the variant first fragment in the composition differ with respect to the sequence of the internal segment. In some embodiments, at least one (z)th fragment is an invariant fragment, wherein all instances of the invariant (z)th fragment in the composition are identical. In some embodiments, at least one (z)th fragment is a variant fragment, wherein two or more instances of the variant (z)th fragment in the composition differ with respect to the sequence of the internal segment. In some embodiments, the (zz)th fragment: is an invariant fragment, wherein all instances of the invariant (zz)th fragment in the composition are identical; or is a variant fragment, wherein two or more instances of the variant (z )th fragment in the composition differ with respect to the sequence of the internal segment. In some embodiments, variant fragments comprise predefined codon variations, optionally codons variations configured to achieve modified and / or improved protein function(s).

[0020] In some embodiments, the composition comprises y sets of n masked fragments. In some embodiments, the value of n is the same between at least two of they sets. In some embodiments, the value of n is different between at least two of the y sets. In some embodiments, the masking region of each set is not complementary to the masking region of any other set. In some embodiments, upon incubation of the y sets together in a single reaction mixture, each set of n masked fragments is capable of, in parallel, joining together via four-way junction (4WJ) intermediates to generate y intermediate products. In some embodiments, the y intermediate products are candidate design variants. In some embodiments, the y intermediate products, or products thereof, are capable of being individually amplified or universally amplified. In some embodiments, y is an integer greater than 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or a number or a range between any two of these values.

[0021] In some embodiments, the final synthetic sequence comprises one or more payload genes, optionally the one or more payload genes encode one or more RNA payload(s) and / or one or more payload protein(s). In some embodiments, the one or more RNA payload(s) are selected from the group comprising a CRISPR single-guide RNA (sgRNA), a small interfering RNA (siRNA), a CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a piwi-interacting RNA (piRNA), an antisense oligonucleotide, an antagomir, an aptamer, a ribozyme, or any combination thereof. A payload protein can comprise: fluorescence activity, polymerase activity, protease activity, phosphatase activity, kinase activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity demyristoylation activity, or any combinationthereof. A payload protein can comprise: nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, adenylation activity, deadenylation activity, or any combination thereof.

[0022] A payload protein can comprise: a biomaterials payload, optionally a structural polypeptide, further optionally silk fibroin, spider silk spidroin, a resilin, a resilin-like polypeptide, an elastin, an elastin-like polypeptide, a collagen, or a collagen-like polypeptide. A payload protein can comprise: a cellular reprogramming factor capable of differentiating a given cell into a desired differentiated state, optionally nerve growth factor (NGF), fibroblast growth factor (FGF), interleukin-6 (IL-6), bone morphogenic protein (BMP), neurogenin3 (Ngn3), pancreatic and duodenal homeobox 1 (Pdxl), Mafa, or any combination thereof. A payload protein can comprise: an agonistic or antagonistic antibody or antigen-binding fragment thereof specific to a checkpoint inhibitor or checkpoint stimulator molecule, optionally PD1, PD-L1, PD-L2, CD27, CD28, CD40, CD137, 0X40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-1, and / or TIM-3. A payload protein can comprise: a secretion tag, optionally the secretion tag is selected from the group comprising AbnA, AmyE, AprE, BglC, BglS, Bpr, Csn, Epr, Ggt, GlpQ, HtrA, Lip A, LytD, MntA, Mpr, NprE, Opp A, PbpA, PbpX, Pel, PelB, PenP, PhoA, PhoB, PhoD, PstS, TasA, Vpr, WapA, WprA, XynA, XynD, YbdN, Ybxl, YcdH, YclQ, YdhF, YdhT, YfkN, YflE, YfmC, Yfnl, YhcR, YlqB, YncM, YnfF, YoaW, YocH, YolA, YqiX, Yqxl, YrpD, YrpE, YuaB, Yuri, YvcE, YvgO, YvpA, YwaD, YweA, YwoF, YwtD, YwtF, YxaLk, YxiA, and YxkC. A payload protein can comprise: a constitutive signal peptide for protein degradation, optionally PEST. A payload protein can comprise: a nuclear localization signal (NLS) or a nuclear export signal (NES). A payload protein can comprise: a dosage indicator protein, optionally the dosage indicator protein is detectable, optionally the dosage indicator protein comprises green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, m Apple, mCherry, mruby3, rsCherry, rsCherryRev, derivatives thereof, or any combination thereof. A payload protein can comprise: a cellular reprogramming factor capable of converting an at least partially differentiated cell to a less differentiated cell, optionally Oct-3, Oct-4, Sox2, c-Myc, Klf4, Nanog, Lin28, ASCL1, MYT1L, TBX3b, SV40 large T, hTERT, miR-291, miR-294, miR-295, or any combinations thereof. A payload proteincan comprise: a programmable nuclease, optionally the programmable nuclease is selected from the group comprising: SpCas9 or a derivative thereof; VRER, VQR, EQR SpCas9; xCas9-3.7; eSpCas9; Cas9-HF1; HypaCas9; evoCas9; ScCas9; StCas9; NmCas9; SaCas9; CjCas9; CasX; Cas9 H940A nickase; Cast 2 and derivatives thereof; dcas9-APOBEC 1 fusion, BE3, and dcas9-deaminase fusions; dcas9-Krab, dCas9-VP64, dCas9-Tetl, and dcas9-transcriptional regulator fusions; Dcas9-fluorescent protein fusions; Cas 13 -fluorescent protein fusions; RCas9-fluorescent protein fusions; Cas 13 -adenosine deaminase fusions, or any combination thereof. A payload protein can comprise: a CRE recombinase, GCaMP, a cell therapy component, a knockdown gene therapy component, a cell-surface exposed epitope, or any combination thereof. A payload protein can comprise: a bispecific T cell engager (BiTE). A payload protein can comprise: a synthetic receptor, optionally a Synthetic Notch (SynNotch) receptor, a Modular Extracellular Sensor Architecture (MESA) receptor, Tango, dCas9-synR, or any combination thereof. A payload protein can comprise: a cytokine, optionally the cytokine is selected from the group consisting of interleukin- 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, granulocyte macrophage colony stimulating factor (GM-CSF), M-CSF, SCF, TSLP, oncostatin M, leukemia-inhibitory factor (LIF), CNTF, Cardiotropin- 1, NNT-l / BSF-3, growth hormone, Prolactin, Erythropoietin, Thrombopoietin, Leptin, G-CSF, or receptor or ligand thereof. A payload protein can comprise: a member of the TGF-p / BMP family selected from the group consisting of TGF-pi, TGF-P2, TGF-P3, BMP-2, BMP-3a, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-15, BMP-16, endometrial bleeding associated factor (EBAF), growth differentiation factor-1 (GDF-1), GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-12, GDF-14, mullerian inhibiting substance (MIS), activin-1, activin-2, activin-3, activin-4, and activin-5. A payload protein can comprise: a member of the TNF family of cytokines selected from the group consisting of TNF-alpha, TNF-beta, LT-beta, CD40 ligand, Fas ligand, CD 27 ligand, CD 30 ligand, and 4-1 BBL. A payload protein can comprise: a member of the immunoglobulin superfamily of cytokines selected from the group consisting of B7.1 (CD80) and B7.2 (B70). A payload protein can comprise: an interferon, optionally the interferon is selected from interferon alpha, interferon beta, or interferon gamma. A payload protein can comprise: a chemokine, optionally the chemokine is selected from CCL1, CCL2, CCL3, CCR4, CCL5, CCL7, CCL8 / MCP-2, CCL11, CCL13 / MCP-4, HCC- 1 / CCL14, CTAC / CCL17, CCL19,CCL22, CCL23, CCL24, CCL26, CCL27, VEGF, PDGF, lymphotactin (XCL1), Eotaxin, FGF, EGF, IP-10, TRAIL, GCP-2 / CXCL6, NAP- 2 / CXCL7, CXCL8, CXCL10, ITAC / CXCL11, CXCL12, CXCL13, or CXCL15. A payload protein can comprise: an interleukin, optionally the interleukin is selected from IL-10 IL-12, IL-1, IL-6, IL-7, IL-15, IL-2, IL-18 or IL-21. A payload protein can comprise: a tumor necrosis factor (TNF), optionally the TNF is selected from TNF- alpha, TNF-beta, TNF-gamma, CD252, CD154, CD178, CD70, CD153, or 4-1BBL. A payload protein can comprise: a factor locally down-regulating the activity of endogenous immune cells. A payload protein can comprise: a factor capable of remodeling a tumor microenvironment and / or reducing immunosuppression at a target site of a subject. A payload protein can comprise: a chimeric antigen receptor (CAR) or T-cell receptor (TCR), optionally the CAR and / or TCR comprises one or more of an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, optionally wherein the intracellular signaling domain comprises a primary signaling domain, a costimulatory domain, or both of a primary signaling domain and a costimulatory domain. A payload protein can comprise: an activity regulator, optionally the activity regulator is capable of reducing T cell activity.

[0023] In some embodiments, a payload protein is associated with an agricultural trait of interest selected from the group consisting of increased yield, increased abiotic stress tolerance, increased drought tolerance, increased flood tolerance, increased heat tolerance, increased cold and frost tolerance, increased salt tolerance, increased heavy metal tolerance, increased low-nitrogen tolerance, increased disease resistance, increased pest resistance, increased herbicide resistance, increased biomass production, male sterility, or any combination thereof. In some embodiments, a payload protein is associated with a biological manufacturing process selected from the group comprising fermentation, distillation, biofuel production, production of a compound, production of a polypeptide, or any combination thereof. In some embodiments, the one or more payload genes are selected from the group comprising a nitrogen fixation gene, a plant stress-induced gene, a nutrient utilization gene, a gene that affects plant pigmentation, a gene that encodes an antisense or ribozyme molecule, a gene encoding an antigen capable of being secreted, a toxin gene, a receptor gene, a ligand gene, a seed storage gene, a hormone gene, an enzyme gene, an interleukin gene, a cytokine gene, a growth factor gene, a transcription factor gene, a transcriptional repressor gene, a DNA-binding protein gene, a recombination gene, a DNA replication gene, a programmed cell death gene, a kinase gene, a phosphatase gene, a G protein gene, a cyclin gene, a cell cycle control gene, a gene involved in transcription, a gene involved in translation, a gene involved in RNA processing, a gene involved in RNAi, an organellar gene, a intracellular trafficking gene, an integral membrane protein gene, a transporter gene, a membrane channel protein gene, a cell wall gene, a geneinvolved in protein processing, a gene involved in protein modification, a gene involved in protein degradation, a gene involved in metabolism, a gene involved in biosynthesis, a gene involved in assimilation of nitrogen or other elements or nutrients, a gene involved in controlling carbon flux, gene involved in respiration, a gene involved in photosynthesis, a gene involved in light sensing, a gene involved in organogenesis, a gene involved in embryogenesis, a gene involved in differentiation, a gene involved in meiotic drive, a gene involved in self incompatibility, a gene involved in development, a gene involved in nutrient, metabolite or mineral transport, a gene involved in nutrient, metabolite or mineral storage, a calcium-binding protein gene, a lipid-binding protein gene, or any combination thereof. In some embodiments, the one or more payload genes are selected from the group comprising a gene encoding an enzyme involved in metabolizing biochemical wastes for use in bioremediation, a gene that encodes an enzyme for modifying pathways that produce secondary plant metabolites, a gene that encodes an enzyme that produces a pharmaceutical, a gene that encodes an enzyme that improves or changes the nutritional content of a plant, a gene that encodes an enzyme involved in vitamin synthesis, a gene that encodes an enzyme involved in carbohydrate, polysaccharide or starch synthesis, a gene that encodes an enzyme involved in mineral accumulation or availability, a gene that encodes a phytase, a gene that encodes an enzyme involved in fatty acid, fat or oil synthesis, a gene that encodes an enzyme involved in synthesis of chemicals or plastics, a gene that encodes an enzyme involved in synthesis of a fuel, a gene that encodes an enzyme involved in synthesis of a fragrance, a gene that encodes an enzyme involved in synthesis of a flavor, a gene that encodes an enzyme involved in synthesis of a pigment or dye, a gene that encodes an enzyme involved in synthesis of a hydrocarbon, a gene that encodes an enzyme involved in synthesis of a structural or fibrous compound, a gene that encodes an enzyme involved in synthesis of a food additive, a gene that encodes an enzyme involved in synthesis of a chemical insecticide, a gene that encodes an enzyme involved in synthesis of an insect repellent, a gene controlling carbon flux in a plant, or any combination thereof. In some embodiments, the one or more payload proteins comprise components of a synthetic protein circuit, optionally payload proteins configured to form one or more logic gates selected from the group comprising an OR logic gate, AND logic gate, NOR logic gate, NAND logic gate, IMPLY logic gate, NIMPLY logic gate, XOR logic gate, and an XNOR logic gate. In some embodiments, a payload protein is capable of modulating the expression, concentration, localization, stability, and / or activity of the one or more endogenous proteins of a cell. In some embodiments, the payload protein is a therapeutic protein or a variant thereof, optionally a therapeutic protein configured to prevent or treat a disease or disorder of a subject, further optionally the subject suffers from a deficiency of said therapeutic protein.

[0024] In some embodiments, one or more of the payload gene(s) comprise: a 5’UTR and / or a 3’UTR; a tandem gene expression element selected from the group an internal ribosomal entry site (IRES), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), porcine teschovirus 2A peptide (P2A) or Thosea asigna virus 2A peptide (T2A), or any combination thereof; and / or a transcript stabilization element, optionally the transcript stabilization element comprises woodchuck hepatitis post-translational regulatory element (WPRE), bovine growth hormone polyadenylation (bGH-polyA) signal sequence, human growth hormone polyadenylation (hGH-polyA) signal sequence, or any combination thereof. In some embodiments, at least one of the payload genes is operably connected to a promoter selected from the group comprising: an RNA pol I promoter; a pol II promoter, optionally CMV, SV40 early region or adenovirus major late promoter; or pol III promoter, optionally a U6 or Hl promoter; a minimal promoter, optionally TATA, miniCMV, and / or miniPromo; a bacteriophage promoter, optionally a bacteriophage T3 promoter, a bacteriophage T7 promoter, a bacteriophage SP6 promoter, or a combination thereof; a tissue-specific promoter and / or a lineage-specific promoter; an inducible promoter, optionally a T7 RNA polymerase promoter, a T3 RNA polymerase promoter, an Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, or a Tetracycline-regulated promoter, a tetracycline-dependent promoter, a lac-dependent promoter, a pB ad-dependent promoter, an AlcA-dependent promoter, a LexA-dependent promoter, or a heat-shock promoter; a ubiquitous promoter, optionally a cytomegalovirus (CMV) immediate early promoter, a CMV promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, an RSV promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pll promoters from vaccinia virus, an elongation factor 1-alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), P-kinesin (P-KIN), the human ROSA 26 locus, a Ubiquitin C promoter (UBC), a phosphoglycerate kinase- 1 (PGK) promoter, 3 -phosphoglycerate kinase promoter, a cytomegalovirus enhancer, human P-actin (HBA) promoter, chicken P-actin (CBA) promoter, a CAG promoter, a CASI promoter, a CBH promoter; or any combination thereof.

[0025] In some embodiments, the final synthetic sequence is or comprises all or a portion of a vector. In some embodiments, the vector is or comprises a viral vector, a plasmid, a transposable element, a naked DNA vector, or any combination thereof. In some embodiments, the vector is or comprises an AAV vector, a lentivirus vector, a retrovirus vector, an adenovirusvector, a herpesvirus vector, a herpes simplex virus vector, a cytomegalovirus vector, a vaccinia virus vector, a MVA vector, a baculovirus vector, a vesicular stomatitis virus vector, a human papillomavirus vector, an avipox virus vector, a Sindbis virus vector, a VEE vector, a Measles virus vector, an influenza virus vector, a hepatitis B virus vector, an integration-deficient lentivirus (IDLV) vector, or any combination thereof. In some embodiments, the transposable element is piggybac transposon or sleeping beauty transposon.

[0026] In some embodiments, the final synthetic sequence is configured for propagation in a eukaryotic or a prokaryotic cell. The final synthetic sequence can comprise: a bacterial origin of replication, optionally ColEl, pl 5 A, pSClOl, and RK2. The final synthetic sequence can comprise: an origin of transfer (oriT) and one or more mobilization genes configured to enable conjugative transfer. The final synthetic sequence can comprise: an autonomously replicating sequence (ARS), a centromeric sequence (CEN), and / or 2p elements. The final synthetic sequence can comprise: a rolling-circle replication origin, optionally derived from pC194, pE194, and pUBHO. The final synthetic sequence can comprise: a mammalian origin of replication, optionally oriP / EBNAl and / or SV40 ori. The final synthetic sequence can comprise: a selection marker, optionally an antibiotic resistance marker and / or a fluorescence marker. The final synthetic sequence can comprise: a counter-selection marker, optionally sacB, rpsL, galK, CYH2, and / or URA3.

[0027] In some embodiments, the final synthetic sequence is configured for insertion into a genome. The final synthetic sequence can comprise: recognition sites for an RNA-guided DNA binding complex, wherein the RNA-guided DNA binding complex comprises one or more Cas proteins, a transposase, one or more crRNAs, or any combination thereof. The final synthetic sequence can comprise: recognition sites for a transposition complex comprising one or more transposases. The final synthetic sequence can comprise: homology arms, optionally targeting a safe-harbor locus selected from AAVS1, ROSA26, CCR5, and Hll. The final synthetic sequence can comprise: one or more recombination sites, optionally loxP, FRT, attB, attP, attL, and attR. The final synthetic sequence can comprise: a reporter cassette. In some embodiments, the final synthetic sequence comprises a digital data storage payload encoded in nucleic acid sequence.

[0028] In some embodiments, each of the n fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. In some embodiments, each of the n masked fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. In some embodiments, the n pairs or n-1 pairs of masking oligonucleotides are housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. In some embodiments, the first polynucleotide strand and the second polynucleotide strand that constitute each of the nfragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber.

[0029] The composition can comprise: a non-thermostable ligase, a thermostable ligase, a chemical coupling agent, a polymerase, a primer capable of binding the first terminal region (or a complement thereof), a primer capable of binding the second terminal region (or a complement thereof), or any combination thereof. The composition can comprise: a ligation buffer. The ligation buffer can comprise: a reaction buffer configured to support annealing, ligation, and / or amplification (e.g., HiFi Taq buffer); one or more of Tris HC1 at about 10 mM to about 200 mM, at a pH of about 7.0 to about 9.5 at the incubation temperature, Mg2+ at about 0.5 mM to about 20 mM, monovalent cation(s) at about 10 mM to about 300 mM, and a reducing agent at about 0.1 mM to about 20 mM; a ligase cofactor, optionally selected from ATP at about 0.05 mM to about 5 mM or NAD+ at about 0.01 mM to about 2 mM; a buffering species selected from Tris, HEPES, Bis Tris, MOPS, and PIPES, optionally configured to maintain pH between 8.3-8.8 at 25°C; and / or one or more additives, optionally selected from bovine serum albumin at about 0.01 mg / mL to about 1 mg / mL, polyethylene glycol at about 1% to about 20% (w / v), betaine at about 0.1 M to about 2.0 M, dimethyl sulfoxide at about 1% to about 20% (v / v), formamide at about 0.5% to about 10% (v / v), glycerol at about 1% to about 20% (v / v), and / or a non-ionic detergent at about 0.001% to about 0.1% (v / v). In some embodiments, the composition does not comprise one or more reagents employed with Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly, optionally an exonuclease, an endonuclease, a single stranded DNA binding protein, a restriction endonuclease, a recombinase, or any combination thereof.

[0030] Disclosed herein include compositions. The composition can comprise: a pre-assembly reaction mixture comprising the n masked fragments disclosed herein at equimolar concentrations, optionally the pre-assembly reaction mixture comprises a ligase or a chemical coupling agent. Disclosed herein include compositions. The composition can comprise: a first intermediate reaction mixture comprising the n masked fragments disclosed herein associated together via four-way junction (4WJ) intermediates, optionally the first intermediate reaction mixture comprises a ligase or a chemical coupling agent. Disclosed herein include compositions. The composition can comprise: a second intermediate reaction mixture comprising the n fragments disclosed herein joined together via hybridization of the first complementary region and the second complementary region to generate an intermediate product. In some embodiments, the intermediate product comprises nicks on the first polynucleotide strand and the second polynucleotide strand. In some embodiments, the four-way junctions (4WJs) are resolved and the intermediate product is not hybridized to an upstream masking oligonucleotideor a downstream masking oligonucleotide. In some embodiments, the second intermediate reaction mixture comprises paired masking oligonucleotides consisting of an upstream masking oligonucleotide hybridized to a downstream masking oligonucleotide. In some embodiments, the second intermediate reaction mixture comprises a ligase or a chemical coupling agent. Disclosed herein include compositions. The composition can comprise: a post-ligation reaction mixture comprising an assembled product wherein the first polynucleotide strand and the second polynucleotide strand do not comprise nicks.

[0031] Disclosed herein include methods. The method can comprise: providing the n masked fragments disclosed herein. The method can comprise: incubating the n masked fragments in a reaction mixture under reaction conditions such that: the first fragment toehold of the (z)th fragment hybridizes to the second fragment toehold of the (z+1 )th fragment; and the masking region of the (z)th upstream masking oligonucleotide hybridizes to the masking region of the (z’)th downstream masking oligonucleotide, thereby joining together the n fragments via four-way junction (4WJ) intermediates to generate an intermediate product. The method can comprise: ligating nicks on the first polynucleotide strands and the second polynucleotide strands to generate an assembled product.

[0032] In some embodiments, association between: (i) the first fragment toehold and second fragment toehold of adjacent fragments; and (ii) the masking regions of an upstream masking oligonucleotide and a downstream masking oligonucleotide, forms a 4WJ. In some embodiments, mismatches between the first complementary region and the second complementary region of incorrectly joined fragments prevent progression of the 4-way strand exchange. In some embodiments, upon resolution of a 4WJ, adjacent fragments are hybridized via the first complementary region and the second complementary region, and the adjacent fragments are separated by a nick in each of the first polynucleotide strand and the second polynucleotide strand. In some embodiments, the resolution of a 4WJ yields a paired masking oligonucleotide consisting of an upstream masking oligonucleotide hybridized to a downstream masking oligonucleotide. In some embodiments, the masking oligonucleotides block hybridization between the first complementary region and the second complementary region when the first complementary region and the second complementary region lack perfect complementarity. In some embodiments, the isothermal strand displacement reaction is driven forward by perfect complementarity between the first complementary region and the second complementary region.

[0033] In some embodiments, the incubation step is performed under isothermal conditions. In some embodiments, the incubation comprises incubation at a first incubation temperature for a first period of time. In some embodiments, the first incubation temperature isabout 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, or a number or a range between any two of these values. In some embodiments, the first period of time is about 10 sec, 20 sec, 30 sec, 40 sec, 50 sec, 60 sec, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, or a number or a range between any two of these values.

[0034] In some embodiments, the ligating step comprises: addition of a ligase to the reaction mixture; and incubation at the second incubation temperature for a second period of time. In some embodiments, the second incubation temperature is about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, or a number or a range between any two of these values. In some embodiments, the second period of time is about 10 sec, 20 sec, 30 sec, 40 sec, 50 sec, 60 sec, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, or a number or a range between any two of these values.

[0035] In some embodiments, the assembly of the n masked fragments occurs independently of the sequence of the internal segments. In some embodiments, the assembly of the n masked fragments is directed by the formation of 4-way junctions (4WJs) between adjacent masked fragments. In some embodiments, the assembled product or a product thereof comprises a final synthetic sequence, wherein the final synthetic sequence comprises the scarless assembly of the n fragments. In some embodiments, the final synthetic sequence is a linear polynucleotide, optionally comprising the structure 5’-[first fragment] -[second fragment]-...-[( / / )th fragment]-3’. In some embodiments, the final synthetic sequence is a circular polynucleotide wherein the 3’ end of the [( / / )th fragment] is linked to the 5’ end of [first fragment] by a phosphodiester bond. In some embodiments, the final synthetic sequence is at least about 500 bases, 750 bases, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50 kb, 75 kb, 100 kb, 250 kb, 500 kb, 750 kb, 1MB, or a number or a range between any two of these values, in length.

[0036] In some embodiments, the ligating step is performed with a ligase, optionally a thermostable ligase, optionally said ligase is selected from the group comprising T3 ligase, T4 ligase, T7 ligase, SplintR, E. coli DNA ligase, Hi-T4 ligase, a ligase suitable for ligating adjacent nucleic acid sequences under hybridization conditions (e.g., HiFi Taq ligase), Taq ligase, 9°N, or any combination thereof. In some embodiments, the ligating step comprisescontacting the intermediate product with a chemical coupling agent effective to form a covalent linkage between adjacent first polynucleotide strands and between adjacent second polynucleotide strands, optionally one or more click chemistry reagents, optionally CuAAC, SPAAC, iEDDA, oxime formation, hydrazone formation, Michael addition, disulfide formation, carbodiimide mediated coupling, native chemical ligation, or any combination thereof. In some embodiments, the incubating step comprises combining the n masked fragments in a single reaction mix at equimolar concentrations, optionally at about 0.1 nM, 0.5 nM, 0.75 nM, 0.9 nM, 1.0 nM, 1.1 nM, 1.25 nM, 1.5 nM, 1.75 nM, 2 nM, 5 nM, or 10 nM, or a number or a range between any two of these values.

[0037] In some embodiments, the providing step comprises: generating the n masked fragments. In some embodiments, said generating step comprises annealing the n fragments with the n pairs or n-1 pairs of masking oligonucleotides to generate the n masked fragments. In some embodiments, the n masked fragments are each generated in separate reactions. In some embodiments, said annealing step comprises an initial denaturation step followed by a gradual decrease in temperature. In some embodiments, the n masked fragments undergo one or more purification steps, optionally: gel electrophoresis, including pulsed-field gel electrophoresis (PFGE); solid or solution phase hybridization / capture; precipitation; dialysis; solid phase reversible immobilization (SPRI) cleanup, optionally performing size selection using SPRI beads, further optionally single-sided or double-sided; and / or column purification.

[0038] In some embodiments, the providing step comprises: generating the n fragments. In some embodiments, said generating step comprises annealing the first polynucleotide strand and the second polynucleotide strand components of each of the n fragments to generate heteroduplexes. In some embodiments, the n fragments are each generated in separate reactions. In some embodiments, the generating step comprises phosphorylation of the first and second polynucleotide strands, further optionally via T4 polynucleotide kinase. In some embodiments, said annealing step comprises an initial denaturation step followed by a gradual decrease in temperature. In some embodiments, the heteroduplexes undergo one or more purification steps, optionally: gel electrophoresis, including pulsed-field gel electrophoresis (PFGE); solid or solution phase hybridization / capture; precipitation; dialysis; solid phase reversible immobilization (SPRI) cleanup, optionally performing size selection using SPRI beads, further optionally single-sided or double-sided; and / or column purification.

[0039] In some embodiments, the method further comprises PCR amplification of the assembled product, or a product thereof, to generate an amplified product. In some embodiments, PCR amplification comprises amplifying the assembled product, or a product thereof, using a primer capable of hybridizing to the first terminal region or a complementthereof, and a primer capable of hybridizing the second terminal region or a complement thereof.

[0040] In some embodiments, the method comprises purification of the assembled product, the amplified product, or products thereof. In some embodiments, said purification step compromises: removal of the paired masking oligonucleotides; gel electrophoresis of the assembled product, the amplified product, or products thereof; solid phase reversible immobilization (SPRI) cleanup, optionally performing size selection using SPRI beads, further optionally single-sided or double-sided; and / or column purification.

[0041] In some embodiments, the method comprises replication of the assembled product, the amplified product, or products thereof, in a cell. In some embodiments, the providing step comprises providing y sets of n masked fragments. In some embodiments, the incubating step comprises incubating the y sets of n masked fragments in a single reaction mixture, wherein the n masked fragments of each set are joined together in parallel via four- way junction (4WJ) intermediates to generate y intermediate products. In some embodiments, the ligating step comprises ligating nicks on the first polynucleotide strands and the second polynucleotide strands of each of the y intermediate products to generate y assembled products.

[0042] In some embodiments, the value of n is the same between at least two of they sets. In some embodiments, the value of n is the different between at least two of the y sets. In some embodiments, the masking region of each set is not complementary to the masking region of any other set. In some embodiments, the y intermediate products are candidate design variants. In some embodiments, the method comprises the y intermediate products, or products thereof, being individually amplified or universally amplified. In some embodiments, y is an integer greater than 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or a number or a range between any two of these values.

[0043] In some embodiments, at least one of the n fragments is a variant fragment, and wherein the assembled products comprise a combinatorial library of at least p variants, wherein p is an integer greater than 1. In some embodiments, wherein p is at least about 10, 50, 100, 250, 500, 750, 1000, 10000, 50000, 100000, 250000, 500000, 750000, 1000000, 5000000, 10000000, or a number or a range between any two of these values. In some embodiments, wherein the combinatorial library achieves a variant coverage of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.99%, or a number or a range between any two of these values, of the theoretical variant library. In some embodiments, every codon mutation profile is represented in the library with an average absolute deviation of less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, or a number or a range between any two of these values, from the theoreticalproportion of occurrence for that codon.

[0044] In some embodiments, at least 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or a number or a range between any two of these values, of the assembled products, or products thereof, comprise all of the intended fragments in the intended order. In some embodiments, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, or a number or a range between any two of these values, of the assembled products, or products thereof, are a partial assembly missing one or more fragments. In some embodiments, less than 1 in 1000, 1 in 10000, 1 in 100000, 1 in 1000000, 1 in 10000000, 1 in 100000000, or a number or a range between any two of these values, of the assembled products are missing one or more fragments or comprise a mis-assembled junction. In some embodiments, the mis-ligation rate at the 4WJ is less than 1 in 1000, 1 in 10000, 1 in 100000, 1 in 1000000, 1 in 10000000, 1 in 100000000, or a number or a range between any two of these values. In some embodiments, n is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 10-25, 10-50, 10-75, 10-100, 10-500, 10-1000, 25-50, 25-75, 25-100, 25-500, 25-1000, 50-75, 50-100, 50-500, 50-1000, 75-100, 75-500, 75-1000, 100-500, 100-1000, 500-1000, or a number or a range between any two of these values. In some embodiments, the yield of correctly assembled products is at least 1-fold, 2-fold, 4-fold, 8-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, or 1000-fold, greater than the yield of a polynucleotide assembly method not comprising 4WJ, optionally Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly. In some embodiments, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a number or a range between any two of these values, of the incubated fragments become a component of an assembled product.

[0045] Disclosed herein include compositions. The composition can comprise: assembled products, or products thereof, generated by a method disclosed herein . In some embodiments, the composition comprises a plurality of cells comprising the assembled products, or products thereof.

[0046] Disclosed herein include methods. The method can comprise: providing a combinatorial library disclosed herein, or a product thereof. The method can comprise: expressing the one or more payload genes in cell(s). The method can comprise: screening for a property of interest. In some embodiments, screening comprises fluorescence-activated cellsorting (FACS), cell viability assay, ELISA, co-immunoprecipitation, a bead-based immunoassay, or any combination thereof. In some embodiments, the property of interest comprises modified enzymatic activity, improved enzymatic activity, modified binding activity, improved binding activity, modified stability, improved stability, modified localization, improved localization, modified solubility, improved solubility, modified expression, improved expression, modified inhibitor resistance, improved inhibitor resistance, modified substrate specificity, improved substrate specificity, or any combination thereof. In some embodiments, the method comprises exposing the cell(s) to one or more agents. In some embodiments, the one or more agents comprise: one or more of a chemical agent, a pharmaceutical, small molecule, a biologic, a CRISPR single-guide RNA (sgRNA), a small interfering RNA (siRNA), CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a piwi-interacting RNA (piRNA), an antisense oligonucleotide, a peptide or peptidomimetic inhibitor, an aptamer, an antibody, an intrabody, or any combination thereof; an expression vector, wherein the expression vector encodes one or more of the following: an mRNA, an antisense nucleic acid molecule, a RNAi molecule, a shRNA, a mature miRNA, a pre-miRNA, a pri-miRNA, an anti-miRNA, a ribozyme, any combination thereof; an infectious agent, an anti-infectious agent, or a mixture thereof; a cytotoxic agent, optionally a chemotherapeutic agent, a biologic agent, a toxin, a radioactive isotope, or any combination thereof; and / or one or more of an epigenetic modifying agent, epigenetic enzyme, a bicyclic peptide, a transcription factor, a DNA or protein modification enzyme, a DNA-intercalating agent, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, a competitive inhibitor for an enzyme, a protein synthesis inhibitor, a nuclease, a protein fragment or domain, a tag or marker, an antigen, an antibody or antibody fragment, a ligand or a receptor, a synthetic or analog peptide from a naturally-bioactive peptide, an anti-microbial peptide, a pore-forming peptide, a targeting or cytotoxic peptide, a degradation or self-destruction peptide, a CRISPR component system or component thereof, DNA, RNA, artificial nucleic acids, a nanoparticle, an oligonucleotide aptamer, a peptide aptamer, or any combination thereof. In some embodiments, the property of interest comprises a property of the cell, optionally improved drug resistance, altered drug sensitivity, improved or modified growth rate under selective pressure, modified or improved cell viability or survival, modified or improved stress tolerance, modified or improved secretion of a compound, altered signaling pathway activation, or any combination thereof. In some embodiments, the method comprises cloning the assembled products, or products thereof, into expression vector(s), optionally prior to an expressing step. In some embodiments, the expression vector is selected from a plasmid, a viral vector, a transposable element, a bacterial artificial chromosome, a yeast artificial chromosome, or any combination thereof. In someembodiments, the cloning step operably connects the final synthetic sequence with one or more regulatory elements selected from a promoter, an enhancer, a polyadenylation signal, a 5’UTR, a 3’ UTR, and a selection marker. In some embodiments, the method comprises transforming or transfecting host cells with the cloned expression vector, optionally bacterial cells for propagation and / or sequence verification and subsequently eukaryotic cells for expression, optionally mammalian, yeast, insect, plant, or fungal cells.

[0047] Disclosed herein include kits. Disclosed herein include systems for synthesizing nucleic acids. The system or kit can comprise: the n masked fragments disclosed herein. The system or kit can comprise: y sets of n masked fragments. In some embodiments, each of the n masked fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. The system or kit can comprise: the n fragments disclosed herein and the n pairs or n-1 pairs of masking oligonucleotides disclosed herein . The system or kit can comprise: y sets of n fragments and j' sets of n pairs or n-1 pairs of masking oligonucleotides. In some embodiments, each of the n fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. In some embodiments, each of the n pairs or n-1 pairs of masking oligonucleotides is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. In some embodiments, the first polynucleotide strand and the second polynucleotide strand that constitute each of the n fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. The system or kit can comprise: a nonthermostable ligase, a thermostable ligase, a chemical coupling agent, a polymerase, a primer capable of binding the first terminal region (or a complement thereof), a primer capable of binding the second terminal region (or a complement thereof), or any combination thereof. The system or kit can comprise: a ligation buffer. The ligation buffer can comprise: a reaction buffer configured to support annealing, ligation, and / or amplification (e.g., HiFi Taq buffer); one or more of Tris HC1 at about 10 mM to about 200 mM, at a pH of about 7.0 to about 9.5 at the incubation temperature, Mg2+ at about 0.5 mM to about 20 mM, monovalent cation(s) at about 10 mM to about 300 mM, and a reducing agent at about 0.1 mM to about 20 mM; a ligase cofactor, optionally selected from ATP at about 0.05 mM to about 5 mM or NAD+ at about 0.01 mM to about 2 mM; a buffering species selected from Tris, HEPES, Bis Tris, MOPS, and PIPES, optionally configured to maintain pH between 8.3-8.8 at 25°C; one or more additives, optionally selected from bovine serum albumin at about 0.01 mg / mL to about 1 mg / mL, polyethylene glycol at about 1% to about 20% (w / v), betaine at about 0.1 M to about 2.0 M, dimethyl sulfoxide at about 1% to about 20% (v / v), formamide at about 0.5% to about 10% (v / v), glycerol at about 1% to about 20% (v / v), and / or a non-ionic detergent at about 0.001% to about 0.1% (v / v). The system or kit can comprise: one or more purification reagent(s),optionally: gel electrophoresis reagent(s), optionally pulsed-field gel electrophoresis (PFGE); solid or solution phase hybridization / capture reagent(s); precipitation reagent(s); dialysis reagent(s); solid phase reversible immobilization (SPRI) cleanup reagent(s), optionally performing size selection using SPRI beads, further optionally single-sided or double-sided; and / or column purification reagent(s). In some embodiments, the system or kit does not comprise one or more reagents employed with Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly, optionally an exonuclease, an endonuclease, a single stranded DNA binding protein, a restriction endonuclease, a recombinase, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 depicts a non-limiting exemplary schematic of all current DNA assembly methods, which use a single-stranded DNA sequence c to “strand anneal” to a complementary single-stranded c* to assemble fragments X and Y together.

[0049] FIG. 2 depicts a non-limiting exemplary schematic of TADA. Masking oligos M and M* can use part of their sequence <X and c*Mto complement regions c* and c on fragments Y and A, “masking” them before the reaction. Barcode pair b &. b * on AT and AT* can constitute the 1stpair of toeholds, and short sequences a &. a* can constitute the 2ndpair ( / ). The TADA reaction can be initiated by mixing masked fragments X and Y. The two pairs of toeholds b* + b and a + a* can initiate the interaction between fragments and associate to form a DNA 4-way junction (ii). The isothermal strand displacement reaction can be driven forward by the perfect homology between masked c and c*; any mismatch between strands can greatly inhibit the forward reaction, favoring the reverse reaction and subsequent dissociation (iii). The strand exchange can proceed to completion, resulting in the formation of the desired joint fragment X + Y with the byproduct of annealed masking oligos MIM* (iv).

[0050] FIG. 3 depicts a non-limiting exemplary schematic of TADA. TADA can occur at both ends of fragments simultaneously and can be scaled to assemble multitudes of building blocks into a defined order with high specificity and yield.

[0051] FIG. 4 depicts a non-limiting exemplary annotated schematic of masking oligonucleotides and fragments.DETAILED DESCRIPTION

[0052] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments maybe utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein and made part of the disclosure herein.

[0053] All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology.Definitions

[0054] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of the present disclosure, the following terms are defined below.

[0055] As used herein, the term “about” shall be being its ordinary meaning, and shall also refer to plus or minus 5% of the provided value.

[0056] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. A polynucleotide can be single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids / triple helices, or a polymer including purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. In some embodiments, a polynucleotide comprises a nucleotide sequence encoding a gene product operably linked to one or more expression control elements (e.g., a promoter), as an expression cassette. Any of the RNA sequences disclosed herein may also be DNA (either single-stranded or double-stranded), e.g., wherein “U” is converted to “T ” Any of the DNA sequences disclosed herein may also be RNA, e.g., wherein “T” is converted to “U.”

[0057] As used herein, the term “binding” refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e g., when a molecule X is said to interact with a molecule Y, it means that the molecule X binds to molecule Y in a non-covalent manner). Binding interactions can be characterized by a dissociation constant (Kd), for example a Kd of, or a Kd less than, 10'6M, 10’7M, IO’8M, IO’9M, IO’10M, 10’11M, IO’12M, IO’13M, IO’14M, 10’15M, or a number or a rangebetween any two of these values. Kd can be dependent on environmental conditions, e.g., pH and temperature. “Affinity” refers to the strength of binding, and increased binding affinity is correlated with a lower Kd.

[0058] The terms “complementarity” and “complementary” can mean that a nucleic acid can form hydrogen bond(s) with another nucleic acid based on traditional Watson-Crick base paring rule, that is, adenine (A) pairs with thymine (U) and guanine (G) pairs with cytosine (C). Complementarity can be perfect (e.g. complete complementarity) or imperfect (e.g. partial complementarity). Perfect or complete complementarity indicates that each and every nucleic acid base of one strand is capable of forming hydrogen bonds according to Watson-Crick canonical base pairing with a corresponding base in another, antiparallel nucleic acid sequence. Partial complementarity indicates that only a percentage of the contiguous residues of a nucleic acid sequence can form Watson-Crick base pairing with the same number of contiguous residues in another, antiparallel nucleic acid sequence. In some embodiments, the complementarity can be at least 70%, 80%, 90%, 100% or a number or a range between any two of these values. In some embodiments, the complementarity is perfect, i.e. 100%. For example, the complementary candidate sequence segment is perfectly complementary to the candidate sequence segment, whose sequence can be deducted from the candidate sequence segment using the Watson-Crick base pairing rules. As used herein, the term “complementary” can refer to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a given position of a nucleic acid is capable of hydrogen bonding with a nucleotide of another nucleic acid, then the two nucleic acids are considered to be complementary to one another at that position. Complementarity between two single-stranded nucleic acid molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules. A first nucleotide sequence can be said to be the “complement” of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence can be said to be the “reverse complement” of a second sequence, if the first nucleotide sequence is complementary to a sequence that is the reverse (i.e., the order of the nucleotides is reversed) of the second sequence. As used herein, the terms “complement”, “complementary”, and “reverse complement” can be used interchangeably. It is understood from the disclosure that if a molecule can hybridize to another molecule it may be the complement of the molecule that is hybridizing.

[0059] As used herein, the term “nucleic acid” refers to a polynucleotide sequence, or fragment thereof. A nucleic acid can comprise nucleotides. A nucleic acid can be exogenous or endogenous to a cell. A nucleic acid can exist in a cell-free environment. A nucleic acid canbe a gene or fragment thereof. A nucleic acid can be DNA. A nucleic acid can be RNA. A nucleic acid can comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. “Nucleic acid”, “polynucleotide, “target polynucleotide”, and “target nucleic acid” can be used interchangeably.

[0060] A nucleic acid can comprise one or more modifications (e.g., a base modification, a backbone modification), to provide the nucleic acid with a new or enhanced feature (e.g., improved stability). A nucleic acid can comprise a nucleic acid affinity tag. A nucleoside can be a base-sugar combination. The base portion of the nucleoside can be a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides can be nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2’, the 3’, or the 5’ hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate groups can covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound; however, linear compounds are generally suitable. Linear compounds can have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially doublestranded compound. Within nucleic acids, the phosphate groups can commonly be referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone can be a 3’ to 5’ phosphodiester linkage.

[0061] A nucleic acid can comprise a modified backbone and / or modified internucleoside linkages. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified nucleic acid backbones containing a phosphorus atom therein can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonate such as 3 ’-alkylene phosphonates, 5’-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates including 3’-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3 ’-5’ linkages, 2’ -5’ linked analogs, andthose having inverted polarity wherein one or more intemucleotide linkages is a 3’ to 3’, a 5’ to 5’ or a 2’ to 2’ linkage.

[0062] A nucleic acid can comprise polynucleotide backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These can include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

[0063] A nucleic acid can comprise a nucleic acid mimetic. The term “mimetic” can be intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with non-furanose groups, replacement of only the furanose ring can also be referred as being a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety can be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid can be a peptide nucleic acid (PNA). In a PNA, the sugar-backbone of a polynucleotide can be replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides can be retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. The backbone in PNA compounds can comprise two or more linked aminoethylglycine units which gives PNA an amide containing backbone. The heterocyclic base moieties can be bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

[0064] A nucleic acid can comprise a morpholino backbone structure. For example, a nucleic acid can comprise a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, a phosphorodiamidate or other non-phosphodiester internucleoside linkage can replace a phosphodiester linkage.

[0065] A nucleic acid can comprise linked morpholino units (e.g., morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. Linking groups can link the morpholino monomeric units in a morpholino nucleic acid. Non-ionic morpholino-based oligomeric compounds can have less undesired interactions with cellular proteins. Morpholinobased polynucleotides can be nonionic mimics of nucleic acids. A variety of compounds within the morpholino class can be joined using different linking groups. A further class of polynucleotide mimetic can be referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a nucleic acid molecule can be replaced with a cyclohexenyl ring.CeNA DMT protected phosphoramidite monomers can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. The incorporation of CeNA monomers into a nucleic acid chain can increase the stability of a DNA / RNA hybrid. CeNA oligoadenylates can form complexes with nucleic acid complements with similar stability to the native complexes. A further modification can include Locked Nucleic Acids (LNAs) in which the 2 ’-hydroxyl group is linked to the 4’ carbon atom of the sugar ring thereby forming a 2’-C, 4’-C-oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2), group bridging the 2’ oxygen atom and the 4’ carbon atom wherein n is 1 or 2. LNA and LNA analogs can display very high duplex thermal stabilities with complementary nucleic acid (Tm=+3 to +10 °C), stability towards 3’-exonucleolytic degradation and good solubility properties.

[0066] A nucleic acid can also include nucleobase (also referred to as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases can include the purine bases, (e.g., adenine (A) and guanine (G)), and the pyrimidine bases, (e.g., thymine (T), cytosine (C) and uracil (U)). Modified nucleobases can include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl ( — C=C — CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5 -trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3 -deazaadenine. Modified nucleobases can include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido(5,4-b)(l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (l,4)benzoxazin-2(3H)-one), phenothiazine cytidine (lH-pyrimido(5,4-b)(l,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (l,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3’,2’:4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0067] As used herein, the term “toehold” can refer to a nucleic acid sequence region that is capable of initiating, promoting, and / or facilitating association between nucleic acid molecules, strands, or complexes through hybridization with a complementary or substantiallycomplementary sequence. A toehold can function as an initial recognition region that nucleates interaction between two nucleic acid species and can thereby promote formation of a more extensive hybridized structure, strand-exchange intermediate, junction intermediate, or assembled product. Unless the context clearly indicates otherwise, a toehold need not alone provide the full stability of a final hybridized product, but instead can serve as an initial point of contact that enables subsequent hybridization of one or more additional sequence regions. As used herein, a “fragment toehold” can refer to a toehold present on a fragment, e.g., a first fragment toehold or a second fragment toehold. In some embodiments, a fragment toehold is located in a 3’ overhang of a fragment. In some embodiments, a fragment toehold is configured to hybridize to a complementary fragment toehold of an adjacent fragment. In some embodiments, such fragment-toehold hybridization can occur after an initial association between adjacent masked fragments has already been established, and can further align the adjacent fragments for formation of a junction intermediate and for subsequent hybridization between a first complementary region of one fragment and a second complementary region of the adjacent fragment. As used herein, a “masking toehold” can refer to a toehold present on a masking oligonucleotide, including a masking toehold of an upstream masking oligonucleotide or a masking toehold of a downstream masking oligonucleotide. In some embodiments, the masking toehold of a masking oligonucleotide associated with one masked fragment is complementary to the masking toehold of a masking oligonucleotide associated with an adjacent masked fragment. In some embodiments, hybridization between such masking toeholds provides an initial interaction between adjacent masked fragments. This initial masking-toehold interaction can bring the adjacent masked fragments into proximity, orient the adjacent masked fragments relative to one another, and / or nucleate the subsequent interaction of the corresponding fragment toeholds. In some embodiments, assembly proceeds through an ordered sequence of interactions involving toeholds. For example, a masking toehold associated with one masked fragment can first hybridize to a complementary masking toehold associated with an adjacent masked fragment. Following that initial interaction, a first fragment toehold of one fragment can hybridize to a complementary second fragment toehold of the adjacent fragment. The combined interactions of the masking toeholds and the fragment toeholds can promote formation of a fourway junction intermediate between the adjacent masked fragments. In some embodiments, the four-way junction then undergoes strand exchange and / or resolution, such that a first complementary region of one fragment hybridizes to a second complementary region of the adjacent fragment, while the masking oligonucleotides become associated with one another. Accordingly, in some embodiments, a toehold is not merely a passive sequence element but is a sequence element that participates in a staged hybridization process. A toehold can providekinetic access to a downstream assembly event, including alignment of adjacent masked fragments, nucleation of fragment-fragment recognition, formation of a four-way junction, promotion of strand exchange, and / or progression toward a more stable hybridized intermediate or assembled product. In some embodiments, toehold interactions contribute to assembly specificity by favoring intended adjacent-fragment interactions over non-intended fragment pairings. A toehold, including a fragment toehold or a masking toehold, can be terminal or internal and can be present in a single-stranded, overhang, partially paired, transiently exposed, or otherwise accessible configuration, provided that the toehold is capable of participating in the intended hybridization-mediated interaction. In some embodiments, a toehold comprises a contiguous sequence of nucleotides. In some embodiments, the functional interaction of a toehold can tolerate one or more mismatches, wobble base pairs, modified nucleotides, nucleic acid analogs, or combinations thereof, so long as the toehold remains capable of initiating or facilitating the intended interaction under the reaction conditions employed.

[0068] As used herein, “melting temperature” or “Tm” of, e.g., a nucleic acid region (such as a toehold), refers to the temperature at which 50% of the molecules are in the duplexed state and 50% are single stranded under a defined set of conditions. Unless otherwise specified, Tm values reported herein are predicted or measured under standard salt and strand conditions and can be adjusted depending on the context. Tm can determined using a nearest neighbor thermodynamic model with salt correction and strand concentration adjustment. In some embodiments, Tm can calculated using a web based calculator provided by Integrated DNA Technologies (IDT OligoAnalyzer), with default parameters for DNA / DNA duplexes (50 mM Na+, no Mg2+, 25°C reference), and a strand concentration of 0.5 pM per strand; in other embodiments, Tm is calculated using IDT OligoAnalyzer with user specified monovalent and divalent ion concentrations and strand concentrations that match the intended reaction conditions. Equivalent calculations can be performed using NUPACK, MELTING, DINAMelt, Primer3, or other software implementing nearest neighbor parameters. For RNA or nucleic acid analogs, the corresponding DNA / RNA or RNA / RNA parameter sets and applicable ion corrections are used. In some embodiments, Tm is measured experimentally by UV absorbance (A260) thermal denaturation using a spectrophotometer with temperature control. Measurements are performed in a buffer comprising, e.g., 10 mM sodium phosphate (pH 7.0) and 100 mM NaCl with an oligonucleotide duplex concentration of 1 pM (strand concentration defined as total single stranded equivalents), using a heating / cooling rate of 0.5-1.0°C / min. Tm is determined as the midpoint of the first derivative of the melting curve. Equivalent buffer systems (e.g., 10 mM Tris HC1, pH 7.5-8.0, with 50-150 mM NaCl and 0-2 mM MgC12) may be used provided that the composition is reported and the Tm is adjusted or recalculated for theintended reaction conditions.

[0069] The term “vector” as used herein, can refer to a vehicle for carrying or transferring a nucleic acid. Non-limiting examples of vectors include plasmids, bacteria, and viruses. The term “construct,” as used herein, can refer to a recombinant nucleic acid that has been generated for the purpose of the expression of a specific nucleotide sequence(s), or that is to be used in the construction of other recombinant nucleotide sequences. As used herein, the term “plasmid” can refer to a nucleic acid that can be used to replicate recombinant DNA sequences within a host organism. The sequence can be a double stranded DNA.

[0070] As used herein, the term “promoter” is a nucleotide sequence that permits binding of RNA polymerase and directs the transcription of a gene. Typically, a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of the gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals (including humans). A promoter can be inducible, repressible, and / or constitutive. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as a change in temperature.

[0071] As used herein, the term “operably linked” is used to describe the connection between regulatory elements and a gene or its coding region. Typically, gene expression is placed under the control of one or more regulatory elements, for example, without limitation, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element. For instance, a promoter is operably linked to a coding sequence if the promoter effects transcription or expression of the coding sequence.

[0072] As used herein in the term “derived from”, in the context of an amino acid sequence or polynucleotide sequence (e.g., an amino acid sequence “derived from” a conjugation system or a transposase system), is meant to indicate that the polypeptide or nucleic acid has a sequence that is based on that of a reference polypeptide or nucleic acid, and is not meant to be limiting as to the source or method in which the protein or nucleic acid is made. By way of example, the term “derived from” includes homologs or variants of reference amino acid or DNA sequences. As used herein, the term “derived from” can also refer to a specified nucleotide sequence that may be obtained from a particular specified source or species, albeit not necessarily directly from that specified source or species.

[0073] Standard techniques can be used for recombinant DNA, oligonucleotidesynthesis, and cell culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose. Unless specific definitions are provided, the nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those commonly known and used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.Toehold Assisted DNA Assembly (TADA) as a Novel Method of DNA Assembly

[0074] To generate longer DNA strands, multiple smaller DNA pieces need to be assembled in the correct order. Provided herein include, in some embodiments, novel methods, compositions, systems, and kits, for DNA assembly using Toehold Assisted DNA Assembly (TADA) to assemble a number of DNA fragments to a bigger size. Current assembly methods share the same intrinsic limitation. Current state-of-the-arts in DNA assembly, in particular Golden Gate, Gibson, and Polymerase Chain Assembly, all rely on “strand annealing” between two single stranded overlapping sequences to direct assembly between fragments (FIG. 1).However, due to fundamental physical and chemical limitations, exposed single-stranded sequences can participate in undesired partial annealing with incomplete complementation, leading to mis-assembled final products. This intrinsically limits the number of pieces, and therefore DNA sizes, that can be assembled with high accuracy, yield, throughput, and sequence complexity.

[0075] To overcome this ubiquitous limitation, provided herein include, in some embodiments, novel methods, compositions, systems, and kits, for DNA assembly employing a novel “TADA” method based on the DNA strand displacement reaction (FIG. 2). Some embodiments provided herein comprise individually “masking” the exposed complementary sequences with “masking oligos”. In the absence of the desired match, the “masking oligos” can stay on and block pairing with sub-optimal ends. In some embodiments, with and only with designated binding partners are the “masking oligos” removed. In some embodiments, this specific reaction is first initiated by matching “toeholds” through an isothermal strand displacement reaction (4-way strand exchange). Even in the unlikely case of incorrect toeholdspairing, the complementary regions can be protected by masking such that any mismatch will block the progression of the 4-way strand exchange by many orders of magnitude. Together, only perfect complementation can effectively drive the completion of the TADA reaction and be eventually joined. As described herein, this principle can be easily scaled up to both ends of a very large number of DNA fragments (FIG. 3).

[0076] TADA compositions, methods, systems, and kits provided herein can exhibit unique properties. The DNA strand displacement principle is intrinsically different from the strand annealing principle utilized in other DNA assembly methods. In some embodiments, and without being bound by any particular theory, the novelty of TADA is to multiplex and apply the DNA strand displacement principles to the field of DNA assembly. In addition, because the matching toeholds b1and b1* that initiate the TADA reaction are not a part of the final assembly sequences (FIG. 3), they can be extensively optimized for maximum mutual specificity within a pair and absolute exclusivity across different pairs in some embodiments. Ultimately, the reduction of unwanted pairings can enable significantly more fragments to be assembled in one reaction, allowing TADA to outperform all current state-of-the-art strand-annealing based methods by several orders or magnitude in both specificity and yield in some embodiments. In some embodiments fragments, masked fragments, and masking oligonucleotides described herein can be designed using the NUPACK web application (See Fomace, M. E., et al. NUPACK: analysis and design of nucleic acid structures, devices, and systems. ChemRxiv (2022), See Zadeh, J. N., et al. NUPACK: analysis and design of nucleic acid systems. J Comput Chem, 32,170-173 (2011), the contents of which are incorporated herein by reference in their entireties).Linear TADA

[0077] Provided herein, in some embodiments, are compositions. The composition can comprise: n fragments. The first fragment can comprise a first fragment toehold and a first complementary region. Each (z)th fragment can comprise a first fragment toehold, a first complementary region, a second complementary region, and a second fragment toehold, wherein 1 < z < n. Each (z)th fragment can comprise a first polynucleotide strand and a second polynucleotide strand. The first polynucleotide strand can comprise a 3’ overhang. The second polynucleotide strand can comprise a 3’ overhang. The 3’ overhang of the first polynucleotide strand can comprise the first fragment toehold and the first complementary region. The 3’ overhang of the second polynucleotide strand can comprise the second fragment toehold and the second complementary region. In some embodiments, for each (z’)th fragment: the first complementary region of the (z’)th fragment is complementary to the second complementary region of the (z+l)th fragment. In some embodiments, for each (z)th fragment: the first fragmenttoehold of the (z)th fragment is complementary to the second fragment toehold of the (z+1 )th fragment. In some embodiments, for each (z)th fragment: the second complementary region of the (z)th fragment is complementary to the first complementary region of the (z-l)th fragment. In some embodiments, for each (z)th fragment: the second fragment toehold of the (z)th fragment is complementary to the first fragment toehold of the (z-l)th fragment. The (zz)th fragment can comprise a second fragment toehold and a second complementary region. The composition can comprise: n-1 pairs of masking oligonucleotides, wherein n is an integer greater than 2. Each pair of masking oligonucleotides can comprise an upstream masking oligonucleotide and a downstream masking oligonucleotide. The upstream masking oligonucleotide can comprise a masking region and a masking toehold. The downstream masking oligonucleotide can comprise a masking region and a masking toehold. In some embodiments, for each (c / )th pair of masking oligonucleotides, wherein q is a positive integer less than zz: the masking region of the (c / )th upstream masking oligonucleotide is complementary to the first complementary region of the (c / )th fragment; the masking region of the (t / )th downstream masking oligonucleotide is complementary to the second complementary region of the (c / +l)th fragment; and the masking toehold of the (c / )th upstream masking oligonucleotide is complementary to the masking toehold of the ( ) th downstream masking oligonucleotide.

[0078] The composition can comprise: n masked fragments. The first masked fragment can comprise the first fragment hybridized to an upstream masking oligonucleotide via the first complementary region of the first fragment. The (zz)th masked fragment can comprise the (zz)th fragment hybridized to a downstream masking oligonucleotide via the second complementary region of the (zz th fragment. The (z)th masked fragment can comprise a (z)th fragment hybridized to: (i) an upstream masking oligonucleotide via the first complementary region of the (z)th fragment; and (ii) a downstream masking oligonucleotide via the second complementary region of the (z)th fragment. The first fragment and / or the (zz)th fragment can be single-stranded. The first fragment and / or the (zz)th fragment can comprise a first polynucleotide strand and a second polynucleotide strand. The first fragment can comprise a first terminal region, optionally a 5’ terminal region. The (zz)th fragment can comprise a second terminal region, optionally a 3’ terminal region.Circular TADA

[0079] Provided herein, in some embodiments, are compositions. The composition can comprise: n fragments. Each fragment can comprise a first fragment toehold, a first complementary region, a second complementary region, and a second fragment toehold. Each fragment can comprise a first polynucleotide strand and a second polynucleotide strand. The first polynucleotide strand can comprise a 3’ overhang. The second polynucleotide strand cancomprise a 3’ overhang. The 3’ overhang of the first polynucleotide strand can comprise the first fragment toehold and the first complementary region. The 3’ overhang of the second polynucleotide strand can comprise the second fragment toehold and the second complementary region. In some embodiments, for each (z)th fragment, wherein 1 < z < n the first complementary region of the (z)th fragment is complementary to the second complementary region of the (z+ 1 )th fragment; the first fragment toehold of the (z)th fragment is complementary to the second fragment toehold of the (z+ l)th fragment; the second complementary region of the (z)th fragment is complementary to the first complementary region of the (z-l)th fragment; and the second fragment toehold of the (z)th fragment is complementary to the first fragment toehold of the (z-l)th fragment. The second complementary region of the first fragment can be complementary to the first complementary region of the (zz)th fragment. The second fragment toehold of the first fragment can be complementary to the first fragment toehold of the (zz)th fragment. The composition can comprise: n pairs of masking oligonucleotides, wherein n is an integer greater than 2. Each pair of masking oligonucleotides can comprise an upstream masking oligonucleotide and a downstream masking oligonucleotide. The upstream masking oligonucleotide can comprise a masking region and a masking toehold. The downstream masking oligonucleotide can comprise a masking region and a masking toehold. In some embodiments, for each (< / )th pair of masking oligonucleotides, wherein q is a positive integer less than zz: the masking region of the (<z)th upstream masking oligonucleotide is complementary to the first complementary region of the (z / )th fragment; the masking region of the (k / )th downstream masking oligonucleotide is complementary to the second complementary region of the (q+ 1 )th fragment; and the masking toehold of the (z / )th upstream masking oligonucleotide is complementary to the masking toehold of the (z / )th downstream masking oligonucleotide. The masking region of the (zz)th upstream masking oligonucleotide can be complementary to the first complementary region of the (zz)th fragment. The masking region of the (zz)th downstream masking oligonucleotide can be complementary to the second complementary region of the first fragment. The masking toehold of the (zz)th upstream masking oligonucleotide can be complementary to the masking toehold of the (zz)th downstream masking oligonucleotide.

[0080] The composition can comprise: n masked fragments, wherein a masked fragment comprises a fragment of the n fragments hybridized to: (i) an upstream masking oligonucleotide via the first complementary region of the fragment; and (ii) a downstream masking oligonucleotide via the second complementary region of the fragment.2-Component TADA

[0081] Provided herein, in some embodiments, are compositions. The composition can comprise: a first fragment and a second fragment. Each of the first fragment and the secondfragment can comprise a first polynucleotide strand and a second polynucleotide strand. The first fragment can comprise a first fragment toehold and a first complementary region. The first polynucleotide strand of the first fragment can comprise a 3’ overhang, and wherein said 3’ overhang of the first polynucleotide strand can comprise the first fragment toehold and the first complementary region. The second fragment can comprise a second complementary region and a second fragment toehold. The second polynucleotide strand of the second fragment can comprise a 3’ overhang, and wherein said 3’ overhang of the second polynucleotide strand can comprise the second fragment toehold and the second complementary region. The first complementary region of the first fragment can be complementary to the second complementary region of the second fragment. The first fragment toehold of the first fragment can be complementary to the second fragment toehold of the second fragment. The composition can comprise: an upstream masking oligonucleotide and a downstream masking oligonucleotide. The upstream masking oligonucleotide can comprise a masking region and a masking toehold. The downstream masking oligonucleotide can comprise a masking region and a masking toehold. The masking region of the upstream masking oligonucleotide can be complementary to the first complementary region of the first fragment. The masking region of the downstream masking oligonucleotide can be complementary to the second complementary region of the second fragment. The masking toehold of the upstream masking oligonucleotide can be complementary to the masking toehold of the downstream masking oligonucleotide.

[0082] The composition can comprise: a first masked fragment and a second masked fragment. The first masked fragment can comprise the first fragment hybridized to the upstream masking oligonucleotide via the first complementary region of the first fragment. The second masked fragment can comprise the second fragment hybridized to the downstream masking oligonucleotide via the second complementary region of the second fragment.Masking Oligonucleotide Configurations

[0083] The upstream masking oligonucleotide and the downstream masking oligonucleotide can be single-stranded. The masking region of the upstream masking oligonucleotide can be 5’ of the masking toehold. The masking region of the downstream masking oligonucleotide can be 3’ of the masking toehold. Less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, or a number or a range between any two of these values, of fragments in the composition can be unmasked fragments, wherein an unmasked fragment comprises: (i) a first complementary region not hybridized to an upstream masking oligonucleotide; and / or (ii) a second complementary region not hybridized to a downstream masking oligonucleotide. In some embodiments, the masking region of each upstream masking oligonucleotide is not complementary to the masking region of any other upstream maskingoligonucleotide. In some embodiments, the masking region of each downstream masking oligonucleotide is not complementary to the masking region of any other downstream masking oligonucleotide. The masking region of the (vr )th upstream masking oligonucleotide can be complementary to the masking region of the (w)th downstream masking oligonucleotide, wherein w is an integer from 1 to n. In some embodiments, the masking region of the ( )th upstream masking oligonucleotide is not complementary to the masking region of the (g)th downstream masking oligonucleotide, wherein fAg, optionally each of f and g is independently an integer from 1 to n or from 1 to n-1. Each pair of masking oligonucleotides can be optimized for maximum mutual specificity within a pair and absolute exclusivity across different pairs.TADA Configurations

[0084] Said complementarity can be or comprises: at least 80%, 85%, 90%, 95%, 99%, 100%, or a number or a range between any two of these values, complementarity; less than five, four, three, two, or one, base pair mismatches; reverse complementarity; canonical Watson-Crick base pairing; wobble base pairing, optionally G-U wobble; and / or DNA nanotechnology interactions, optionally Hoogsteen base pairing, G-quadruplex(es), DNA origami, aptamer-ligand interactions, or any combination thereof.

[0085] One or more of the n fragments can comprise an internal segment. In some embodiments, the internal segment does not comprise any of the first fragment toehold, the second fragment toehold, the first complementary region, and the second complementary region. The internal segment can be 5’ of the first fragment toehold and the first complementary region. The internal segment can be 3’ of the second fragment toehold and the second complementary region. The internal segment can be double-stranded. The first polynucleotide strand and / or second polynucleotide strand of one or more of the n fragments can comprise a 5’ phosphate.

[0086] In some embodiments, the first fragment, the (z)th fragment, the ( / / )th fragment, one or more of the n fragments, the first fragment toehold, the second fragment toehold, the first complementary region, the second complementary region, the internal segment, one or more upstream masking oligonucleotides, one or more downstream masking oligonucleotides, the masking region, the masking toehold, the first polynucleotide strand, the second polynucleotide strand, and / or the terminal region: (a) is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 1-5, 1-10, 10-100, 10-250, 25-50, 25-100, 25-250, 50-100, 50-200, 50-250, 75-100, 75-200, 75-250, 100-150, 100-200, 100-250, 150-200, 150-250, 200-250, or a number or a range between any two of these values, nucleotides in length; (b) comprises a GC content of about 20%, 21%, 22%, 23%, 24%, 25%,26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%.42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 20%-50%, 20%-75%, 20%- 100%, 30%-60%, 30%-75%, 30%-100%, 40%-60%, 40%-75%, 40%-100%, 50%-75%, 50%-100%, 60%-75%, 60%-100%, 75%-100%, or a number or a range between any two of these values; (c) comprises a melting temperature (Tm) of about 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 35°C-55°C, 35°C-75°C, 35°C-100°C, 45°C-55°C, 45°C-75°C, 45°C-100°C, 55°C-75°C, 55°C-100°C, 65°C-75°C, 65°C-100°C, 75°C-100°C, or a number or a range between any two of these values; (d) comprises DNA; (e) comprises RNA; and / or (f) comprises one or more nucleic acid analogs, optionally selected from the group consisting of RNA, 2’-O-methyl RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino, phosphorodiamidate morpholino oligomer (PMO), HNA, FANA, TNA, ANA, GNA, CeNA, UNA, L-DNA, or any combination thereof.

[0087] In some embodiments, the fragments, upstream masking oligonucleotides, downstream masking oligonucleotides, the first polynucleotide strand, and / or the second polynucleotide strand: comprise or are derived from synthetic oligonucleotides; and / or comprise or are derived from rolling circle amplification products, restriction enzyme digestion products, reverse transcription products, CRISPR-excised products, PCR amplification products, templateindependent polymerase products, recombinase-generated products, phage-derived products, or any combination thereof. In some embodiments, n can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 10-25, 10-50, 10-75, 10-100, 10-500, 10-1000, 25-50, 25-75, 25-100, 25-500, 25-1000, 50-75, 50-100, 50-500, 50-1000, 75-100, 75-500, 75-1000, 100-500, 100-1000, 500-1000, or a number or a range between any two of these values.TADA Assembly Configurations

[0088] Upon incubation in a reaction mixture, the n masked fragments can be capable of joining together via at least one four- way junction (4WJ) intermediate to generate an intermediate product. A ligase can be capable of ligating nicks on the first polynucleotide strands and the second polynucleotide strands of said intermediate product to generate anassembled product. A ligase and / or a chemical coupling agent can be capable of forming a covalent linkage between adjacent first polynucleotide strands and between adjacent second polynucleotide strands of said intermediate product to generate an assembled product. The covalent linkage can be formed by a click ligation between complementary reactive handles on adjacent first polynucleotide strands and / or second polynucleotide strands, optionally copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), strain promoted azide-alkyne cycloaddition (SPAAC), or inverse electron demand Diels-Alder (iEDDA) reaction between a trans cyclooctene and a tetrazine oxime formation, hydrazone formation, Michael addition, disulfide formation, carbodiimide-mediated coupling, native chemical ligation, or any combination thereof. The first polynucleotide strands and / or second polynucleotide strands can comprise synthetic modifications and / or modified synthetic nucleotides, optionally selected a 5’ alkyne, a 3’ azide, a trans-cyclooctene, a tetrazine, a 5’ amine, an aldehyde, an aminooxy group, a thiol, a maleimide, or a phosphorothioate, or any combination thereof. The chemical coupling agent can comprise a click chemistry reagent, a copper(I) source, a copper(I)-stabilizing ligand, a strain-promoted cycloaddition reagent, a tetrazine, an EDC or other carbodiimide, an aniline or p-phenylenediamine catalyst, or any combination thereof.

[0089] The assembled product or a product thereof can comprise a final synthetic sequence. The final synthetic sequence can comprise the scarless assembly of the n fragments. The final synthetic sequence can be at least about 500 bases, 750 bases, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50 kb, 75 kb, 100 kb, 250 kb, 500 kb, 750 kb, 1MB, or a number or a range between any two of these values, in length.

[0090] In some embodiments, the first fragment, the (z)th fragment, the ( / / )th fragment, one or more of the n fragments, the first fragment toehold, the second fragment toehold, the first complementary region, the second complementary region, and / or the internal segment: (a) comprises an elevated GC content of at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values; (b) comprises a reduced GC content of less about 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 40%-30%, 40%-20%, 40%-10%, 40%-5%, 40%-l%, 30%-20%, 30%-10%, 30%-5%, 30%-l%, 20%-10%, 20%-5%, 20%-l%, 10%-5%, 10%-l%, 5%-l%, or a number or a range between any two of these values; (c) comprises two or more repeats, optionally tandem repeats, optionally at least 4 nt in length, optionally occurring at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a number or a range between any two of these values, times withinthe final synthetic sequence; and / or (d) comprises two or more mononucleotide stretches, optionally at least 4 nt in length, optionally occurring at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a number or a range between any two of these values, times within the final synthetic sequence.

[0091] In some embodiments, for each (z’)th fragment, the first fragment toehold of the (z)th fragment is not complementary to the second fragment toehold of any (Zr)th fragment, wherein k is an integer not equal to (z+1). In some embodiments, for at least one (z)th fragment, the first fragment toehold of the (z)th fragment is complementary to the second fragment toehold of one or more (A)th fragments, wherein k is an integer not equal to (z+1).Combinatorial Libraries

[0092] In some embodiments, the first fragment: is an invariant fragment, wherein all instances of the invariant first fragment in the composition are identical; or is a variant fragment, wherein two or more instances of the variant first fragment in the composition differ with respect to the sequence of the internal segment. At least one (z)th fragment can be an invariant fragment, wherein all instances of the invariant (z)th fragment in the composition are identical. At least one (z)th fragment can be a variant fragment, wherein two or more instances of the variant (z)th fragment in the composition differ with respect to the sequence of the internal segment. In some embodiments, the (zz)th fragment: is an invariant fragment, wherein all instances of the invariant (zz)th fragment in the composition are identical; or is a variant fragment, wherein two or more instances of the variant (zz)th fragment in the composition differ with respect to the sequence of the internal segment. In some embodiments, variant fragments can comprise predefined codon variations, optionally codons variations configured to achieve modified and / or improved protein function(s).Parallel TADA Assembly

[0093] The composition can comprise y sets of n masked fragments. The value of n can be the same between at least two of the y sets. The value of n can be different between at least two of the y sets. In some embodiments, the masking region of each set is not complementary to the masking region of any other set. In some embodiments, upon incubation of the sets together in a single reaction mixture, each set of n masked fragments is capable of, in parallel joining together via four- way junction (4WJ) intermediates to generate y intermediate products. The y intermediate products can be candidate design variants. The y intermediate products, or products thereof, can be capable of being individually amplified or universally amplified. In some embodiments, y can be an integer greater than 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or a number or a range between any two of these values.Payloads

[0094] The final synthetic sequence can comprise one or more payload genes. The one or more payload genes can encode one or more RNA payload(s) and / or one or more payload protein(s). The one or more RNA payload(s) can be selected from the group comprising a CRISPR single-guide RNA (sgRNA), a small interfering RNA (siRNA), a CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a piwi-interacting RNA (piRNA), an antisense oligonucleotide, an antagomir, an aptamer, a ribozyme, or any combination thereof. A payload protein can comprise: fluorescence activity, polymerase activity, protease activity, phosphatase activity, kinase activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity demyristoylation activity, or any combination thereof. A payload protein can comprise: nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, adenylation activity, deadenylation activity, or any combination thereof.

[0095] A payload protein can comprise: a biomaterials payload, optionally a structural polypeptide, further optionally silk fibroin, spider silk spidroin, a resilin, a resilin-like polypeptide, an elastin, an elastin-like polypeptide, a collagen, or a collagen-like polypeptide. A payload protein can comprise: a cellular reprogramming factor capable of differentiating a given cell into a desired differentiated state, optionally nerve growth factor (NGF), fibroblast growth factor (FGF), interleukin-6 (IL-6), bone morphogenic protein (BMP), neurogenin3 (Ngn3), pancreatic and duodenal homeobox 1 (Pdxl), Mafa, or any combination thereof. A payload protein can comprise: an agonistic or antagonistic antibody or antigen-binding fragment thereof specific to a checkpoint inhibitor or checkpoint stimulator molecule, optionally PD1, PD-L1, PD-L2, CD27, CD28, CD40, CD137, 0X40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-1, and / or TIM-3. A payload protein can comprise: a secretion tag, optionally the secretion tag is selected from the group comprising AbnA, AmyE, AprE, BglC, BglS, Bpr, Csn, Epr, Ggt, GlpQ, HtrA, Lip A, LytD, MntA, Mpr, NprE, Opp A, PbpA, PbpX, Pel, PelB, PenP, PhoA, PhoB, PhoD, PstS, TasA, Vpr, WapA, WprA, XynA, XynD, YbdN, Ybxl, YcdH, YclQ, YdhF, YdhT, YfkN, YflE, YfmC, Yfnl, YhcR, YlqB, YncM, YnfF, YoaW, YocH, YolA, YqiX, Yqxl, YrpD, YrpE, YuaB, Yuri, YvcE, YvgO, YvpA, YwaD, YweA, YwoF, YwtD, YwtF, YxaLk, YxiA, and YxkC. A payload protein can comprise: a constitutive signal peptide for protein degradation, optionally PEST. A payload protein cancomprise: a nuclear localization signal (NLS) or a nuclear export signal (NES). A payload protein can comprise: a dosage indicator protein, optionally the dosage indicator protein is detectable, optionally the dosage indicator protein comprises green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, m Apple, mCherry, mruby3, rsCherry, rsCherryRev, derivatives thereof, or any combination thereof.

[0096] A payload protein can comprise: a cellular reprogramming factor capable of converting an at least partially differentiated cell to a less differentiated cell, optionally Oct-3, Oct-4, Sox2, c-Myc, Klf4, Nanog, Lin28, ASCL1, MYT1L, TBX3b, SV40 large T, hTERT, miR-291, miR-294, miR-295, or any combinations thereof. A payload protein can comprise: a programmable nuclease, optionally the programmable nuclease is selected from the group comprising: SpCas9 or a derivative thereof; VRER, VQR, EQR SpCas9; xCas9-3.7; eSpCas9; Cas9-HF1; HypaCas9; evoCas9; ScCas9; StCas9; NmCas9; SaCas9; CjCas9; CasX; Cas9 H940A nickase; Cast 2 and derivatives thereof; dcas9-APOBECl fusion, BE3, and dcas9-deaminase fusions; dcas9-Krab, dCas9-VP64, dCas9-Tetl, and dcas9-transcriptional regulator fusions; Dcas9-fluorescent protein fusions; Cas 13 -fluorescent protein fusions; RCas9-fluorescent protein fusions; Cas 13 -adenosine deaminase fusions, or any combination thereof. A payload protein can comprise: a CRE recombinase, GCaMP, a cell therapy component, a knockdown gene therapy component, a cell-surface exposed epitope, or any combination thereof. A payload protein can comprise: a bispecific T cell engager (BiTE). A payload protein can comprise: a synthetic receptor, optionally a Synthetic Notch (SynNotch) receptor, a Modular Extracellular Sensor Architecture (MESA) receptor, Tango, dCas9-synR, or any combination thereof.

[0097] A payload protein can comprise: a cytokine, optionally the cytokine is selected from the group consisting of interleukin- 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, granulocyte macrophage colony stimulating factor (GM-CSF), M-CSF, SCF, TSLP, oncostatin M, leukemia-inhibitory factor (LIF), CNTF, Cardiotropin- 1, NNT-l / BSF-3, growth hormone, Prolactin, Erythropoietin, Thrombopoietin, Leptin, G-CSF, or receptor or ligand thereof. A payload protein can comprise: a member of the TGF-p / BMP family selected from the group consisting of TGF-pi, TGF-P2,TGF-P3, BMP-2, BMP-3a, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-15, BMP-16, endometrial bleeding associated factor (EBAF), growth differentiation factor-1 (GDF-1), GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-12, GDF-14, mullerian inhibiting substance (MIS), activin-1, activin-2, activin-3, activin-4, and activin-5. A payload protein can comprise: a member of the TNF family of cytokines selected from the group consisting of TNF-alpha, TNF-beta, LT-beta, CD40 ligand, Fas ligand, CD 27 ligand, CD 30 ligand, and 4-1 BBL. A payload protein can comprise: a member of the immunoglobulin superfamily of cytokines selected from the group consisting of B7.1 (CD80) andB7.2 (B70).

[0098] A payload protein can comprise: an interferon, optionally the interferon is selected from interferon alpha, interferon beta, or interferon gamma. A payload protein can comprise: a chemokine, optionally the chemokine is selected from CCL1, CCL2, CCL3, CCR4, CCL5, CCL7, CCL8 / MCP-2, CCL11, CCL13 / MCP-4, HCC- 1 / CCL14, CTAC / CCL17, CCL19, CCL22, CCL23, CCL24, CCL26, CCL27, VEGF, PDGF, lymphotactin (XCL1), Eotaxin, FGF, EGF, IP-10, TRAIL, GCP-2 / CXCL6, NAP- 2 / CXCL7, CXCL8, CXCL10, ITAC / CXCL11, CXCL12, CXCL13, or CXCL15. A payload protein can comprise: an interleukin, optionally the interleukin is selected from IL-10 IL-12, IL-1, IL-6, IL-7, IL-15, IL-2, IL-18 or IL-21. A payload protein can comprise: a tumor necrosis factor (TNF), optionally the TNF is selected from TNF- alpha, TNF-beta, TNF-gamma, CD252, CD154, CD178, CD70, CD153, or 4-1BBL. A payload protein can comprise: a factor locally down-regulating the activity of endogenous immune cells. A payload protein can comprise: a factor capable of remodeling a tumor microenvironment and / or reducing immunosuppression at a target site of a subject. A payload protein can comprise: a chimeric antigen receptor (CAR) or T-cell receptor (TCR), optionally the CAR and / or TCR comprises one or more of an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, optionally wherein the intracellular signaling domain comprises a primary signaling domain, a costimulatory domain, or both of a primary signaling domain and a costimulatory domain. A payload protein can comprise: an activity regulator, optionally the activity regulator is capable of reducing T cell activity.

[0099] A payload protein can be associated with an agricultural trait of interest selected from the group consisting of increased yield, increased abiotic stress tolerance, increased drought tolerance, increased flood tolerance, increased heat tolerance, increased cold and frost tolerance, increased salt tolerance, increased heavy metal tolerance, increased low-nitrogen tolerance, increased disease resistance, increased pest resistance, increased herbicide resistance, increased biomass production, male sterility, or any combination thereof. A payload protein can be associated with a biological manufacturing process selected from the groupcomprising fermentation, distillation, biofuel production, production of a compound, production of a polypeptide, or any combination thereof. The one or more payload genes can be selected from the group comprising a nitrogen fixation gene, a plant stress-induced gene, a nutrient utilization gene, a gene that affects plant pigmentation, a gene that encodes an antisense or ribozyme molecule, a gene encoding an antigen capable of being secreted, a toxin gene, a receptor gene, a ligand gene, a seed storage gene, a hormone gene, an enzyme gene, an interleukin gene, a cytokine gene, a growth factor gene, a transcription factor gene, a transcriptional repressor gene, a DNA-binding protein gene, a recombination gene, a DNA replication gene, a programmed cell death gene, a kinase gene, a phosphatase gene, a G protein gene, a cyclin gene, a cell cycle control gene, a gene involved in transcription, a gene involved in translation, a gene involved in RNA processing, a gene involved in RNAi, an organellar gene, a intracellular trafficking gene, an integral membrane protein gene, a transporter gene, a membrane channel protein gene, a cell wall gene, a gene involved in protein processing, a gene involved in protein modification, a gene involved in protein degradation, a gene involved in metabolism, a gene involved in biosynthesis, a gene involved in assimilation of nitrogen or other elements or nutrients, a gene involved in controlling carbon flux, gene involved in respiration, a gene involved in photosynthesis, a gene involved in light sensing, a gene involved in organogenesis, a gene involved in embryogenesis, a gene involved in differentiation, a gene involved in meiotic drive, a gene involved in self incompatibility, a gene involved in development, a gene involved in nutrient, metabolite or mineral transport, a gene involved in nutrient, metabolite or mineral storage, a calcium-binding protein gene, a lipid-binding protein gene, or any combination thereof.

[0100] The one or more payload genes can be selected from the group comprising a gene encoding an enzyme involved in metabolizing biochemical wastes for use in bioremediation, a gene that encodes an enzyme for modifying pathways that produce secondary plant metabolites, a gene that encodes an enzyme that produces a pharmaceutical, a gene that encodes an enzyme that improves or changes the nutritional content of a plant, a gene that encodes an enzyme involved in vitamin synthesis, a gene that encodes an enzyme involved in carbohydrate, polysaccharide or starch synthesis, a gene that encodes an enzyme involved in mineral accumulation or availability, a gene that encodes a phytase, a gene that encodes an enzyme involved in fatty acid, fat or oil synthesis, a gene that encodes an enzyme involved in synthesis of chemicals or plastics, a gene that encodes an enzyme involved in synthesis of a fuel, a gene that encodes an enzyme involved in synthesis of a fragrance, a gene that encodes an enzyme involved in synthesis of a flavor, a gene that encodes an enzyme involved in synthesis of a pigment or dye, a gene that encodes an enzyme involved in synthesis of a hydrocarbon, agene that encodes an enzyme involved in synthesis of a structural or fibrous compound, a gene that encodes an enzyme involved in synthesis of a food additive, a gene that encodes an enzyme involved in synthesis of a chemical insecticide, a gene that encodes an enzyme involved in synthesis of an insect repellent, a gene controlling carbon flux in a plant, or any combination thereof. The one or more payload proteins can comprise components of a synthetic protein circuit, optionally payload proteins configured to form one or more logic gates selected from the group comprising an OR logic gate, AND logic gate, NOR logic gate, NAND logic gate, IMPLY logic gate, NIMPLY logic gate, XOR logic gate, and an XNOR logic gate. A payload protein can be capable of modulating the expression, concentration, localization, stability, and / or activity of the one or more endogenous proteins of a cell. The payload protein can be a therapeutic protein or a variant thereof, optionally a therapeutic protein configured to prevent or treat a disease or disorder of a subject, further optionally the subject suffers from a deficiency of said therapeutic protein.

[0101] In some embodiments, one or more of the payload gene(s) comprise: a 5’UTR and / or a 3’UTR; a tandem gene expression element selected from the group an internal ribosomal entry site (IRES), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), porcine teschovirus 2A peptide (P2A) or Thosea asigna virus 2A peptide (T2A), or any combination thereof; and / or a transcript stabilization element, optionally the transcript stabilization element comprises woodchuck hepatitis post-translational regulatory element (WPRE), bovine growth hormone polyadenylation (bGH-polyA) signal sequence, human growth hormone polyadenylation (hGH-polyA) signal sequence, or any combination thereof. At least one of the payload genes can be operably connected to a promoter selected from the group comprising: an RNA pol I promoter; a pol II promoter, optionally CMV, SV40 early region or adenovirus major late promoter; or pol III promoter, optionally a U6 or Hl promoter; a minimal promoter, optionally TATA, miniCMV, and / or miniPromo; a bacteriophage promoter, optionally a bacteriophage T3 promoter, a bacteriophage T7 promoter, a bacteriophage SP6 promoter, or a combination thereof; a tissue-specific promoter and / or a lineage-specific promoter; an inducible promoter, optionally a T7 RNA polymerase promoter, a T3 RNA polymerase promoter, an Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, or a Tetracycline-regulated promoter, a tetracycline-dependent promoter, a lac-dependent promoter, a pB ad-dependent promoter, an AlcA-dependent promoter, a LexA-dependent promoter, or a heat-shock promoter; a ubiquitous promoter, optionally a cytomegalovirus (CMV) immediate early promoter, a CMV promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, an RSV promoter, a herpes simplex virus(HSV) (thymidine kinase) promoter, H5, P7.5, and Pll promoters from vaccinia virus, an elongation factor 1-alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), P-kinesin (P-KIN), the human ROSA 26 locus, a Ubiquitin C promoter (UBC), a phosphoglycerate kinase-1 (PGK) promoter, 3 -phosphoglycerate kinase promoter, a cytomegalovirus enhancer, human P-actin (HBA) promoter, chicken P-actin (CBA) promoter, a CAG promoter, a CASI promoter, a CBH promoter; or any combination thereof.

[0102] The final synthetic sequence can be or can comprise all or a portion of a vector. The vector can be or can comprise a viral vector, a plasmid, a transposable element, a naked DNA vector, or any combination thereof. The vector can be or can comprise an AAV vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, a herpesvirus vector, a herpes simplex virus vector, a cytomegalovirus vector, a vaccinia virus vector, a MVA vector, a baculovirus vector, a vesicular stomatitis virus vector, a human papillomavirus vector, an avipox virus vector, a Sindbis virus vector, a VEE vector, a Measles virus vector, an influenza virus vector, a hepatitis B virus vector, an integration-deficient lentivirus (IDLV) vector, or any combination thereof. The transposable element can be piggybac transposon or sleeping beauty transposon.

[0103] The final synthetic sequence can be configured for propagation in a eukaryotic or a prokaryotic cell. The final synthetic sequence can comprise: a bacterial origin of replication, optionally ColEl, pl5A, pSClOl, and RK2. The final synthetic sequence can comprise: an origin of transfer (oriT) and one or more mobilization genes configured to enable conjugative transfer. The final synthetic sequence can comprise: an autonomously replicating sequence (ARS), a centromeric sequence (CEN), and / or 2p elements. The final synthetic sequence can comprise: a rolling-circle replication origin, optionally derived from pC194, pE194, and pUBHO. The final synthetic sequence can comprise: a mammalian origin of replication, optionally oriP / EBNAl and / or SV40 ori. The final synthetic sequence can comprise: a selection marker, optionally an antibiotic resistance marker and / or a fluorescence marker. The final synthetic sequence can comprise: a counter- sei ection marker, optionally sacB, rpsL, galK, CYH2, and / or URA3.

[0104] The final synthetic sequence can be configured for insertion into a genome. The final synthetic sequence can comprise: recognition sites for an RNA-guided DNA binding complex, wherein the RNA-guided DNA binding complex comprises one or more Cas proteins, a transposase, one or more crRNAs, or any combination thereof. The final synthetic sequencecan comprise: recognition sites for a transposition complex comprising one or more transposases. The final synthetic sequence can comprise: homology arms, optionally targeting a safe-harbor locus selected from AAVS1, ROSA26, CCR5, and Hll. The final synthetic sequence can comprise: one or more recombination sites, optionally loxP, FRT, attB, attP, attL, and attR. The final synthetic sequence can comprise: a reporter cassette. The final synthetic sequence can comprise a digital data storage payload encoded in nucleic acid sequence.TADA Compositions

[0105] Each of the n fragments can be housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. Each of the n masked fragments can be housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. The n pairs or n-1 pairs of masking oligonucleotides can be housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. The first polynucleotide strand and the second polynucleotide strand that constitute each of the n fragments can be housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. In some embodiments, the composition does not comprise one or more reagents employed with Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly, optionally an exonuclease, an endonuclease, a single stranded DNA binding protein, a restriction endonuclease, a recombinase, or any combination thereof.

[0106] The composition can comprise: a non-thermostable ligase, a thermostable ligase, a chemical coupling agent, a polymerase, a primer capable of binding the first terminal region (or a complement thereof), a primer capable of binding the second terminal region (or a complement thereof), or any combination thereof. The composition can comprise: a ligation buffer. The ligation buffer can comprise: a reaction buffer configured to support annealing, ligation, and / or amplification (e.g., HiFi Taq buffer); one or more of Tris HC1 at about 10 mM to about 200 mM, at a pH of about 7.0 to about 9.5 at the incubation temperature, Mg2+ at about 0.5 mM to about 20 mM, monovalent cation(s) at about 10 mM to about 300 mM, and a reducing agent at about 0.1 mM to about 20 mM; a ligase cofactor, optionally selected from ATP at about 0.05 mM to about 5 mM or NAD+ at about 0.01 mM to about 2 mM; a buffering species selected from Tris, HEPES, Bis Tris, MOPS, and PIPES, optionally configured to maintain pH between 8.3-8.8 at 25°C; and / or one or more additives, optionally selected from bovine serum albumin at about 0.01 mg / mL to about 1 mg / mL, polyethylene glycol at about 1% to about 20% (w / v), betaine at about 0.1 M to about 2.0 M, dimethyl sulfoxide at about 1% to about 20% (v / v), formamide at about 0.5% to about 10% (v / v), glycerol at about 1% to about 20% (v / v), and / or a non-ionic detergent at about 0.001% to about 0.1% (v / v).

[0107] Provided herein, in some embodiments, are compositions. The compositioncan comprise: assembled products, or products thereof, generated by a method disclosed herein. The composition can comprise a plurality of cells comprising the assembled products, or products thereof. The composition can comprise a cell comprising the final synthetic sequence or a portion thereof. The composition can comprise a nucleic acid comprising the final synthetic sequence or a portion thereof generated by a method disclosed herein.TAD A Reaction Mixtures

[0108] Provided herein, in some embodiments, are compositions. The composition can comprise: a pre-assembly reaction mixture comprising the n masked fragments disclosed herein at equimolar concentrations. In some embodiments, the pre-assembly reaction mixture comprises a ligase or a chemical coupling agent.

[0109] Provided herein, in some embodiments, are compositions. The composition can comprise: a first intermediate reaction mixture comprising the n masked fragments disclosed herein associated together via four-way junction (4WJ) intermediates. In some embodiments, the first intermediate reaction mixture comprises a ligase or a chemical coupling agent.

[0110] Provided herein, in some embodiments, are compositions. The composition can comprise: a second intermediate reaction mixture comprising the n fragments disclosed herein joined together via hybridization of the first complementary region and the second complementary region to generate an intermediate product. The intermediate product can comprise nicks on the first polynucleotide strand and the second polynucleotide strand. In some embodiments, the four-way junctions (4WJs) are resolved and the intermediate product is not hybridized to an upstream masking oligonucleotide or a downstream masking oligonucleotide. The second intermediate reaction mixture can comprise paired masking oligonucleotides consisting of an upstream masking oligonucleotide hybridized to a downstream masking oligonucleotide. The second intermediate reaction mixture can comprise a ligase or a chemical coupling agent.[OHl] Provided herein, in some embodiments, are compositions. The composition can comprise: a post-ligation reaction mixture comprising an assembled product wherein the first polynucleotide strand and the second polynucleotide strand do not comprise nicks.TADA Methods

[0112] Provided herein, in some embodiments, are methods. The method can comprise: providing the n masked fragments disclosed herein. The method can comprise: incubating the n masked fragments in a reaction mixture under reaction conditions such that: the first fragment toehold of the (z)th fragment hybridizes to the second fragment toehold of the (z+l)th fragment; and the masking region of the (z)th upstream masking oligonucleotide hybridizes to the masking region of the (z)th downstream masking oligonucleotide, therebyjoining together the n fragments via four- way junction (4WJ) intermediates to generate an intermediate product. The method can comprise: ligating nicks on the first polynucleotide strands and the second polynucleotide strands to generate an assembled product.

[0113] In some embodiments, assembly is initiated when two adjacent masked fragments come into proximity and the masking toehold of an upstream masking oligonucleotide of one masked fragment hybridizes to the complementary masking toehold of a downstream masking oligonucleotide of an adjacent masked fragment. This initial masking-toehold interaction can nucleate association between the adjacent masked fragments and can align the two masked fragments for subsequent strand exchange. Following this initial interaction, the first fragment toehold of one fragment can hybridize to the complementary second fragment toehold of the adjacent fragment. The combined interaction of the masking toeholds and the fragment toeholds can promote formation of a four-way junction intermediate between the adjacent masked fragments. In some embodiments, once the four- way junction has formed, strand exchange can proceed such that the first complementary region of one fragment becomes hybridized to the second complementary region of the adjacent fragment. Where the complementary regions are correctly matched, the four-way junction can resolve to yield adjacent fragments joined through hybridization of their complementary regions, while the upstream masking oligonucleotide and the downstream masking oligonucleotide can be released from the fragments as a paired masking oligonucleotide complex. In some embodiments, this process occurs successively for multiple adjacent fragment pairs in the same reaction mixture, thereby generating an intermediate product in which the fragments are arranged in the intended order and are separated by nicks at the junctions between adjacent fragments. A ligase and / or chemical coupling agent can then be used to form covalent linkages across the junctions and thereby generate an assembled product.

[0114] In some embodiments, association between: (i) the first fragment toehold and second fragment toehold of adjacent fragments; and (ii) the masking regions of an upstream masking oligonucleotide and a downstream masking oligonucleotide, forms a 4WJ. In some embodiments, mismatches between the first complementary region and the second complementary region of incorrectly joined fragments prevent progression of the 4-way strand exchange. In some embodiments, upon resolution of a 4WJ, adjacent fragments are hybridized via the first complementary region and the second complementary region, and the adjacent fragments are separated by a nick in each of the first polynucleotide strand and the second polynucleotide strand. In some embodiments, the resolution of a 4WJ yields a paired masking oligonucleotide consisting of an upstream masking oligonucleotide hybridized to a downstream masking oligonucleotide. In some embodiments, the masking oligonucleotides blockhybridization between the first complementary region and the second complementary region when the first complementary region and the second complementary region lack perfect complementarity. The isothermal strand displacement reaction can be driven forward by perfect complementarity between the first complementary region and the second complementary region. In some embodiments the method comprises filing a gap with a DNA polymerase lacking at least one of 5’ to 3’ exonuclease activity and 3’ to 5’ exonuclease activity.

[0115] The incubation step can be performed under isothermal conditions. The incubation can comprise incubation at a first incubation temperature for a first period of time. The first incubation temperature can be about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21 °C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, or a number or a range between any two of these values. The first period of time can be about 10 sec, 20 sec, 30 sec, 40 sec, 50 sec, 60 sec, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, or a number or a range between any two of these values.

[0116] In some embodiments, the ligating step comprises: addition of a ligase to the reaction mixture; and incubation at the second incubation temperature for a second period of time. The second incubation temperature can be about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, or a number or a range between any two of these values. The second period of time can be about 10 sec, 20 sec, 30 sec, 40 sec, 50 sec, 60 sec, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, or a number or a range between any two of these values.

[0117] In some embodiments, the assembly of the n masked fragments occurs independently of the sequence of the internal segments. The assembly of the n masked fragments can be directed by the formation of 4-way junctions (4WJs) between adjacent masked fragments. The assembled product or a product thereof can comprise a final synthetic sequence. The final synthetic sequence can comprise the scarless assembly of the n fragments. The final synthetic sequence can be a linear polynucleotide, optionally comprising the structure 5’-[first fragment] -[second fragmen t]-...-[( / 7)th fragment]-3’. The final synthetic sequence can be a circular polynucleotide wherein the 3’ end of the [( / / )th fragment] is linked to the 5’ end of [first fragment] by a phosphodiester bond.

[0118] The final synthetic sequence can be at least about 500 bases, 750 bases, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50 kb, 75 kb, 100 kb,250 kb, 500 kb, 750 kb, 1MB, or a number or a range between any two of these values, in length. The ligating step can be performed with a ligase, optionally a thermostable ligase, optionally said ligase is selected from the group comprising T3 ligase, T4 ligase, T7 ligase, SplintR, E. coli DNA ligase, Hi-T4 ligase, a ligase suitable for ligating adjacent nucleic acid sequences under hybridization conditions (e.g., HiFi Taq ligase), Taq ligase, 9°N, or any combination thereof. The ligating step can comprise contacting the intermediate product with a chemical coupling agent effective to form a covalent linkage between adjacent first polynucleotide strands and between adjacent second polynucleotide strands, optionally one or more click chemistry reagents, optionally CuAAC, SPAAC, iEDDA, oxime formation, hydrazone formation, Michael addition, disulfide formation, carbodiimide mediated coupling, native chemical ligation, or any combination thereof. The incubating step can comprise combining the n masked fragments in a single reaction mix at equimolar concentrations, optionally at about 0.1 nM, 0.5 nM, 0.75 nM, 0.9 nM, 1.0 nM, 1.1 nM, 1.25 nM, 1.5 nM, 1.75 nM, 2 nM, 5 nM, or 10 nM, or a number or a range between any two of these values.

[0119] In some embodiments, the providing step comprises: generating the n masked fragments. Said generating step can comprise annealing the n fragments with the n pairs or n-1 pairs of masking oligonucleotides to generate the n masked fragments. The n masked fragments can be each generated in separate reactions. Said annealing step can comprise an initial denaturation step followed by a gradual decrease in temperature. In some embodiments, the n masked fragments undergo one or more purification steps, optionally: gel electrophoresis, including pulsed-field gel electrophoresis (PFGE); solid or solution phase hybridization / capture; precipitation; dialysis; solid phase reversible immobilization (SPRI) cleanup, optionally performing size selection using SPRI beads, further optionally single-sided or double-sided; and / or column purification.

[0120] In some embodiments, the providing step comprises: generating the n fragments. Said generating step can comprise annealing the first polynucleotide strand and the second polynucleotide strand components of each of the n fragments to generate heteroduplexes. The n fragments can be each generated in separate reactions. The generating step can comprise phosphorylation of the first and second polynucleotide strands, further optionally via T4 polynucleotide kinase. Said annealing step can comprise an initial denaturation step followed by a gradual decrease in temperature. In some embodiments, the heteroduplexes undergo one or more purification steps, optionally: gel electrophoresis, including pulsed-field gel electrophoresis (PFGE); solid or solution phase hybridization / capture; precipitation; dialysis; solid phase reversible immobilization (SPRI) cleanup, optionally performing size selection using SPRI beads, further optionally single-sided or double-sided; and / or column purification.

[0121] In some embodiments, the method further comprises PCR amplification of the assembled product, or a product thereof, to generate an amplified product. PCR amplification can comprise amplifying the assembled product, or a product thereof, using a primer capable of hybridizing to the first terminal region or a complement thereof, and a primer capable of hybridizing the second terminal region or a complement thereof. The method can comprise purification of the assembled product, the amplified product, or products thereof. In some embodiments, said purification step compromises: removal of the paired masking oligonucleotides; gel electrophoresis of the assembled product, the amplified product, or products thereof; solid phase reversible immobilization (SPRI) cleanup, optionally performing size selection using SPRI beads, further optionally single-sided or double-sided; and / or column purification. The method can comprise replication of the assembled product, the amplified product, or products thereof, in a cell.

[0122] The providing step can comprise providing y sets of n masked fragments. The incubating step can comprise incubating the y sets of n masked fragments in a single reaction mixture, wherein the n masked fragments of each set can be joined together in parallel via fourway junction (4WJ) intermediates to generate y intermediate products. The ligating step can comprise ligating nicks on the first polynucleotide strands and the second polynucleotide strands of each of the y intermediate products to generate y assembled products. The value of n can be the same between at least two of the y sets. The value of n can be the different between at least two of the sets. In some embodiments, the masking region of each set is not complementary to the masking region of any other set. The y intermediate products can be candidate design variants. The method can comprise the y intermediate products, or products thereof, being individually amplified or universally amplified. In some embodiments, y is an integer greater than 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or a number or a range between any two of these values.

[0123] At least one of the n fragments can be a variant fragment, and wherein the assembled products can comprise a combinatorial library of at least p variants, wherein p is an integer greater than 1. In some embodiments, wherein p is at least about 10, 50, 100, 250, 500, 750, 1000, 10000, 50000, 100000, 250000, 500000, 750000, 1000000, 5000000, 10000000, or a number or a range between any two of these values. In some embodiments, wherein the combinatorial library achieves a variant coverage of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.99%, or a number or a range between any two of these values, of the theoretical variant library. Every codon mutation profile can be represented in the library with an average absolute deviation of less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, or a number or arange between any two of these values, from the theoretical proportion of occurrence for that codon.

[0124] At least 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or a number or a range between any two of these values, of the assembled products, or products thereof, can comprise all of the intended fragments in the intended order. Less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, or a number or a range between any two of these values, of the assembled products, or products thereof, can be a partial assembly missing one or more fragments. Less than 1 in 1000, 1 in 10000, 1 in 100000, 1 in 1000000, 1 in 10000000, 1 in 100000000, or a number or a range between any two of these values, of the assembled products can be missing one or more fragments or comprise a mis-assembled junction. The mis-ligation rate at the 4WJ can be less than 1 in 1000, 1 in 10000, 1 in 100000, 1 in 1000000, 1 in 10000000, 1 in 100000000, or a number or a range between any two of these values. In some embodiments, n is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 10-25, 10-50, 10-75, 10-100, 10-500, 10-1000, 25-50, 25-75, 25-100, 25-500, 25-1000, 50-75, 50- 100, 50-500, 50-1000, 75-100, 75-500, 75-1000, 100-500, 100-1000, 500-1000, or a number or a range between any two of these values. The yield of correctly assembled products can be at least 1-fold, 2-fold, 4-fold, 8-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, or 1000-fold, greater than the yield of a polynucleotide assembly method not comprising 4WJ, optionally Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly. In some embodiments, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a number or a range between any two of these values, of the incubated fragments become a component of an assembled product.

[0125] Provided herein, in some embodiments, are methods. The method can comprise: providing a combinatorial library disclosed herein, or a product thereof. The method can comprise: expressing the one or more payload genes in cell(s). The method can comprise: screening for a property of interest. Screening can comprise fluorescence-activated cell sorting (FACS), cell viability assay, ELISA, co-immunoprecipitation, a bead-based immunoassay, or any combination thereof. The property of interest can comprise modified enzymatic activity, improved enzymatic activity, modified binding activity, improved binding activity, modified stability, improved stability, modified localization, improved localization, modified solubility,improved solubility, modified expression, improved expression, modified inhibitor resistance, improved inhibitor resistance, modified substrate specificity, improved substrate specificity, or any combination thereof. The method can comprise exposing the cell(s) to one or more agents. In some embodiments, the one or more agents comprise: one or more of a chemical agent, a pharmaceutical, small molecule, a biologic, a CRISPR single-guide RNA (sgRNA), a small interfering RNA (siRNA), CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a piwi-interacting RNA (piRNA), an antisense oligonucleotide, a peptide or peptidomimetic inhibitor, an aptamer, an antibody, an intrabody, or any combination thereof; an expression vector, wherein the expression vector encodes one or more of the following: an mRNA, an antisense nucleic acid molecule, a RNAi molecule, a shRNA, a mature miRNA, a pre-miRNA, a pri-miRNA, an anti-miRNA, a ribozyme, any combination thereof; an infectious agent, an anti-infectious agent, or a mixture thereof; a cytotoxic agent, optionally a chemotherapeutic agent, a biologic agent, a toxin, a radioactive isotope, or any combination thereof; and / or one or more of an epigenetic modifying agent, epigenetic enzyme, a bicyclic peptide, a transcription factor, a DNA or protein modification enzyme, a DNA-intercalating agent, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, a competitive inhibitor for an enzyme, a protein synthesis inhibitor, a nuclease, a protein fragment or domain, a tag or marker, an antigen, an antibody or antibody fragment, a ligand or a receptor, a synthetic or analog peptide from a naturally-bioactive peptide, an anti-microbial peptide, a pore-forming peptide, a targeting or cytotoxic peptide, a degradation or selfdestruction peptide, a CRISPR component system or component thereof, DNA, RNA, artificial nucleic acids, a nanoparticle, an oligonucleotide aptamer, a peptide aptamer, or any combination thereof. The property of interest can comprise a property of the cell, optionally improved drug resistance, altered drug sensitivity, improved or modified growth rate under selective pressure, modified or improved cell viability or survival, modified or improved stress tolerance, modified or improved secretion of a compound, altered signaling pathway activation, or any combination thereof.

[0126] The method can comprise cloning the assembled products, or products thereof, into expression vector(s), optionally prior to an expressing step. The expression vector can be selected from a plasmid, a viral vector, a transposable element, a bacterial artificial chromosome, a yeast artificial chromosome, or any combination thereof. In some embodiments, the cloning step operably connects the final synthetic sequence with one or more regulatory elements selected from a promoter, an enhancer, a polyadenylation signal, a 5’UTR, a 3’ UTR, and a selection marker. The method can comprise transforming or transfecting host cells with the cloned expression vector, optionally bacterial cells for propagation and / or sequenceverification and subsequently eukaryotic cells for expression, optionally mammalian, yeast, insect, plant, or fungal cells.Systems and Kits

[0127] Provided herein, in some embodiments, are kits. Provided herein, in some embodiments, are systems for synthesizing nucleic acids. The system or kit can comprise: the n masked fragments disclosed herein. The system or kit can comprise: y sets of n masked fragments. Each of the n masked fragments can be housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. The system or kit can comprise: the n fragments disclosed herein and the n pairs or n-1 pairs of masking oligonucleotides disclosed herein. The system or kit can comprise: y sets of n fragments and y sets of n pairs or n-1 pairs of masking oligonucleotides. Each of the n fragments can be housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. Each of the n pairs or n-1 pairs of masking oligonucleotides can be housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber. The first polynucleotide strand and the second polynucleotide strand that constitute each of the n fragments can be housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber.

[0128] The system or kit can comprise: a non-thermostable ligase, a thermostable ligase, a chemical coupling agent, a polymerase, a primer capable of binding the first terminal region (or a complement thereof), a primer capable of binding the second terminal region (or a complement thereof), or any combination thereof. The system or kit can comprise: a ligation buffer. The ligation buffer can comprise: a reaction buffer configured to support annealing, ligation, and / or amplification (e.g., HiFi Taq buffer); one or more of Tris HC1 at about 10 mM to about 200 mM, at a pH of about 7.0 to about 9.5 at the incubation temperature, Mg2+ at about 0.5 mM to about 20 mM, monovalent cation(s) at about 10 mM to about 300 mM, and a reducing agent at about 0.1 mM to about 20 mM; a ligase cofactor, optionally selected from ATP at about 0.05 mM to about 5 mM or NAD+ at about 0.01 mM to about 2 mM; a buffering species selected from Tris, HEPES, Bis Tris, MOPS, and PIPES, optionally configured to maintain pH between 8.3-8.8 at 25°C; one or more additives, optionally selected from bovine serum albumin at about 0.01 mg / mL to about 1 mg / mL, polyethylene glycol at about 1% to about 20% (w / v), betaine at about 0.1 M to about 2.0 M, dimethyl sulfoxide at about 1% to about 20% (v / v), formamide at about 0.5% to about 10% (v / v), glycerol at about 1% to about 20% (v / v), and / or a non-ionic detergent at about 0.001% to about 0.1% (v / v).

[0129] The system or kit can comprise: one or more purification reagent(s), optionally: gel electrophoresis reagent(s), optionally pulsed-field gel electrophoresis (PFGE); solid or solution phase hybridization / capture reagent(s); precipitation reagent(s); dialysisreagent(s); solid phase reversible immobilization (SPRI) cleanup reagent(s), optionally performing size selection using SPRI beads, further optionally single-sided or double-sided; and / or column purification reagent(s). In some embodiments, the system or kit does not comprise one or more reagents employed with Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly, optionally an exonuclease, an endonuclease, a single stranded DNA binding protein, a restriction endonuclease, a recombinase, or any combination thereof.Sidewinder

[0130] The systems, methods, compositions, and kits provided herein can, in some embodiments, be employed in concert with the systems, methods, compositions, and kits for Sidewinder-based polynucleotide assembly described in U.S. Patent Application No.19 / 441,759, entitled, “SIDEWINDER THREE-WAY IUNCTION DNA ASSEMBLY,” filed lanuary 6, 2026, the content of which is incorporated herein by reference in its entirety. Sidewinder-based methods, compositions, systems, and kits can employ a strategy which implements highly specific external barcodes that are not incorporated into the final assembled product. In some embodiments, a highly specific DNA barcode pair forms an external third helix to hold synthetic fragments together at a temperature prohibiting interactions of short complementary toehold sequences alone before enzymatically ligating nicks in the lower strand to covalently fix the connection between fragments. The method can comprise removal of the external third helix either enzymatically, or by PCR amplification of the lower strand without the external third helix, to form a seamless connection. In some embodiments the fragments and / or masked fragments are derived from Sidewinder assembly methods. In some embodiments the assembled products, or products thereof, generated by methods disclosed herein can be used as inputs for further assembly using Sidewinder assembly methods.

[0131] The Sidewinder composition can comprise: n fragments, wherein n is an integer greater than 2. Each fragment can comprise a first polynucleotide strand and a second polynucleotide strand. Each (i)th fragment can comprise a first barcode, a first toehold, a second barcode, and a second toehold, wherein 1 < z < n. The first fragment can comprise a first terminal region, a second barcode, and a first toehold, optionally the first terminal region is a 5’ first terminal region. The (zz)th fragment can comprise a first barcode, a second toehold, and a second terminal region, optionally the second terminal region is a 3’ second terminal region. In some embodiments, for each (z)th fragment, wherein 1 < z < n the first polynucleotide strand comprises a 5’ overhang and a 3’ overhang; the 5’ overhang of the first polynucleotide strand comprises the first barcode; the 3’ overhang of the first polynucleotide strand comprises the second barcode; the first barcode of the (z)th fragment is complementary to the second barcodeof the (z-l)th fragment; the first toehold of the (z)th fragment is complementary to the second toehold of the (z+l)th fragment; the second barcode of the (z)th fragment is complementary to the first barcode of the (z+l)th fragment; and the second toehold of the (z)th fragment is complementary to the first toehold of the (z- 1 )th fragment. The methods, compositions, systems, and kits provided herein can comprise the generation of a linear product.

[0132] The Sidewinder composition can comprise: n fragments, wherein n is an integer greater than 2. Each fragment can comprise a first barcode, a first toehold, a second barcode, and a second toehold. Each fragment can comprise a first polynucleotide strand and a second polynucleotide strand. The first polynucleotide strand can comprise a 5’ overhang and a 3’ overhang. The 5’ overhang of the first polynucleotide strand can comprise the first barcode. The 3’ overhang of the first polynucleotide strand can comprise the second barcode. In some embodiments, for each (z)th fragment, wherein 1 < z < n the first barcode of the (z)th fragment is complementary to the second barcode of the (z-l)th fragment; the first toehold of the (z)th fragment is complementary to the second toehold of the (z+ l)th fragment; the second barcode of the (z)th fragment is complementary to the first barcode of the (z+l)th fragment; and the second toehold of the (z)th fragment is complementary to the first toehold of the (z-l)th fragment. The first barcode of the first fragment can be complementary to the second barcode of the (zz)th fragment. The second toehold of the first fragment can be complementary to the first toehold of the (zz)th fragment. The methods, compositions, systems, and kits provided herein can comprise the generation of a circular product.

[0133] In some embodiments of Sidewinder, for each (z)th fragment, wherein 1 < z < n the first barcode of the (z)th fragment is not complementary to the first barcode of any of the n fragments; and the first barcode of the (z)th fragment is not complementary to the second barcode of any (A)th fragment, wherein k is an integer not equal to (z-1). In some embodiments, the 3’ overhang of the first polynucleotide strand comprises the first toehold, the first toehold is 5’ of the second barcode, the second polynucleotide strand comprises a 3’ overhang, and the 3’ overhang of the second polynucleotide strand comprises the second toehold. In some embodiments, the 5’ overhang of the first polynucleotide strand comprises the second toehold, the second toehold is 3’ of the first barcode, the second polynucleotide strand comprises a 5’ overhang, and the 5’ overhang of the second polynucleotide strand comprises the first toehold.

[0134] In some embodiments of Sidewinder, upon incubation in a reaction mixture, the n fragments can be capable of joining together via at least one three-way junction (3WJ) intermediate to generate an intermediate product. A ligase can be capable of ligating nicks on the second polynucleotide strands of said intermediate product to generate an assembled product. A ligase and / or a chemical coupling agent can be capable of forming a covalent linkagebetween adjacent second polynucleotide strands of said intermediate product to generate an assembled product. In some embodiments, hybridization of a first barcode and a second barcode of adjacent fragments forms a helix, wherein said 3WJ intermediates each comprise a helix. In some embodiments, (i) the hybridization of the first toehold and the second toehold of adjacent fragments further stabilizes the 3WJ intermediates; and / or (ii) one or more fragments do not comprise a toehold and the intermediate product is sufficiently stabilized by hybridization between first and second barcodes. In some embodiments, the formation of the helix holds adjacent fragments together at a temperature prohibiting interactions of the first toehold and second toehold of adjacent fragments alone. In some embodiments, the helix orthogonally winds up on the side of the final assembled sequence, thereby joining adjacent fragments together via the 3WJ intermediate. The association of the first toehold and second toehold of adjacent fragments can be unstable at the temperature(s) of the incubation step in the absence of the formation of the helix. The Sidewinder method can comprise: providing n fragments, wherein n is an integer greater than 2. Each fragment can comprise a first polynucleotide strand and a second polynucleotide strand. Each (z)th fragment can comprise a first barcode and a second barcode on the first polynucleotide strand, wherein 1 < z < n. In some embodiments, the first barcode of the (z’)th fragment forms a pair with the second barcode of the (z-l)th fragment. In some embodiments, the second barcode of the (z’)th fragment forms a pair with the first barcode of the (z+l)th fragment. The method can comprise: incubating the n fragments in a reaction mixture under reaction conditions such that: the first barcode of the (i)th fragment hybridizes to the second barcode of the (i-l)th fragment; and the second barcode of the (z’)th fragment hybridizes to the first barcode of the (z+l)th fragment, thereby joining together the n fragments via three-way junction (3WJ) intermediates to generate an intermediate product. The method can comprise: ligating nicks on the second polynucleotide strands to generate an assembled product. In some embodiments, hybridization of a first barcode and a second barcode of adjacent fragments forms a helix, wherein said 3WJ intermediates each comprise a helix. In some embodiments, (i) the hybridization of the first toehold and the second toehold of adjacent fragments further stabilizes the 3WJ intermediates; and / or (ii) one or more fragments do not comprise a toehold and the intermediate product is sufficiently stabilized by hybridization between first and second barcodes. In some embodiments of the methods, compositions, systems, and kits provided herein, some or all of the fragments do not comprise a first toehold and / or a second toehold, and the hybridization of first and second barcodes to form 3WJs are sufficient to generate an intermediate product suitable for ligation to generate an assembled product.. In some embodiments, the formation of the helix holds adjacent fragments together at a temperature prohibiting interactions of the first toehold and second toehold of adjacentfragments alone. In some embodiments, the helix orthogonally winds up on the side of the final assembled sequence, thereby joining adjacent fragments together via the 3WJ intermediate. The association of the first toehold and second toehold of adjacent fragments can be unstable at the temperature(s) of the incubation step in the absence of the formation of the helix.Super USER and HighT Assembly

[0135] The systems, methods, compositions, and kits provided herein can, in some embodiments, be employed in concert with the systems, methods, compositions, and kits for Super USER and High Temperature (HighT) assembly described in U.S. Provisional Patent Application Ser. No. 63 / 777,887, entitled, “Super USER Cloning as a Novel Method of DNA Assembly,” filed March 26, 2025, and in a PCT Application entitled, “Super USER Cloning as a Novel Method of DNA Assembly,” filed March 25, 2026, the contents of which are incorporated herein by reference in their entireties. In some embodiments the fragments or masked fragments are derived from Super USER and / or HighT assembly methods. In some embodiments the assembled products, or products thereof, generated by methods disclosed herein can be used as inputs for further assembly using Super USER and / or HighT assembly methods.

[0136] The Super USER method can comprise: providing m assembly precursors, wherein m is a positive integer. The assembly precursors can be double-stranded nucleic acid molecules (e.g., double-stranded DNA molecules) comprising one or two 5’ overhang-forming regions. Each 5’ overhang-forming region can comprise two or more 5’ terminal cleavage regions. Each of the 5’ terminal cleavage regions can comprise one or more non-canonical nucleotide(s). The method can comprise: contacting the m assembly precursors with cleavage agent(s) configured to remove the non-canonical nucleotide(s) to generate m assembly fragments. Each of the m assembly fragments can comprise one or two 3’ overhang(s) exposed by removal of the corresponding 5' terminal cleavage region(s) of the 5’ overhang-forming region. In some embodiments, each 3’ overhang begins at a position corresponding to the 3’ most non-canonical nucleotide of the corresponding 5’ terminal cleavage region.

[0137] The HighT assembly method can comprise: providing n assembly fragments, wherein n is an integer greater than 1. The n assembly fragments can be double-stranded DNA molecules comprising one or two 3’ overhang(s) configured to hybridize to a complementary 3’ overhang of another of the n assembly fragments. The method can comprise: incubating the n assembly fragments in the presence of a ligase at a temperature greater than the melting temperature of the 3’ overhang(s), thereby generating an assembled product.

[0138] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the artthat various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0139] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0140] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g, “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to“at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms.

[0141] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0142] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0143] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A composition, comprising:(a) n fragments,wherein the first fragment comprises a first fragment toehold and a first complementary region,wherein each (z)th fragment comprises a first fragment toehold, a first complementary region, a second complementary region, and a second fragment toehold, wherein 1 < z < «,wherein each (z)th fragment comprises a first polynucleotide strand and a second polynucleotide strand,wherein the first polynucleotide strand comprises a 3’ overhang, wherein the second polynucleotide strand comprises a 3’ overhang,wherein the 3’ overhang of the first polynucleotide strand comprises the first fragment toehold and the first complementary region, andwherein the 3’ overhang of the second polynucleotide strand comprises the second fragment toehold and the second complementary region,wherein for each (z)th fragment:the first complementary region of the (z)th fragment is complementary to the second complementary region of the (z+l)th fragment;the first fragment toehold of the (z)th fragment is complementary to the second fragment toehold of the (z+ 1 )th fragment;the second complementary region of the (z)th fragment is complementary to the first complementary region of the (z-l)th fragment; andthe second fragment toehold of the (z)th fragment is complementary to the first fragment toehold of the (z-l)th fragment, and wherein the (zz)th fragment comprises a second fragment toehold and a second complementary region; and(b) n-1 pairs of masking oligonucleotides, wherein n is an integer greater than 2, wherein each pair of masking oligonucleotides comprises an upstream masking oligonucleotide and a downstream masking oligonucleotide,wherein the upstream masking oligonucleotide comprises a masking region and a masking toehold, andwherein the downstream masking oligonucleotide comprises a masking region and a masking toehold,wherein for each ( ) th pair of masking oligonucleotides, wherein q is a positive integer less than nthe masking region of the (< / )th upstream masking oligonucleotide is complementary to the first complementary region of the (< / )th fragment;the masking region of the (k / Jth downstream masking oligonucleotide is complementary to the second complementary region of the ( +l)th fragment; andthe masking toehold of the (t / ) th upstream masking oligonucleotide is complementary to the masking toehold of the (^)th downstream masking oligonucleotide.

2. The composition of claim 1, wherein the composition comprises n masked fragments,wherein the first masked fragment comprises the first fragment hybridized to an upstream masking oligonucleotide via the first complementary region of the first fragment,wherein the ( / / )th masked fragment comprises the (zz)th fragment hybridized to a downstream masking oligonucleotide via the second complementary region of the (with fragment, andwherein the (z)th masked fragment comprises a (z)th fragment hybridized to:(i) an upstream masking oligonucleotide via the first complementary region of the (z)th fragment; and(ii) a downstream masking oligonucleotide via the second complementary region of the (z)th fragment.

3. The composition of any one of claims 1-2, wherein:the first fragment and / or the (zz)th fragment is single-stranded;the first fragment and / or the (zz)th fragment comprises a first polynucleotide strand and a second polynucleotide strand;the first fragment comprises a first terminal region, optionally a 5’ terminal region; and / orthe (z )th fragment comprises a second terminal region, optionally a 3’ terminal region.A composition, comprising:(a) n fragments,wherein each fragment comprises a first fragment toehold, a first complementary region, a second complementary region, and a second fragment toehold,wherein each fragment comprises a first polynucleotide strand and a second polynucleotide strand,wherein the first polynucleotide strand comprises a 3’ overhang, wherein the second polynucleotide strand comprises a 3’ overhang,wherein the 3’ overhang of the first polynucleotide strand comprises the first fragment toehold and the first complementary region, andwherein the 3’ overhang of the second polynucleotide strand comprises the second fragment toehold and the second complementary region,wherein for each (z)th fragment, wherein 1 <i <nthe first complementary region of the (z)th fragment is complementary to the second complementary region of the (z+l)th fragment;the first fragment toehold of the (z)th fragment is complementary to the second fragment toehold of the (z+ 1 )th fragment;the second complementary region of the (z)th fragment is complementary to the first complementary region of the (z-l)th fragment; andthe second fragment toehold of the (z)th fragment is complementary to the first fragment toehold of the (z-l)th fragment; wherein the second complementary region of the first fragment is complementary to the first complementary region of the (zz)th fragment, wherein the second fragment toehold of the first fragment is complementary to the first fragment toehold of the (zz)th fragment; and(b) n pairs of masking oligonucleotides, wherein n is an integer greater than 2, wherein each pair of masking oligonucleotides comprises an upstream masking oligonucleotide and a downstream masking oligonucleotide, wherein the upstream masking oligonucleotide comprises a maskingregion and a masking toehold, andwherein the downstream masking oligonucleotide comprises a masking region and a masking toehold;wherein for each ( ) th pair of masking oligonucleotides, wherein q is a positive integer less than nthe masking region of the (< / )th upstream masking oligonucleotide is complementary to the first complementary region of the (< / )th fragment;the masking region of the (k / Jth downstream masking oligonucleotide is complementary to the second complementary region of the ( +l)th fragment; andthe masking toehold of the (t / ) th upstream masking oligonucleotide is complementary to the masking toehold of the (^)th downstream masking oligonucleotide,wherein the masking region of the ( / r)th upstream masking oligonucleotide is complementary to the first complementary region of the ( / / )th fragment,wherein the masking region of the («)th downstream masking oligonucleotide is complementary to the second complementary region of the first fragment, andwherein the masking toehold of the («)th upstream masking oligonucleotide is complementary to the masking toehold of the («)th downstream masking oligonucleotide.

5. The composition of claim 4, wherein the composition comprises n masked fragments, wherein a masked fragment comprises a fragment of the n fragments hybridized to:(i) an upstream masking oligonucleotide via the first complementary region of the fragment; and(ii) a downstream masking oligonucleotide via the second complementary region of the fragment.

6. A composition, comprising:(a) a first fragment and a second fragment,wherein each of the first fragment and the second fragment comprises a first polynucleotide strand and a second polynucleotide strand,wherein the first fragment comprises a first fragment toehold and a first complementary region,wherein the first polynucleotide strand of the first fragment comprises a 3’overhang, and wherein said 3’ overhang of the first polynucleotide strand comprises the first fragment toehold and the first complementary region, wherein the second fragment comprises a second complementary region and a second fragment toehold,wherein the second polynucleotide strand of the second fragment comprises a 3’ overhang, and wherein said 3’ overhang of the second polynucleotide strand comprises the second fragment toehold and the second complementary region,wherein the first complementary region of the first fragment is complementary to the second complementary region of the second fragment, and wherein the first fragment toehold of the first fragment is complementary to the second fragment toehold of the second fragment; and(b) an upstream masking oligonucleotide and a downstream masking oligonucleotide,wherein the upstream masking oligonucleotide comprises a masking region and a masking toehold,wherein the downstream masking oligonucleotide comprises a masking region and a masking toehold,wherein the masking region of the upstream masking oligonucleotide is complementary to the first complementary region of the first fragment, wherein the masking region of the downstream masking oligonucleotide is complementary to the second complementary region of the second fragment, andwherein the masking toehold of the upstream masking oligonucleotide is complementary to the masking toehold of the downstream masking oligonucleotide.

7. The composition of claim 6, wherein the composition comprises a first masked fragment and a second masked fragment,wherein the first masked fragment comprises the first fragment hybridized to the upstream masking oligonucleotide via the first complementary region of the first fragment; andwherein the second masked fragment comprises the second fragment hybridized to the downstream masking oligonucleotide via the second complementary region of the second fragment.

8. The composition of any one of claims 1-7, wherein:the upstream masking oligonucleotide and the downstream masking oligonucleotide are single-stranded;the masking region of the upstream masking oligonucleotide is 5’ of the masking toehold; andthe masking region of the downstream masking oligonucleotide is 3’ of the masking toehold.

9. The composition of any one of claims 1-8, wherein less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, or 0.01%, of fragments in the composition are unmasked fragments, wherein an unmasked fragment comprises:(i) a first complementary region not hybridized to an upstream masking oligonucleotide; and / or(ii) a second complementary region not hybridized to a downstream masking oligonucleotide.

10. The composition of any one of claims 1-9,wherein the masking region of each upstream masking oligonucleotide is not complementary to the masking region of any other upstream masking oligonucleotide; wherein the masking region of each downstream masking oligonucleotide is not complementary to the masking region of any other downstream masking oligonucleotide;wherein the masking region of the (ii’)th upstream masking oligonucleotide is complementary to the masking region of the (ii’)th downstream masking oligonucleotide, wherein w is an integer from 1 to / / ; andwherein the masking region of the ( )th upstream masking oligonucleotide is not complementary to the masking region of the (g)th downstream masking oligonucleotide, wherein f Ag, optionally each of f and g is independently an integer from 1 to n or from 1 to H-l,optionally each pair of masking oligonucleotides is optimized for maximum mutual specificity within a pair and absolute exclusivity across different pairs.

11. The composition of any one of claims 1-10, wherein said complementarity is or comprises:at least 80%, 85%, 90%, 95%, 99%, or 100% complementarity;less than five, four, three, two, or one, base pair mismatches;reverse complementarity;canonical Watson-Crick base pairing;wobble base pairing, optionally G-U wobble; and / orDNA nanotechnology interactions, optionally Hoogsteen base pairing,G-quadruplex(es), DNA origami, aptamer-ligand interactions, or any combination thereof.

12. The composition of any one of claims 1-11, wherein one or more of the n fragments comprise an internal segment, and wherein:the internal segment does not comprise any of the first fragment toehold, the second fragment toehold, the first complementary region, and the second complementary region;the internal segment is 5’ of the first fragment toehold and the first complementary region;the internal segment is 3’ of the second fragment toehold and the second complementary region; and / orthe internal segment is double-stranded.

13. The composition of any one of claims 1-12, wherein the first polynucleotide strand and / or second polynucleotide strand of one or more of the n fragments comprises a 5’ phosphate.

14. The composition of any one of claims 1-13, wherein the first fragment, the (z)th fragment, the ( / z)th fragment, one or more of the n fragments, the first fragment toehold, the second fragment toehold, the first complementary region, the second complementary region, the internal segment, one or more upstream masking oligonucleotides, one or more downstream masking oligonucleotides, the masking region, the masking toehold, the first polynucleotide strand, the second polynucleotide strand, and / or the terminal region:is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 1-5, 1-10, 10-100, 10-250, 25-50, 25-100, 25- 250, 50-100, 50-200, 50-250, 75-100, 75-200, 75-250, 100-150, 100-200, 100-250, 150- 200, 150-250, or 200-250, nucleotides in length;comprises a GC content of about 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 20%-50%, 20%-75%, 20%-100%, 30%-60%, 30%-75%, 30%-100%, 40%-60%, 40%-75%, 40%- 100%, 50%-75%, 50%-100%, 60%-75%, 60%-100%, or 75%-100%;comprises a melting temperature (Tm) of about 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 35°C-55°C, 35°C-75°C, 35°C-100°C, 45°C-55°C, 45°C-75°C, 45°C-100°C, 55°C-75°C, 55°C-100°C, 65°C-75°C, 65°C- 100°C, or 75°C-100°C;comprises DNA;comprises RNA; and / orcomprises one or more nucleic acid analogs, optionally selected from the group consisting of RNA, 2’-O-methyl RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino, phosphorodiamidate morpholino oligomer (PMO), HNA, FANA, TNA, ANA, GNA, CeNA, UNA, L-DNA, or any combination thereof.

15. The composition of any one of claims 1-14, wherein the fragments, upstream masking oligonucleotides, downstream masking oligonucleotides, the first polynucleotide strand, and / or the second polynucleotide strand:comprise or are derived from synthetic oligonucleotides; and / orcomprise or are derived from rolling circle amplification products, restriction enzyme digestion products, reverse transcription products, CRISPR-excised products, PCR amplification products, template-independent polymerase products, recombinasegenerated products, phage-derived products, or any combination thereof.

16. The composition of any one of claims 1-15, wherein n is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 10-25, 10-50, 10-75, 10-100, 10-500, 10-1000, 25-50, 25-75, 25-100, 25-500, 25-1000, 50-75, 50-100, 50-500, 50-1000, 75-100, 75-500, 75-1000, 100-500, 100-1000, or 500-1000.

17. The composition of any one of claims 2, 5, or 7-16,wherein, upon incubation in a reaction mixture, the n masked fragments are capable of joining together via at least one four-way junction (4WJ) intermediate to generate an intermediate product.

18. The composition of claim 17, wherein a ligase is capable of ligating nicks on the first polynucleotide strands and the second polynucleotide strands of said intermediate product to generate an assembled product.

19. The composition of claim 17, wherein a ligase and / or a chemical coupling agentis capable of forming a covalent linkage between adjacent first polynucleotide strands and between adjacent second polynucleotide strands of said intermediate product to generate an assembled product.

20. The composition of claim 19,wherein the covalent linkage is formed by a click ligation between complementary reactive handles on adjacent first polynucleotide strands and / or second polynucleotide strands, optionally copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), strain promoted azide-alkyne cycloaddition (SPAAC), or inverse electron demand Diels-Alder (iEDDA) reaction between a trans cyclooctene and a tetrazine oxime formation, hydrazone formation, Michael addition, disulfide formation, carbodiimide-mediated coupling, native chemical ligation, or any combination thereof;wherein the first polynucleotide strands and / or second polynucleotide strands comprise synthetic modifications and / or modified synthetic nucleotides, optionally selected a 5’ alkyne, a 3’ azide, a trans-cyclooctene, a tetrazine, a 5’ amine, an aldehyde, an aminooxy group, a thiol, a maleimide, or a phosphorothioate, or any combination thereof; and / orwherein the chemical coupling agent comprises a click chemistry reagent, a copper(I) source, a copper(I)-stabilizing ligand, a strain-promoted cycloaddition reagent, a tetrazine, an EDC or other carbodiimide, an aniline or p-phenylenediamine catalyst, or any combination thereof.

21. The composition of any one of claims 1-20, wherein the assembled product or a product thereof comprises a final synthetic sequence, wherein the final synthetic sequence comprises the scarless assembly of the n fragments.

22. The composition of any one of claims 1-21, wherein the final synthetic sequence is at least about 500 bases, 750 bases, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50 kb, 75 kb, 100 kb, 250 kb, 500 kb, 750 kb, or 1MB, in length.

23. The composition of any one of claims 1-22, wherein the first fragment, the (z)th fragment, the ( / z)th fragment, one or more of the n fragments, the first fragment toehold, the second fragment toehold, the first complementary region, the second complementary region, and / or the internal segment:comprises an elevated GC content of at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%;comprises a reduced GC content of less about 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%,19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 40%-30%, 40%-20%, 40%-10%, 40%-5%, 40%-l%, 30%-20%, 30%-10%, 30%-5%, 30%-l%, 20%-10%, 20%-5%, 20%-l%, 10%-5%, 10%-l%, or 5%-l%;comprises two or more repeats, optionally tandem repeats, optionally at least 4 nt in length, optionally occurring at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times within the final synthetic sequence; and / orcomprises two or more mononucleotide stretches, optionally at least 4 nt in length, optionally occurring at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times within the final synthetic sequence.

24. The composition of any one of claims 1-23,wherein for each (z)th fragment, the first fragment toehold of the (z)th fragment is not complementary to the second fragment toehold of any (&)th fragment, wherein k is an integer not equal to (z+1); orwherein for at least one (z)th fragment, the first fragment toehold of the (z)th fragment is complementary to the second fragment toehold of one or more (A)th fragments, wherein Aus an integer not equal to (z+1).

25. The composition of any one of claims 1-24,(a) wherein the first fragment:is an invariant fragment, wherein all instances of the invariant first fragment in the composition are identical; oris a variant fragment, wherein two or more instances of the variant first fragment in the composition differ with respect to the sequence of the internal segment;(b) wherein at least one (z)th fragment is an invariant fragment, wherein all instances of the invariant (z)th fragment in the composition are identical;(c) wherein at least one (z)th fragment is a variant fragment, wherein two or more instances of the variant (z)th fragment in the composition differ with respect to the sequence of the internal segment; and / or(d) wherein the (zz)th fragment:is an invariant fragment, wherein all instances of the invariant (zz)th fragment in the composition are identical; oris a variant fragment, wherein two or more instances of the variant (zz)th fragment in the composition differ with respect to the sequence of the internal segment.

26. The composition of any one of claims 1-25, wherein variant fragments comprisepredefined codon variations, optionally codons variations configured to achieve modified and / or improved protein function(s).

27. The composition of any one of claims 2, 5, or 7-26, wherein the composition comprises sets of n masked fragments, and wherein:the value of n is the same between at least two of they sets;the value of n is different between at least two of they sets;the masking region of each set is not complementary to the masking region of any other set;upon incubation of the y sets together in a single reaction mixture, each set of n masked fragments is capable of, in parallel joining together via four- way junction (4WJ) intermediates to generate y intermediate products;the intermediate products are candidate design variants;the intermediate products, or products thereof, are capable of being individually amplified or universally amplified; and / ory is an integer greater than 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

28. The composition of any one of claims 1-27, wherein the final synthetic sequence comprises one or more payload genes, optionally the one or more payload genes encode one or more RNA payload(s) and / or one or more payload protein(s).

29. The composition of any one of claims 1-28, wherein the one or more RNA payload(s) are selected from the group comprising a CRISPR single-guide RNA (sgRNA), a small interfering RNA (siRNA), a CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a piwi-interacting RNA (piRNA), an antisense oligonucleotide, an antagomir, an aptamer, a ribozyme, or any combination thereof.

30. The composition of any one of claims 1-29, wherein a payload protein comprises:fluorescence activity, polymerase activity, protease activity, phosphatase activity, kinase activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity demyristoylation activity, or any combination thereof;nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, glycosylase activity, acetyltransferase activity, deacetylase activity, adenylation activity, deadenylation activity, or anycombination thereof;a biomaterials payload, optionally a structural polypeptide, further optionally silk fibroin, spider silk spidroin, a resilin, a resilin-like polypeptide, an elastin, an elastin-like polypeptide, a collagen, or a collagen-like polypeptide;a cellular reprogramming factor capable of differentiating a given cell into a desired differentiated state, optionally nerve growth factor (NGF), fibroblast growth factor (FGF), interleukin-6 (IL-6), bone morphogenic protein (BMP), neurogenin3 (Ngn3), pancreatic and duodenal homeobox 1 (Pdxl), Mafa, or any combination thereof;an agonistic or antagonistic antibody or antigen-binding fragment thereof specific to a checkpoint inhibitor or checkpoint stimulator molecule, optionally PD1, PD-L1, PD-L2, CD27, CD28, CD40, CD137, 0X40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA4, IDO, KIR, LAG3, PD-1, and / or TIM-3;a secretion tag, optionally the secretion tag is selected from the group comprising AbnA, AmyE, AprE, BglC, BglS, Bpr, Csn, Epr, Ggt, GlpQ, HtrA, LipA, LytD, MntA, Mpr, NprE, OppA, PbpA, PbpX, Pel, PelB, PenP, PhoA, PhoB, PhoD, PstS, TasA, Vpr, WapA, WprA, XynA, XynD, YbdN, Ybxl, YcdH, YclQ, YdhF, YdhT, YfkN, YflE, YfmC, Yfnl, YhcR, YlqB, YncM, YnfF, YoaW, YocH, YolA, YqiX, Yqxl, YrpD, YrpE, YuaB, Yuri, YvcE, YvgO, YvpA, YwaD, YweA, YwoF, YwtD, YwtF, YxaLk, YxiA, and YxkC;a constitutive signal peptide for protein degradation, optionally PEST;a nuclear localization signal (NLS) or a nuclear export signal (NES);a dosage indicator protein, optionally the dosage indicator protein is detectable, optionally the dosage indicator protein comprises green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), blue fluorescent protein (BFP), red fluorescent protein (RFP), TagRFP, Dronpa, Padron, m Apple, mCherry, mruby3, rsCherry, rsCherryRev, derivatives thereof, or any combination thereof;a cellular reprogramming factor capable of converting an at least partially differentiated cell to a less differentiated cell, optionally Oct-3, Oct-4, Sox2, c-Myc, Klf4, Nanog, Lin28, ASCL1, MYT1L, TBX3b, SV40 large T, hTERT, miR-291, miR-294, miR-295, or any combinations thereof;a programmable nuclease, optionally the programmable nuclease is selected from the group comprising: SpCas9 or a derivative thereof; VRER, VQR, EQR SpCas9; xCas9-3.7; eSpCas9; Cas9-HF1; HypaCas9; evoCas9; ScCas9; StCas9; NmCas9; SaCas9; CjCas9; CasX; Cas9 H940A nickase; Cast 2 and derivatives thereof; dcas9-AP0BEC1 fusion, BE3, and dcas9-deaminase fusions; dcas9-Krab, dCas9-VP64, dCas9-Tetl, and dcas9-transcriptional regulator fusions; Dcas9-fluorescent protein fusions; Cast 3 -fluorescent protein fusions; RCas9-fluorescent protein fusions; Cas 13 -adenosine deaminase fusions, or any combination thereof;a CRE recombinase, GCaMP, a cell therapy component, a knock-down gene therapy component, a cell-surface exposed epitope, or any combination thereof;a bispecific T cell engager (BiTE);a synthetic receptor, optionally a Synthetic Notch (SynNotch) receptor, a Modular Extracellular Sensor Architecture (MESA) receptor, Tango, dCas9-synR, or any combination thereof;a cytokine, optionally the cytokine is selected from the group consisting of interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, granulocyte macrophage colony stimulating factor (GM-CSF), M-CSF, SCF, TSLP, oncostatin M, leukemia-inhibitory factor (LIF), CNTF, Cardiotropin- 1, NNT-l / BSF-3, growth hormone, Prolactin, Erythropoietin, Thrombopoietin, Leptin, G-CSF, or receptor or ligand thereof;a member of the TGF-p / BMP family selected from the group consisting of TGF-Pl, TGF-P2, TGF-P3, BMP-2, BMP-3a, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8a, BMP-8b, BMP-9, BMP-10, BMP-11, BMP-15, BMP-16, endometrial bleeding associated factor (EBAF), growth differentiation factor-1 (GDF-1), GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-12, GDF-14, mullerian inhibiting substance (MIS), activin-1, activin-2, activin-3, activin-4, and activin-5;a member of the TNF family of cytokines selected from the group consisting of TNF-alpha, TNF-beta, LT-beta, CD40 ligand, Fas ligand, CD 27 ligand, CD 30 ligand, and 4-1 BBL;a member of the immunoglobulin superfamily of cytokines selected from the group consisting of B7.1 (CD80) and B7.2 (B70);an interferon, optionally the interferon is selected from interferon alpha, interferon beta, or interferon gamma;a chemokine, optionally the chemokine is selected from CCL1, CCL2, CCL3, CCR4, CCL5, CCL7, CCL8 / MCP-2, CCL11, CCL13 / MCP-4, HCC- 1 / CCL14,CTAC / CCL17, CCL19, CCL22, CCL23, CCL24, CCL26, CCL27, VEGF, PDGF, lymphotactin (XCL1), Eotaxin, FGF, EGF, IP- 10, TRAIL, GCP-2 / CXCL6, NAP- 2 / CXCL7, CXCL8, CXCL10, ITAC / CXCL11, CXCL12, CXCL13, or CXCL15;an interleukin, optionally the interleukin is selected from IL-10 IL-12, IL-1, IL-6, IL-7, IL-15, IL-2, IL-18 or IL-21;a tumor necrosis factor (TNF), optionally the TNF is selected from TNF- alpha, TNF-beta, TNF-gamma, CD252, CD154, CD178, CD70, CD153, or 4-1BBL;a factor locally down-regulating the activity of endogenous immune cells;a factor capable of remodeling a tumor microenvironment and / or reducing immunosuppression at a target site of a subject;a chimeric antigen receptor (CAR) or T-cell receptor (TCR), optionally the CAR and / or TCR comprises one or more of an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, optionally wherein the intracellular signaling domain comprises a primary signaling domain, a costimulatory domain, or both of a primary signaling domain and a costimulatory domain; and / oran activity regulator, optionally the activity regulator is capable of reducing T cell activity.

31. The composition of any one of claims 1-30, wherein a payload protein is associated with an agricultural trait of interest selected from the group consisting of increased yield, increased abiotic stress tolerance, increased drought tolerance, increased flood tolerance, increased heat tolerance, increased cold and frost tolerance, increased salt tolerance, increased heavy metal tolerance, increased low-nitrogen tolerance, increased disease resistance, increased pest resistance, increased herbicide resistance, increased biomass production, male sterility, or any combination thereof.

32. The composition of any one of claims 1-31, wherein a payload protein is associated with a biological manufacturing process selected from the group comprising fermentation, distillation, biofuel production, production of a compound, production of a polypeptide, or any combination thereof.

33. The composition of any one of claims 1-32, wherein the one or more payload genes are selected from the group comprising a nitrogen fixation gene, a plant stress-induced gene, a nutrient utilization gene, a gene that affects plant pigmentation, a gene that encodes an antisense or ribozyme molecule, a gene encoding an antigen capable of being secreted, a toxin gene, a receptor gene, a ligand gene, a seed storage gene, a hormone gene, an enzyme gene, an interleukin gene, a cytokine gene, a growth factor gene, a transcription factor gene, a transcriptional repressor gene, a DNA-binding protein gene, a recombination gene, a DNAreplication gene, a programmed cell death gene, a kinase gene, a phosphatase gene, a G protein gene, a cyclin gene, a cell cycle control gene, a gene involved in transcription, a gene involved in translation, a gene involved in RNA processing, a gene involved in RNAi, an organellar gene, a intracellular trafficking gene, an integral membrane protein gene, a transporter gene, a membrane channel protein gene, a cell wall gene, a gene involved in protein processing, a gene involved in protein modification, a gene involved in protein degradation, a gene involved in metabolism, a gene involved in biosynthesis, a gene involved in assimilation of nitrogen or other elements or nutrients, a gene involved in controlling carbon flux, gene involved in respiration, a gene involved in photosynthesis, a gene involved in light sensing, a gene involved in organogenesis, a gene involved in embryogenesis, a gene involved in differentiation, a gene involved in meiotic drive, a gene involved in self incompatibility, a gene involved in development, a gene involved in nutrient, metabolite or mineral transport, a gene involved in nutrient, metabolite or mineral storage, a calcium-binding protein gene, a lipid-binding protein gene, or any combination thereof.

34. The composition of any one of claims 1-33, wherein the one or more payload genes are selected from the group comprising a gene encoding an enzyme involved in metabolizing biochemical wastes for use in bioremediation, a gene that encodes an enzyme for modifying pathways that produce secondary plant metabolites, a gene that encodes an enzyme that produces a pharmaceutical, a gene that encodes an enzyme that improves or changes the nutritional content of a plant, a gene that encodes an enzyme involved in vitamin synthesis, a gene that encodes an enzyme involved in carbohydrate, polysaccharide or starch synthesis, a gene that encodes an enzyme involved in mineral accumulation or availability, a gene that encodes a phytase, a gene that encodes an enzyme involved in fatty acid, fat or oil synthesis, a gene that encodes an enzyme involved in synthesis of chemicals or plastics, a gene that encodes an enzyme involved in synthesis of a fuel, a gene that encodes an enzyme involved in synthesis of a fragrance, a gene that encodes an enzyme involved in synthesis of a flavor, a gene that encodes an enzyme involved in synthesis of a pigment or dye, a gene that encodes an enzyme involved in synthesis of a hydrocarbon, a gene that encodes an enzyme involved in synthesis of a structural or fibrous compound, a gene that encodes an enzyme involved in synthesis of a food additive, a gene that encodes an enzyme involved in synthesis of a chemical insecticide, a gene that encodes an enzyme involved in synthesis of an insect repellent, a gene controlling carbon flux in a plant, or any combination thereof.

35. The composition of any one of claims 1-34, wherein the one or more payload proteins comprise components of a synthetic protein circuit, optionally payload proteins configured to form one or more logic gates selected from the group comprising an OR logic -n-gate, AND logic gate, NOR logic gate, NAND logic gate, IMPLY logic gate, NIMPLY logic gate, XOR logic gate, and an XNOR logic gate.

36. The composition of any one of claims 1-35, wherein a payload protein is capable of modulating the expression, concentration, localization, stability, and / or activity of the one or more endogenous proteins of a cell.

37. The composition of any one of claims 1-36, wherein the payload protein is a therapeutic protein or a variant thereof, optionally a therapeutic protein configured to prevent or treat a disease or disorder of a subject, further optionally the subject suffers from a deficiency of said therapeutic protein.

38. The composition of any one of claims 1-37, wherein one or more of the payload gene(s) comprise:a 5’UTR and / or a 3’UTR;a tandem gene expression element selected from the group an internal ribosomal entry site (IRES), foot-and-mouth disease virus 2A peptide (F2A), equine rhinitis A virus 2A peptide (E2A), porcine teschovirus 2A peptide (P2A) or Thosea asigna virus 2A peptide (T2A), or any combination thereof; and / ora transcript stabilization element, optionally the transcript stabilization element comprises woodchuck hepatitis post-translational regulatory element (WPRE), bovine growth hormone polyadenylation (bGH-polyA) signal sequence, human growth hormone polyadenylation (hGH-polyA) signal sequence, or any combination thereof.

39. The composition of any one of claims 1-38, wherein at least one of the payload genes is operably connected to a promoter selected from the group comprising:an RNA pol I promoter;a pol II promoter, optionally CMV, SV40 early region or adenovirus major late promoter;or pol III promoter, optionally a U6 or Hl promoter;a minimal promoter, optionally TATA, miniCMV, and / or miniPromo;a bacteriophage promoter, optionally a bacteriophage T3 promoter, a bacteriophage T7 promoter, a bacteriophage SP6 promoter, or a combination thereof; a tissue-specific promoter and / or a lineage-specific promoter;an inducible promoter, optionally a T7 RNA polymerase promoter, a T3 RNA polymerase promoter, an Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, a lactose induced promoter, a heat shock promoter, or a Tetracycline-regulated promoter, a tetracycline-dependent promoter, a lac-dependent promoter, a pB ad- dependent promoter, an AlcA-dependent promoter, a LexA-dependent promoter, or aheat-shock promoter;a ubiquitous promoter, optionally a cytomegalovirus (CMV) immediate early promoter, a CMV promoter, a viral simian virus 40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV) LTR promoter, a Rous sarcoma virus (RSV) LTR, an RSV promoter, a herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5, and Pll promoters from vaccinia virus, an elongation factor 1-alpha (EFla) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), Glyceraldehyde 3 -phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), P-kinesin (P-KIN), the human ROSA 26 locus, a Ubiquitin C promoter (UBC), a phosphoglycerate kinase- 1 (PGK) promoter, 3 -phosphoglycerate kinase promoter, a cytomegalovirus enhancer, human P-actin (HBA) promoter, chicken P-actin (CBA) promoter, a CAG promoter, a CASI promoter, a CBH promoter;or any combination thereof.

40. The composition of any one of claims 1-39, wherein the final synthetic sequence is or comprises all or a portion of a vector,optionally a viral vector, a plasmid, a transposable element, a naked DNA vector, or any combination thereof,optionally an AAV vector, a lentivirus vector, a retrovirus vector, an adenovirus vector, a herpesvirus vector, a herpes simplex virus vector, a cytomegalovirus vector, a vaccinia virus vector, a MVA vector, a baculovirus vector, a vesicular stomatitis virus vector, a human papillomavirus vector, an avipox virus vector, a Sindbis virus vector, a VEE vector, a Measles virus vector, an influenza virus vector, a hepatitis B virus vector, an integration-deficient lentivirus (IDLV) vector, or any combination thereof, and optionally the transposable element is piggybac transposon or sleeping beauty transposon.

41. The composition of any one of claims 1-40, wherein the final synthetic sequence is configured for propagation in a eukaryotic or a prokaryotic cell, optionally the final synthetic sequence comprises:a bacterial origin of replication, optionally ColEl, pl 5 A, pSClOl, and RK2; an origin of transfer (oriT) and one or more mobilization genes configured to enable conjugative transfer;an autonomously replicating sequence (ARS), a centromeric sequence (CEN), and / or 2p elements;a rolling-circle replication origin, optionally derived from pC194, pE194, and pUBUO;a mammalian origin of replication, optionally oriP / EBNAl and / or SV40 ori; a selection marker, optionally an antibiotic resistance marker and / or a fluorescence marker; and / ora counter-selection marker, optionally sacB, rpsL, galK, CYH2, and / or URA3.

42. The composition of any one of claims 1-41, wherein the final synthetic sequence is configured for insertion into a genome, optionally the final synthetic sequence comprises:recognition sites for an RNA-guided DNA binding complex, wherein the RNA- guided DNA binding complex comprises one or more Cas proteins, a transposase, one or more crRNAs, or any combination thereof;recognition sites for a transposition complex comprising one or more transposases;homology arms, optionally targeting a safe-harbor locus selected from AAVS1, ROSA26, CCR5, and Hl 1;one or more recombination sites, optionally loxP, FRT, attB, attP, attL, and attR; and / ora reporter cassette.

43. The composition of any one of claims 1-42, wherein the final synthetic sequence comprises a digital data storage payload encoded in nucleic acid sequence.

44. The composition of any one of claims 2, 5, or 7-43,wherein each of the n fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber;wherein each of the n masked fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber;wherein the n pairs or n-1 pairs of masking oligonucleotides are housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber; and / or wherein the first polynucleotide strand and the second polynucleotide strand that constitute each of the n fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber.

45. The composition of any one of claims 1-44, wherein the composition further comprises:a non-thermostable ligase, a thermostable ligase, a chemical coupling agent, a polymerase, a primer capable of binding the first terminal region (or a complement thereof), a primer capable of binding the second terminal region (or a complementthereof), or any combination thereof; and / ora ligation buffer, optionally comprising:a reaction buffer configured to support annealing, ligation, and / or amplification;one or more of Tris HC1 at about 10 mM to about 200 mM, at a pH of about 7.0 to about 9.5 at the incubation temperature, Mg2+ at about 0.5 mM to about 20 mM, monovalent cation(s) at about 10 mM to about 300 mM, and a reducing agent at about 0.1 mM to about 20 mM;a ligase cofactor, optionally selected from ATP at about 0.05 mM to about 5 mM or NAD+ at about 0.01 mM to about 2 mM;a buffering species selected from Tris, HEPES, Bis Tris, MOPS, and PIPES, optionally configured to maintain pH between 8.3-8.8 at 25°C; and / or one or more additives, optionally selected from bovine serum albumin at about 0.01 mg / mL to about 1 mg / mL, polyethylene glycol at about 1% to about 20% (w / v), betaine at about 0.1 M to about 2.0 M, dimethyl sulfoxide at about 1% to about 20% (v / v), formamide at about 0.5% to about 10% (v / v), glycerol at about 1% to about 20% (v / v), and / or a non-ionic detergent at about 0.001% to about 0.1% (v / v).

46. The composition of any one of claims 1-45, wherein the composition does not comprise one or more reagents employed with Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly, optionally an exonuclease, an endonuclease, a single stranded DNA binding protein, a restriction endonuclease, a recombinase, or any combination thereof.

47. A composition, comprising:a pre-assembly reaction mixture comprising the n masked fragments of any one of claims 2, 5, or 7-46 at equimolar concentrations, optionally the pre-assembly reaction mixture comprises a ligase or a chemical coupling agent.

48. A composition, comprising:a first intermediate reaction mixture comprising the n masked fragments of any one of claims 2, 5, or 7-46 associated together via four-way junction (4WJ) intermediates, optionally the first intermediate reaction mixture comprises a ligase or a chemical coupling agent.

49. A composition, comprising:a second intermediate reaction mixture comprising the n fragments of any one of claims 1-46 joined together via hybridization of the first complementary region and thesecond complementary region to generate an intermediate product, wherein the intermediate product comprises nicks on the first polynucleotide strand and the second polynucleotide strand,wherein the four-way junctions (4WJs) are resolved and the intermediate product is not hybridized to an upstream masking oligonucleotide or a downstream masking oligonucleotide,wherein the second intermediate reaction mixture comprises paired masking oligonucleotides consisting of an upstream masking oligonucleotide hybridized to a downstream masking oligonucleotide,optionally the second intermediate reaction mixture comprises a ligase or a chemical coupling agent.

50. A composition, comprising:a post-ligation reaction mixture comprising an assembled product wherein the first polynucleotide strand and the second polynucleotide strand do not comprise nicks.

51. A method, compri sing :providing the n masked fragments of any one of claims 2, 5, or 7-46; incubating the n masked fragments in a reaction mixture under reaction conditions such that:the first fragment toehold of the (z)th fragment hybridizes to the second fragment toehold of the (z+ 1 )th fragment; andthe masking region of the (z)th upstream masking oligonucleotide hybridizes to the masking region of the (z)th downstream masking oligonucleotide,thereby joining together the n fragments via four- way junction (4WJ) intermediates to generate an intermediate product; andligating nicks on the first polynucleotide strands and the second polynucleotide strands to generate an assembled product.

52. The method of claim 51,wherein association between:(i) the first fragment toehold and second fragment toehold of adjacent fragments; and(ii) the masking regions of an upstream masking oligonucleotide and a downstream masking oligonucleotide,forms a 4WJ.

53. The method of any one of claims 51-52, wherein mismatches between the firstcomplementary region and the second complementary region of incorrectly joined fragments prevent progression of the 4-way strand exchange.

54. The method of any one of claims 51-53, wherein, upon resolution of a 4WJ, adjacent fragments are hybridized via the first complementary region and the second complementary region, and the adjacent fragments are separated by a nick in each of the first polynucleotide strand and the second polynucleotide strand.

55. The method of any one of claims 51-54, wherein the resolution of a 4WJ yields a paired masking oligonucleotide consisting of an upstream masking oligonucleotide hybridized to a downstream masking oligonucleotide.

56. The method of any one of claims 51-55, wherein the masking oligonucleotides block hybridization between the first complementary region and the second complementary region when the first complementary region and the second complementary region lack perfect complementarity.

57. The method of any one of claims 51-56, wherein the isothermal strand displacement reaction is driven forward by perfect complementarity between the first complementary region and the second complementary region.

58. The method of any one of claims 51-57, wherein the incubation step is performed under isothermal conditions.

59. The method of any one of claims 51-58, wherein the incubation comprises incubation at a first incubation temperature for a first period of time, optionally:the first incubation temperature is about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C; andthe first period of time is about 10 sec, 20 sec, 30 sec, 40 sec, 50 sec, 60 sec, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, or 12 hr.

60. The method of any one of claims 51-59, wherein the ligating step comprises: addition of a ligase to the reaction mixture; andincubation at the second incubation temperature for a second period of time, optionally the second incubation temperature is about 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C, andoptionally the second period of time is about 10 sec, 20 sec, 30 sec, 40 sec, 50sec, 60 sec, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, or 12 hr.

61. The method of any one of claims 51-60,wherein the assembly of the n masked fragments occurs independently of the sequence of the internal segments, andwherein the assembly of the n masked fragments is directed by the formation of 4-way junctions (4WJs) between adjacent masked fragments.

62. The method of any one of claims 51-61, wherein the assembled product or a product thereof comprises a final synthetic sequence, wherein the final synthetic sequence comprises the scarless assembly of the n fragments.

63. The method of any one of claims 51-62, wherein:the final synthetic sequence is a linear polynucleotide, optionally comprising the structure 5 ’ -[first fragment]-[second fragment]-.. ,-[( / / )th fragment]-3’; orthe final synthetic sequence is a circular polynucleotide wherein the 3’ end of the [( / / )th fragment] is linked to the 5’ end of [first fragment] by a phosphodiester bond.

64. The method of any one of claims 51-63, wherein the final synthetic sequence is at least about 500 bases, 750 bases, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50 kb, 75 kb, 100 kb, 250 kb, 500 kb, 750 kb, or 1MB, in length.

65. The method of any one of claims 51-64, wherein the ligating step is performed with a ligase, optionally a thermostable ligase, optionally said ligase is selected from the group comprising T3 ligase, T4 ligase, T7 ligase, SplintR, E. coli DNA ligase, Hi-T4 ligase, a ligase suitable for ligating adjacent nucleic acid sequences under hybridization conditions, Taq ligase, 9°N, or any combination thereof.

66. The method of any one of claims 51-65, wherein the ligating step comprises contacting the intermediate product with a chemical coupling agent effective to form a covalent linkage between adjacent first polynucleotide strands and between adjacent second polynucleotide strands, optionally one or more click chemistry reagents, optionally CuAAC, SPAAC, iEDDA, oxime formation, hydrazone formation, Michael addition, disulfide formation, carbodiimide mediated coupling, native chemical ligation, or any combination thereof.

67. The method of any one of claims 51-66, wherein the incubating step comprises combining the n masked fragments in a single reaction mix at equimolar concentrations, optionally at about 0.1 nM, 0.5 nM, 0.75 nM, 0.9 nM, 1.0 nM, 1.1 nM, 1.25 nM, 1.5 nM, 1.75 nM, 2 nM, 5 nM, or 10 nM.

68. The method of any one of claims 51-67, wherein the providing step comprises: generating the n masked fragments,optionally said generating step comprises annealing the n fragments with the n pairs or n-1 pairs of masking oligonucleotides to generate the n masked fragments,optionally the n masked fragments are each generated in separate reactions,optionally said annealing step comprises an initial denaturation step followed by a gradual decrease in temperature,optionally the n masked fragments undergo one or more purification steps, further optionally:gel electrophoresis, including pulsed-field gel electrophoresis (PFGE);solid or solution phase hybridization / capture;precipitation;dialysis;solid phase reversible immobilization (SPRI) cleanup, optionally performing size selection using SPRI beads, further optionally singlesided or double-sided; and / orcolumn purification.

69. The method of any one of claims 51-68, wherein the providing step comprises: generating the n fragments,optionally said generating step comprises annealing the first polynucleotide strand and the second polynucleotide strand components of each of the n fragments to generate heteroduplexes,optionally the n fragments are each generated in separate reactions, optionally the generating step comprises phosphorylation of the first and second polynucleotide strands, further optionally via T4 polynucleotide kinase, optionally said annealing step comprises an initial denaturation step followed by a gradual decrease in temperature,optionally the heteroduplexes undergo one or more purification steps, further optionally:gel electrophoresis, including pulsed-field gel electrophoresis (PFGE);solid or solution phase hybridization / capture;precipitation;dialysis;solid phase reversible immobilization (SPRI) cleanup, optionally performing size selection using SPRI beads, further optionally singlesided or double-sided; and / orcolumn purification.

70. The method of any one of claims 51-68, wherein the method further comprises PCR amplification of the assembled product, or a product thereof, to generate an amplified product, optionally:PCR amplification comprises amplifying the assembled product, or a product thereof, using a primer capable of hybridizing to the first terminal region or a complement thereof, and a primer capable of hybridizing the second terminal region or a complement thereof.

71. The method of any one of claims 51-70, wherein the method comprises purification of the assembled product, the amplified product, or products thereof, optionally said purification step compromises:removal of the paired masking oligonucleotides;gel electrophoresis of the assembled product, the amplified product, or products thereof;solid phase reversible immobilization (SPRI) cleanup, optionally performing size selection using SPRI beads, further optionally single-sided or double-sided; and / or column purification.

72. The method of any one of claims 51-71, wherein the method comprises replication of the assembled product, the amplified product, or products thereof, in a cell.

73. The method of any one of claims 51-72,wherein the providing step comprises providing y sets of n masked fragments; wherein the incubating step comprises incubating the y sets of n masked fragments in a single reaction mixture, wherein the n masked fragments of each set are joined together in parallel via four- way junction (4WJ) intermediates to generate y intermediate products; andwherein the ligating step comprises ligating nicks on the first polynucleotide strands and the second polynucleotide strands of each of the y intermediate products to generate assembled products.

74. The method of any one of claims 51-73, wherein:the value of n is the same between at least two of they sets;the value of n is the different between at least two of they sets;the masking region of each set is not complementary to the masking region ofany other set;the intermediate products are candidate design variants;the method comprises the y intermediate products, or products thereof, being individually amplified or universally amplified; and / ory is an integer greater than 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

75. The method of any one of claims 51-74, wherein at least one of the n fragments is a variant fragment, and wherein the assembled products comprise a combinatorial library of at least p variants, wherein p is an integer greater than 1.

76. The method of any one of claims 51-75,wherein p is at least about 10, 50, 100, 250, 500, 750, 1000, 10000, 50000, 100000, 250000, 500000, 750000, 1000000, 5000000, or 10000000;wherein the combinatorial library achieves a variant coverage of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.99%, of the theoretical variant library; and / orevery codon mutation profile is represented in the library with an average absolute deviation of less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, or 0.01%, from the theoretical proportion of occurrence for that codon.

77. The method of any one of claims 51-76,wherein at least 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999%, of the assembled products, or products thereof, comprise all of the intended fragments in the intended order;wherein less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, or 0.01%, of the assembled products, or products thereof, are a partial assembly missing one or more fragments;wherein less than 1 in 1000, 1 in 10000, 1 in 100000, 1 in 1000000, 1 in 10000000, or 1 in 100000000, of the assembled products are missing one or more fragments or comprise a mis-assembled junction; and / orwherein the mis-ligation rate at the 4WJ is less than 1 in 1000, 1 in 10000, 1 in 100000, 1 in 1000000, 1 in 10000000, or 1 in 100000000.

78. The method of any one of claims 51-77, wherein n is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 10-25, 10-50, 10-75, 10-100, 10-500, 10-1000, 25-50, 25-75, 25-100,25-500, 25-1000, 50-75, 50-100, 50-500, 50-1000, 75-100, 75-500, 75-1000, 100-500, 100-1000, or 500-1000.

79. The method of any one of claims 51-78, wherein the yield of correctly assembled products is at least 1-fold, 2-fold, 4-fold, 8-fold, 10-fold, 20-fold, 50-fold, 100-fold, 500-fold, or 1000-fold, greater than the yield of a polynucleotide assembly method not comprising 4WJ, optionally Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly.

80. The method of any one of claims 51-79, wherein at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, of the incubated fragments become a component of an assembled product.

81. A composition, comprising: assembled products, or products thereof, generated by the method of any one of claims 51-80.

82. The composition of claim 81, wherein the composition comprises a plurality of cells comprising the assembled products, or products thereof.

83. A method, comprising:providing the combinatorial library of any one of claims 75-76, or a product thereof;expressing the one or more payload genes in cell(s); andscreening for a property of interest.

84. The method of claim 83, wherein screening comprises fluorescence-activated cell sorting (FACS), cell viability assay, ELISA, co-immunoprecipitation, a bead-based immunoassay, or any combination thereof.

85. The method of any one of claims 83-84, wherein the property of interest comprises modified enzymatic activity, improved enzymatic activity, modified binding activity, improved binding activity, modified stability, improved stability, modified localization, improved localization, modified solubility, improved solubility, modified expression, improved expression, modified inhibitor resistance, improved inhibitor resistance, modified substrate specificity, improved substrate specificity, or any combination thereof.

86. The method of any one of claims 83-85, wherein the method comprises exposing the cell(s) to one or more agents, optionally the one or more agents comprise:one or more of a chemical agent, a pharmaceutical, small molecule, a biologic, a CRISPR single-guide RNA (sgRNA), a small interfering RNA (siRNA), CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), a piwi-interacting RNA (piRNA), an antisense oligonucleotide, a peptide or peptidomimetic inhibitor, anaptamer, an antibody, an intrabody, or any combination thereof;an expression vector, wherein the expression vector encodes one or more of the following: an mRNA, an antisense nucleic acid molecule, a RNAi molecule, a shRNA, a mature miRNA, a pre-miRNA, a pri-miRNA, an anti-miRNA, a ribozyme, any combination thereof;an infectious agent, an anti-infectious agent, or a mixture thereof;a cytotoxic agent, optionally a chemotherapeutic agent, a biologic agent, a toxin, a radioactive isotope, or any combination thereof; and / orone or more of an epigenetic modifying agent, epigenetic enzyme, a bicyclic peptide, a transcription factor, a DNA or protein modification enzyme, a DNA- intercalating agent, an efflux pump inhibitor, a nuclear receptor activator or inhibitor, a proteasome inhibitor, a competitive inhibitor for an enzyme, a protein synthesis inhibitor, a nuclease, a protein fragment or domain, a tag or marker, an antigen, an antibody or antibody fragment, a ligand or a receptor, a synthetic or analog peptide from a naturally- bioactive peptide, an anti-microbial peptide, a pore-forming peptide, a targeting or cytotoxic peptide, a degradation or self-destruction peptide, a CRISPR component system or component thereof, DNA, RNA, artificial nucleic acids, a nanoparticle, an oligonucleotide aptamer, a peptide aptamer, or any combination thereof.

87. The method of any one of claims 83-86, wherein the property of interest comprises a property of the cell, optionally improved drug resistance, altered drug sensitivity, improved or modified growth rate under selective pressure, modified or improved cell viability or survival, modified or improved stress tolerance, modified or improved secretion of a compound, altered signaling pathway activation, or any combination thereof.

88. The method of any one of claims 83-87, wherein the method comprises cloning the assembled products, or products thereof, into expression vector(s), optionally prior to an expressing step, optionally:the expression vector is selected from a plasmid, a viral vector, a transposable element, a bacterial artificial chromosome, a yeast artificial chromosome, or any combination thereof;the cloning step operably connects the final synthetic sequence with one or more regulatory elements selected from a promoter, an enhancer, a polyadenylation signal, a 5’UTR, a 3’ UTR, and a selection marker; and / orthe method comprises transforming or transfecting host cells with the cloned expression vector, optionally bacterial cells for propagation and / or sequence verification and subsequently eukaryotic cells for expression, optionally mammalian, yeast, insect,plant, or fungal cells.

89. A kit, comprising:(al) the n masked fragments of any one of claims 2, 5, or 7-46,optionally y sets of n masked fragments,optionally each of the n masked fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber;(a2) the n fragments of any one of claims 1-46 and the n pairs or n-1 pairs of masking oligonucleotides of any one of claims 1-46,optionally y sets of n fragments and y sets of n pairs or n-1 pairs of masking oligonucleotides,optionally each of the n fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber,optionally each of the n pairs or n-1 pairs of masking oligonucleotides is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber, optionally the first polynucleotide strand and the second polynucleotide strand that constitute each of the n fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber;(b) a non-thermostable ligase, a thermostable ligase, a chemical coupling agent, a polymerase, a primer capable of binding the first terminal region (or a complement thereof), a primer capable of binding the second terminal region (or a complement thereof), or any combination thereof; and / or(c) a ligation buffer, optionally comprising:a reaction buffer configured to support annealing, ligation, and / or amplification;one or more of Tris HC1 at about 10 mM to about 200 mM, at a pH of about 7.0 to about 9.5 at the incubation temperature, Mg2+ at about 0.5 mM to about 20 mM, monovalent cation(s) at about 10 mM to about 300 mM, and a reducing agent at about 0.1 mM to about 20 mM;a ligase cofactor, optionally selected from ATP at about 0.05 mM to about 5 mM or NAD+ at about 0.01 mM to about 2 mM;a buffering species selected from Tris, HEPES, Bis Tris, MOPS, and PIPES, optionally configured to maintain pH between 8.3-8.8 at 25°C; and / or one or more additives, optionally selected from bovine serum albumin at about 0.01 mg / mL to about 1 mg / mL, polyethylene glycol at about 1% to about 20% (w / v), betaine at about 0.1 M to about 2.0 M, dimethyl sulfoxide at about1% to about 20% (v / v), formamide at about 0.5% to about 10% (v / v), glycerol at about 1% to about 20% (v / v), and / or a non-ionic detergent at about 0.001% to about 0.1% (v / v); and / or(d) one or more purification reagent(s), optionally:gel electrophoresis reagent(s), optionally pulsed-field gel electrophoresis (PFGE);solid or solution phase hybridization / capture reagent(s);precipitation reagent(s);dialysis reagent(s);solid phase reversible immobilization (SPRI) cleanup reagent(s), optionally performing size selection using SPRI beads, further optionally singlesided or double-sided; and / orcolumn purification reagent(s),optionally the kit does not comprise one or more reagents employed with Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly, optionally an exonuclease, an endonuclease, a single stranded DNA binding protein, a restriction endonuclease, a recombinase, or any combination thereof.

90. A system for synthesizing nucleic acids, comprising:(al) the n masked fragments of any one of claims 2, 5, or 7-46,optionally sets of n masked fragments,optionally each of the n masked fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber;(a2) the n fragments of any one of claims 1-46 and the n pairs or n-1 pairs of masking oligonucleotides of any one of claims 1-46,optionally y sets of n fragments and y sets of n pairs or n-1 pairs of masking oligonucleotides,optionally each of the n fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber,optionally each of the n pairs or n-1 pairs of masking oligonucleotides is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber, optionally the first polynucleotide strand and the second polynucleotide strand that constitute each of the n fragments is housed in a separate vessel, optionally a tube, a well, or a microfluidic chamber;(b) a non-thermostable ligase, a thermostable ligase, a chemical coupling agent, apolymerase, a primer capable of binding the first terminal region (or a complement thereof), a primer capable of binding the second terminal region (or a complement thereof), or any combination thereof;(c) a ligation buffer, optionally comprising:a reaction buffer configured to support annealing, ligation, and / or amplification;one or more of Tris HC1 at about 10 mM to about 200 mM, at a pH of about 7.0 to about 9.5 at the incubation temperature, Mg2+ at about 0.5 mM to about 20 mM, monovalent cation(s) at about 10 mM to about 300 mM, and a reducing agent at about 0.1 mM to about 20 mM;a ligase cofactor, optionally selected from ATP at about 0.05 mM to about 5 mM or NAD+ at about 0.01 mM to about 2 mM;a buffering species selected from Tris, HEPES, Bis Tris, MOPS, and PIPES, optionally configured to maintain pH between 8.3-8.8 at 25°C; and / or one or more additives, optionally selected from bovine serum albumin at about 0.01 mg / mL to about 1 mg / mL, polyethylene glycol at about 1% to about 20% (w / v), betaine at about 0.1 M to about 2.0 M, dimethyl sulfoxide at about 1% to about 20% (v / v), formamide at about 0.5% to about 10% (v / v), glycerol at about 1% to about 20% (v / v), and / or a non-ionic detergent at about 0.001% to about 0.1% (v / v); and / or(d) one or more purification reagent(s), optionally:gel electrophoresis reagent(s), optionally pulsed-field gel electrophoresis (PFGE);solid or solution phase hybridization / capture reagent(s);precipitation reagent(s);dialysis reagent(s);solid phase reversible immobilization (SPRI) cleanup reagent(s), optionally performing size selection using SPRI beads, further optionally singlesided or double-sided; and / orcolumn purification reagent(s),optionally the system does not comprise one or more reagents employed with Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly, optionally an exonuclease, an endonuclease, a single stranded DNA binding protein, a restriction endonuclease, a recombinase, or any combination thereof.

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