Super user cloning 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 ligation at temperatures significantly higher than the melting temperature of assembly overhangs. Methods, compositions, systems, and kits provided herein can employ a USER cloning strategy which implements primers having 2 (or more) deoxyuridine bases.

Description

CIT 8985-PCT / 30KJ-810010-WO PATENT SUPER USER CLONING 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 / 777,887, filed March 26, 2025, the content of this related application is incorporated herein by reference in its entirety for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0002] This invention was made with government support under Grant No. GM 140937 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 30KJ-810010-WO, created March 22, 2026, which is 793,256 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUNDField

[0004] The present disclosure relates generally to the field of polynucleotide assembly.Description of the Related Art

[0005] 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. The current USER Cloning technique is limited to 4-5 DNA fragments assembledwith high efficiency, leaving the technique scarcely used. There is a need for compositions, methods, systems, and kits for polynucleotide assembly.SUMMARY

[0006] Disclosed herein include methods. The method can comprise: providing m assembly precursors, wherein m is a positive integer. In some embodiments, the assembly precursors are double-stranded DNA molecules comprising one or two 5’ overhang-forming regions. In some embodiments, each 5’ overhang-forming region comprises two or more 5’ terminal cleavage regions. In some embodiments, each of the 5’ terminal cleavage regions comprises 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. In some embodiments, each of the m assembly fragments comprises 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.

[0007] In some embodiments, the non-canonical nucleotide(s) comprises deoxyuridine, deoxyinosine, deoxy-7-m ethylguanosine, deoxy-5,6-dihydroxythymidine, deoxy-3 -methyladenosine, 5-methyl-deoxycytidine, O-6-methyl-deoxyguanosine, 5-iodo-deoxyuridine, 8-oxy-deoxy guanine, l,N6-ethenoadenine, 8-oxo-guanine (8oxoG), or any combination thereof. In some embodiments, the non-canonical nucleotide(s) comprise deoxyuridine. In some embodiments, the cleavage agent(s) comprise DNA glycosylase-lyase Endonuclease VIII, a DNA glycosylase, an AP cleaving agent, APE 1 (AP Endonuclease 1), Endo III (Endonuclease III), Endo IV (Endonuclease IV), Endo V (Endonuclease V), Endo VIII (Endonuclease VIII), Fpg (formamido-pyrimidine-DNA glycosylase), OGGI (8-oxoguanine DNA glycosylase 1), NEIL1 (Endonuclease Vlll-like 1), T7 Endo I (T7 Endonuclease I), T4 PDG (T4 pyrimidine dimer DNA glycosylase), UDG (uracil DNA glycosylase), SMUG1 (Single-strand selective monofunctional uracil DNA glycosylase), AAG (methylpurine DNA glycosylase), or any combination thereof. In some embodiments, the contacting step is performed in the presence of a 5’ ssDNA exonuclease configured to remove waste products derived from the 5’ terminal cleavage regions, optionally selected from the group comprising Red, RecJF, Exo VII, Dna2, T5 Exo, or any combination thereof.

[0008] In some embodiments, the 5’ terminal cleavage region comprises 1, 2, 3, 4, or 5 non-canonical nucleotide(s), optionally the step of providing m assembly precursors comprises PCR amplification reach on(s) comprising precursor primer(s) having a 5’ terminal cleavage region(s) comprising said non-canonical nucleotide(s), optionally m sets of precursor primer(s).In some embodiments, at least two of the m assembly precursors are contacted with the one or more cleavage agents in separate reaction vessels. In some embodiments, at least two of the m assembly precursors are contacted with the one or more cleavage agents in a common reaction mixture. In some embodiments, the method comprises purifying the assembly precursors and / or assembly fragments, optionally one or more purification steps comprising: gel electrophoresis, optionally 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, optionally size exclusion column purification.

[0009] In some embodiments, the 3’ overhang comprises the sequence ANxWNyWNzT, wherein x, y, and z are positive integers. In some embodiments, the 3’ overhang exhibits no secondary structure (NSS), optionally at 30°C. In some embodiments, the 3’ overhang is optimized to minimize cross reactivity with non-complementary overhangs. In some embodiments, the 3’ overhang is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, nucleotides in length. In some embodiments, the 3’ overhang 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%. In some embodiments, the 3’ overhang comprises a melting temperature (Tm) of about 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, 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, or 75°C.

[0010] In some embodiments, the contacting step is performed at a first incubation temperature for a first period of time. In some embodiments, the first incubation temperature is about 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 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, or 40°C, optionally 37°C. In some embodiments, the first period of time is about 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, optionally one hour.

[0011] In some embodiments, one or more of the m assembly precursors comprises an Uracil Primer Dimer Overhang Generation (UPDOG) product. In some embodiments,providing the m assembly precursors comprises: providing a first UPDOG primer and a second UPDOG primer capable of hybridizing each other, wherein the first UPDOG primer and second UPDOG primer each comprise 5’ terminal cleavage region(s) comprising two or more non-canonical nucleotide(s); and contacting the first UPDOG primer and the second UPDOG primer in the presence of a polymerase to generate the UPDOG product.

[0012] The method can comprise: providing n assembly fragments, wherein n is an integer greater than 1. In some embodiments, at least two of the n assembly fragments comprise or are derived from the m assembly fragments generated by a method disclosed herein. In some embodiments, the n assembly fragments are 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, thereby generating an assembled product.

[0013] Disclosed herein include methods. The method can comprise: providing n assembly fragments, wherein n is an integer greater than 1. In some embodiments, the n assembly fragments are 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.

[0014] The method can comprise: generating one or more of the n assembly fragments according to a method disclosed herein. In some embodiments, contacting the m assembly precursors with the cleavage agent(s) and incubating the n assembly fragments in the presence of the ligase are performed simultaneously. In some embodiments, the assembly fragments comprise or are derived from Golden Gate assembly fragments, optionally the method further comprises contacting assembly precursors with Type-IIS and / or Type-IIP cleaving agents.

[0015] In some embodiments, the ligase is capable of ligating nicks at junctions of hybridized assembly fragments to generate the assembled product. In some embodiments, the ligase is selected from the group comprising a thermostable ligase 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.

[0016] In some embodiments, the assembly fragment is at least 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600,2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000, nucleotides in length. In some embodiments, the assembly fragment 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%, or 100%. In some embodiments, the assembly fragment comprises a melting temperature (Tm) of about 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, 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, or 75°C.

[0017] In some embodiments, the incubating step is performed at second incubation temperature for a second period of time. In some embodiments, the second incubation temperature is about 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 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, 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, 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, or 75°C, optionally 50°C. In some embodiments, the second period of time is about 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, optionally 16 hr.

[0018] In some embodiments, the second incubation temperature is at least about 0.5°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 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, or 50°C, higher than the median 3’ overhang Tm. In some embodiments, n or m 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.

[0019] In some embodiments, n is 3 or more, and the assembled product is linear. Insome embodiments, the first assembly fragment of the n assembly fragments comprises a second 3’ overhang. In some embodiments, each (z)th assembly fragment of the n assembly fragments comprises a first 3’ overhang and a second 3’ overhang, wherein 1 < z < n. In some embodiments, the (zz)th assembly of the n assembly fragments fragment comprises a first 3’ overhang. In some embodiments, the first 3’ overhang of each (z)th assembly fragment is complementary to the second 3’ overhang of the (z-l)th assembly fragment. In some embodiments, the second 3’ overhang of each (z)th assembly fragment is complementary to the first 3’ overhang of the (z+l)th assembly fragment.

[0020] In some embodiments, n is 3 or more, and the assembled product is circular. In some embodiments, each assembly fragment of the n assembly fragments comprises a first 3’ overhang and a second 3’ overhang. In some embodiments, the first 3’ overhang of the first assembly fragment of the n assembly fragments is complementary to the second 3’ overhang of the (zz)th assembly fragment of the n assembly fragments.

[0021] In some embodiments, for each (z)th assembly fragment, wherein 1 < z < n the first 3’ overhang of the (z)th assembly fragment is complementary to the second 3’ overhang of the (z-l)th assembly fragment, and the second 3’ overhang of the (z)th assembly fragment is complementary to the first 3’ overhang of the (z+l)th assembly fragment. In some embodiments, the first assembly fragment of the n assembly fragments comprises a first terminal region, optionally a 5’ terminal region. In some embodiments, the (zz)th assembly fragment of the n assembly fragments comprises a second terminal region, optionally a 3’ terminal region. In some embodiments, one or more of the n assembly fragments comprise an internal segment, wherein the internal segment does not comprise the 3’ overhang(s), and wherein the internal segment is double-stranded. In some embodiments, the assembly precursors and / or the assembly fragments: comprise or are derived from synthetic oligonucleotides; and / or comprise or are derived from Sidewinder products, TADA products, rolling circle amplification products, restriction enzyme digestion products, reverse transcription products, CRISPR-excised products, PCR amplification products, template-independent polymerase products, recombinase-generated products, phage-derived products, or any combination thereof.

[0022] 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 complement thereof, and a primer capable of hybridizing the second terminal region or a complement thereof. In some embodiments, PCR amplification comprises PCR primer(s) having an overhang, and wherein the final synthetic sequence comprises the sequence of said overhang. Insome embodiments, the method comprises purification of the assembled product, the amplified product, or products thereof. In some embodiments, said purification step compromises: 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.

[0023] In some embodiments, the assembled product, the amplified product, or products thereof, comprises a final synthetic sequence, and wherein the final synthetic sequence is at least about 500 bp, 750 bp, 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. In some embodiments, the final synthetic sequence 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%. In some embodiments, the final synthetic sequence 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%. In some embodiments, the final synthetic sequence 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. In some embodiments, the final synthetic sequence 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, or 20 times within the final synthetic sequence.

[0024] The final synthetic sequence can comprise 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). 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.

[0025] 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.

[0026] 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; Casl2 and derivatives thereof; dcas9-AP0BECl 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 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.

[0027] 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).

[0028] 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.

[0029] 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 group comprising 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 geneinvolved 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.

[0030] 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, 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. 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.

[0031] In some embodiments, a payload gene encodes an isoform of a payload protein comprising two or more exons, optionally a therapeutically relevant isoform. In some embodiments, providing n assembly fragments comprises: PCR amplification of one or more one exons from genomic DNA, optionally patient-derived genomic DNA. In some embodiments, at least one of the two or more exons is encoded in the 5’ end of an amplification primer. In some embodiments, at least one assembly fragment of the n assembly fragments is an UPDOG product comprising two or more exons.

[0032] In some embodiments, one or more of the payload gene(s) comprise: (a) a 5’UTR and / or a 3’UTR; (b) 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 (c) 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.

[0033] In some embodiments, at least one of the payload genes is operably connected to a promoter selected from the group comprising: (a) an RNA pol I promoter; (b) a pol II promoter, optionally CMV, SV40 early region or adenovirus major late promoter; (c) pol III promoter, optionally a U6 or Hl promoter; (d) a minimal promoter, optionally TATA, miniCMV, and / or miniPromo; (e) a bacteriophage promoter, optionally a bacteriophage T3 promoter, a bacteriophage T7 promoter, a bacteriophage SP6 promoter, or a combination thereof; (f) a tissue-specific promoter and / or a lineage-specific promoter; (g) 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; (h) 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.

[0034] In some embodiments, the method comprises replication of the assembled product, the amplified product, or products thereof, in a cell, optionally episomal replicons. In some embodiments, the method comprises delivery of the assembled product, the amplified product, or products thereof, to a cell. In some embodiments, delivery comprises one or more of transformation, transfection, transduction, conjugation, electroporation, lipid-mediated transfection, liposome-mediated delivery, nanoparticle-mediated delivery, viral vector-mediated delivery, microinjection, biolistic delivery, calcium phosphate-mediated transfection, PEG-mediated transformation or transfection. In some embodiments, the method comprises conjugative transfer from a donor cell to a recipient cell. In some embodiments, the final synthetic sequence is or comprises all or a portion of a vector, The vector can comprise a viral vector, a plasmid, a transposable element, a naked DNA vector, or any combination thereof. The vector 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. In some embodiments, the transposable element is piggybac transposon or sleeping beauty transposon.

[0035] In some embodiments, the final synthetic sequence is configured for propagation in a eukaryotic or a prokaryotic cell. In some embodiments, the final synthetic sequence comprises a bacterial origin of replication, optionally ColEl, pl 5 A, pSClOl, and RK2. In some embodiments, the final synthetic sequence comprises an origin of transfer (oriT) and one or more mobilization genes configured to enable conjugative transfer. In some embodiments, the final synthetic sequence comprises an autonomously replicating sequence (ARS), a centromeric sequence (CEN), and / or 2p elements. In some embodiments, the final synthetic sequence comprises a rolling-circle replication origin, optionally derived from pC194, pE194, and pUBHO. In some embodiments, the final synthetic sequence comprises a mammalian origin of replication, optionally oriP / EBNAl and / or SV40 ori. In some embodiments, the final synthetic sequence comprises a selection marker, optionally an antibiotic resistance marker and / or a fluorescence marker. In some embodiments, the final synthetic sequence comprises a counter-selection marker, optionally sacB, rpsL, galK, CYH2, and / orUR A3.

[0036] In some embodiments, the method comprises integration of the final synthetic sequence, or a portion thereof, into the genome of a cell, optionally via a site-specific recombinase. In some embodiments, the site-specific recombinase is a tyrosine recombinase, optionally selected from the group comprising HK022 recombinase, Cre, lambda phage, phage 186, phi80, P21, Flp, XerC / D, XerA, lambda integrase (Int), P2 integrase, FimB, FimE, HbiF, Rci, or any combination thereof. In some embodiments, the site-specific recombinase is a serine recombinase, optionally selected from the group comprising Bxbl, Tn3, y6 resolvase, Gin, Hin, TP901-1, C31, TGI, Rvl, C.IS607-like serine transposases, or any combination thereof. In some embodiments, the method comprises integration without episomal replicon intermediate(s).

[0037] In some embodiments, the final synthetic sequence is configured for insertion into a genome. In some embodiments, 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. In some embodiments, the final synthetic sequence comprises recognition sites for a transposition complex comprising one or more transposases. In some embodiments, the final synthetic sequence comprises homology arms, optionally targeting a safe-harbor locus selected from AAVS1, ROSA26, CCR5, and Hll. In some embodiments, the final synthetic sequence comprises one or more recombination sites, optionally loxP, FRT, attB, attP, attL, and attR. In some embodiments, the final synthetic sequence comprises a reporter cassette. In some embodiments, the assembled product, the amplified product, or product thereof, comprises a circular DNA molecule comprising: a recombination site, optionally an attB recombination site; terminator(s); and / or a transcriptionally inactive first selection marker gene configured to be transcriptionally active upon correct integration. In some embodiments, the method comprises introducing the circular DNA molecule into a recipient cell comprising: (a) a recombinase protein, optionally a tyrosine recombinase; and (b) an integration site comprising a complementary recombination site, optionally an attP recombination site. In some embodiments, the integration site comprises an upstream constitutive promoter and / or a transcriptionally active second selection marker gene. In some embodiments, the terminator(s) render the second selection marker gene transcriptionally inactive upon correct integration. In some embodiments, the introduction into the recipient cell comprises conjugative transfer from a donor cell. In some embodiments, the assembled product, the amplified product, or product thereof, comprises a linear DNA molecule comprising: (a) a first recombination site and a second recombination site, optionally situated on the 5’ and 3’ terminal ends, respectively, further optionally LoxP (LI) andLox2272 (L2) recombination sites; and / or (b) a first selection marker gene. In some embodiments, the method comprises introducing the linear DNA molecule into a recipient cell comprising: (a) a recombinase protein, optionally a serine recombinase; and (b) an integration site comprising the first recombination site and the second recombination site, optionally LoxP (LI) and Lox2272 (L2) recombination sites. In some embodiments, the integration site comprises one or more selection marker genes situated between the first recombination site and the second recombination site, In some embodiments, the introduction into the recipient cell comprises conjugative transfer from a donor cell.

[0038] In some embodiments, the first assembly fragment is an invariant fragment, wherein all instances of the invariant first assembly fragment are identical. In some embodiments, the first assembly fragment is a variant fragment, wherein two or more instances of the variant first assembly fragment differ with respect to the sequence of the internal segment. In some embodiments, at least one (z)th assembly fragment is an invariant fragment, wherein all instances of the invariant (z)th assembly fragment are identical. In some embodiments, at least one (z)th assembly fragment is a variant fragment, wherein two or more instances of the variant (z)th assembly fragment differ with respect to the sequence of the internal segment. In some embodiments, the (z )th assembly fragment is an invariant fragment, wherein all instances of the invariant (z )th assembly fragment are identical. In some embodiments, the (z )th assembly fragment is a variant fragment, wherein two or more instances of the variant (z )th assembly fragment 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). In some embodiments, at least one of the n assembly 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, p is at least about 10, 50, 100, 250, 500, 750, 1000, 10000, 50000, 100000, 250000, 500000, 750000, 1000000, 5000000, or 10000000. In some embodiments, 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. 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%, or 0.01%, from the theoretical proportion of occurrence for that codon.

[0039] In some embodiments, 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 assembly fragments in the intended order. In some embodiments, 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 assembly fragments. In some embodiments, 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 assembly fragments or comprise a misassembled junction. In some embodiments, the mis-ligation rate at a junction is less than 1 in 1000, 1 in 10000, 1 in 100000, 1 in 1000000, 1 in 10000000, or 1 in 100000000. 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 a ligation temperature higher than the median melting temperature of the 3’ overhangs, 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%, or 99%, of the incubated assembly fragments become a component of an assembled product.

[0040] 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.

[0041] 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 cell sorting (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: (a) 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, anantibody, an intrabody, or any combination thereof; (b) 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; (c) an infectious agent, an anti-infectious agent, or a mixture thereof; (d) a cytotoxic agent, optionally a chemotherapeutic agent, a biologic agent, a toxin, a radioactive isotope, or any combination thereof; and / or (e) 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 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. 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.

[0042] Disclosed herein include system for synthesizing nucleic acids. Disclosed herein include kits. The system or kit can comprise: one or more of the m assembly precursors, m sets of precursor primer(s), and n assembly fragments disclosed herein. The system or kit can comprise: one or more cleavage agent(s), optionally DNA glycosylase-lyase Endonuclease VIII, a DNA glycosylase, an AP cleaving agent, APE 1 (AP Endonuclease 1), Endo III (Endonuclease III), Endo IV (Endonuclease IV), Endo V (Endonuclease V), Endo VIII (Endonuclease VIII),Fpg (formamido-pyrimidine-DNA glycosylase), OGGI (8-oxoguanine DNA glycosylase 1), NEIL1 (Endonuclease Vlll-like 1), T7 Endo I (T7 Endonuclease I), T4 PDG (T4 pyrimidine dimer DNA glycosylase), UDG (uracil DNA glycosylase), SMUG1 (Single-strand selective monofunctional uracil DNA glycosylase), AAG (methylpurine DNA glycosylase), or any combination thereof. The system or kit can comprise: a non-thermostable ligase, a thermostable ligase, 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); 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). 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 doublesided; and / or column purification reagent(s). In some embodiments, the kit or 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIGS. 1A-1J depict non-limiting exemplary schematics and data related to high temperature ligation enabling improved accuracy and yield. FIG. 1A. Schematic depicting the advantages offered by High Temperature (HighT) ligation in comparison to low temperature ligation. FIG. IB. Schematic representation of multi-fragment assemblies and subsequent PCR amplification for generating Assembly Amplicons. FIG. 1C. DNA agarose gel depicting IpL of PCR product comparing ligation temperatures with increasing assembly size and number of fragments for 3’ overhang lengths of 4 bp and 10 bp. A segment of the LuxABCDE cassette was assembled with 5, 10, and 20-piece assemblies for all conditions. FIG. ID. Violin plots depicting the distribution of the predicted Tmof assembly overhangs for the 20-piece assemblies for 3’ overhang lengths of 4 bp (Min=8, Max=16, Ql=10, Median=12, Q3=12, Lower bound=7,Upper bound=15) and 10 bp (Min=24, Max=34, Ql=26, Median=28, Q3=31, Lower bound=18.5, Upper bound=38.5). FIG. IE. Schematic depicting the improved 2WJ cloning mechanism with corresponding modifications to the protocol which can improve the reaction accuracy and efficiency. FIG. IF. Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 4-piece GFP assembly: : -control (Average accuracy=50.0%, Standard deviation=4.54%, Average yield=2.01 x io5GFP + colonies / pmol, Standard deviation= 1.69 x io4GFP + colonies / pmol) a. (Average accuracy=51.2%, Standard deviation=2.83%, Average yield=2.79 x 106GFP + colonies / pmol, Standard deviation=5.99 x io5GFP + colonies / pmol) p. (Average accuracy=91.8%, Standard deviation=1.8%, Average yield=1.82 x io6GFP + colonies / pmol, Standard deviation=8.23 x io5GFP + colonies / pmol), y. (Average accuracy=88.3%, Standard deviation=2.8%, Average yield=5.61 x 106GFP + colonies / pmol, Standard deviation=2.42 x 106GFP + colonies / pmol), 6. (Average accuracy=96.7%, Standard deviation=0.49%, Average yield=3.14 x 106GFP + colonies / pmol, Standard deviation=5.81 x 105GFP + colonies / pmol), E. (Average accuracy=88.7%, Standard deviation=1.6%, Average yield=4.47 x io7GFP + colonies / pmol, Standard deviation=3.39 x io7GFP + colonies / pmol), . (Average accuracy=86.5%, Standard deviation=2.9%, Average yield=1.03 x 108GFP + colonies / pmol, Standard deviation=9.03 x io7GFP + colonies / pmol), T|. (Average accuracy=89.9%, Standard deviation=3.3%, Average yield=7.41 x io8GFP + colonies / pmol, Standard deviation=1.10 x io8GFP + colonies / pmol), 0. (Average accuracy=82.7%, Standard deviation=4.5%, Average yield=1.27 x 109GFP + colonies / pmol, Standard deviation=1.70 x 108GFP + colonies / pmol), positive control (Average accuracy=100%, Standard deviation=0%, Average yield= 1.42 x io10GFP + colonies / pmol, Standard deviation=9.98 x 109GFP + colonies / pmol). FIG. 1G. DNA agarose gel depicting IpL of PCR product comparing the Assembly Amplicon of the LuxABCDE cassette assembled at 22°C, 30°C, and 50°C. FIG. 1H. Coverage plot depicting the consensus sequence of the LuxABCDE Assembly Amplicon at different assembly temperatures.FIG. II. Bar plots depicting analysis of all possible fragment connections in the LuxABCDE Assembly Amplicon Nanopore sequencing data, comparing the number of correct and incorrect connections at 22°C, 30°C and 50°C. FIG. 1J. Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the Lux cassette 20-piece at 22°C (Average accuracy=9.8%, Standard deviation=2.28%, Average yield=2.17 x 105Lux + colonies / pmol, Standard deviation=2.10 x 105Lux + colonies / pmol) and 20-piece at 50°C (Average accuracy=76.4%, Standard deviation=5.0%, Average yield=3.48 x 105Lux + colonies / pmol, Standard deviation=8.97 x 104Lux + colonies / pmol) and 20-piece 50°C Assembly amplicon (Average accuracy=66.4%, Standard deviation=5.5%, Average yield=1.18 x 107Lux + colonies / pmol, Standard deviation=9.22 x 106Lux + colonies / pmol).

[0044] FIGS. 2A-2F depict non-limiting exemplary schematics and data related to HighT assembly coupled with two independent genome integration schemes. FIG. 2A.Schematic depicting circular HighT assembly genomically integrated using a tyrosine recombinase. LuxABCDE is divided into 9 fragments, which are HighT ligated to an integration backbone fragment containing two terminators, an attB sequence, and a transcriptionally inactive antibiotic resistance marker +1 (i). LuxABCDE is divided into 19 fragments, which are HighT ligated and amplified into an Assembly Amplicon that is subsequently assembled with the same integration backbone (ii). The final assembly product of both constructions (iii) are electroporated into recipient cells with a plasmid containing the attB integrase, antibiotic resistance marker +2 and a pre-engineered genome containing a promoter, attP site, transcriptionally active GFP, and antibiotic resistance marker +3 (iv). Through protein mediated DNA crossover (v) the recipient genome gains transcriptionally active antibiotic resistance marker +1, the cargo insert, and losses fluorescence expression in the post-integrated genome (vi). FIG. 2B. Phenotypic spot assay where pre-integration attBP recipient is compared to postintegration attBP cells on various growth media (+1 = kanamycin, +2 = carbenicillin, +3 = chloramphenicol). FIG. 2C. Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 10-piece scheme (i) Average accuracy=19.1%, Standard deviation=10.7%, Average yield=1.37 x 103Lux + colonies / pg DNA, Standard deviation= 5.51 x 102Lux + colonies / pg DNA) and the 19-piece Assembly Amplicon scheme (ii) (Average accuracy=30.6%, Standard deviation=2.8%, Average yield=1.43 x 104Lux + colonies / pg DNA, Standard deviation=1.33x 103Lux + colonies / pg DNA). FIG. 2D. Schematic depicting LoxP (LI) and Lox2272 (L2) recombination sites onto distal ends of integrated cargo to enable Cre recombinase mediated genome integration. FIG. 2E. Phenotypic spot assay where pre-integration LoxP recipient is compared to post-integration LoxP cells on various growth media (+1* = chloramphenicol, +2* = tetracycline, +3* = spectinomycin, -1* = sacB sucrose sensitivity). FIG. 2F. Transformation results for LoxP dz 10-piece assemblies (blue) relative to dzz 20-piece Assembly Amplicon (pink). Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for 3 biological replicates of the 10-piece scheme (i) (Average accuracy=58.6%, Standard deviation=5.1%, Average yield=1.22x 103Lux + colonies / pg DNA, Standard deviation=5.61x 102Lux + colonies / pg DNA) and the 19-piece Assembly Amplicon scheme (ii) (Average accuracy=76.6%, Standard deviation=4.5%, Averageyield=3.22* 103Lux + colonies / pg DNA, Standard deviation=2.68x 102Lux + colonies / pg DNA).

[0045] FIGS. 3A-3F depict non-limiting exemplary schematics and data related to low abundance human protein isoform variants constructed with HighT. FIG. 3A. Isoform splicing variant information can be isolated from cellular transcripts by reverse transcribing mRNA transcripts into a cDNA library where primers can selectively isolate genetic variants filled with SNPs (red lines). Alternatively specific exons can be amplified from gDNA and HighT assembled into the desired isoform. FIG. 3B. Coverage plot of cDNA MAPT amplicon reads aligned to MAPT genomic chromosomal location. FIG. 3C. Nanopore sequencing to compare isoform variation in amplicons of MAPT generated from human heart cDNA verse HighT assembly of gDNA amplicons. Target isoform denoted in blue, all isoforms or sequencing artifacts with >1% representation are given unique identification numbers. FIG. 3D.Comparison of isoform variation in amplicons of VEGFA generated from human heart cDNA compared to two different HighT assemblies, one from gDNA amplicon HighT and the second entirely from oligo based HighT assembly. FIG. 3E. Comparison of isoform variation in amplicons of BRCA1 generated from human heart cDNA verse a hybrid gDNA amplicon and oligo based HighT assembly. FIG. 3F. DNA agarose gel depicting 50 ng of either cDNA amplicon or HighT Assembly Amplicons from panels c-e for MAPT, VEGFA, and BRCA1.

[0046] FIGS. 4A-4E depict non-limiting exemplary schematics and data related to high fidelity assembly enabling in vitro hierarchical assembly of large and complex linear DNA constructs. FIG. 4A. Schematic depicting the de novo assembly pipeline integrating Sidewinder with HighT 2WJ assembly for in vitro hierarchical assembly of large constructs. FIG. 4B. Plot depicting the local GC content in a 20-base window across the genomic sequence of the Sonic Hedgehog gene (SHH) in the northern white rhino. The average GC content of the 10 Sidewinder assemblies is listed above the region in the plot. FIG. 4C. DNA agarose gel depicting 50 ng of the ten 1 kb Sidewinder assemblies post PCR amplification with dU incorporation. FIG. 4D. DNA agarose gel depicting 50 ng of the final 10 kb Assembly Amplicon of the genomic Sonic Hedgehog from northern white rhino assembled entirely de novo and in vitro. FIG. 4E. Nanopore sequence alignment of reads to expected 10-kB reference.

[0047] FIGS. 5A-5D depict non-limiting exemplary schematics and data related to oligo based assembly conducted via varying length overhangs. FIG. 5A. Oligos are designed to be annealed with a largely homologous region (dark grey) and an exposed 3’ overhang (light grey) of varying length 4 bp, 10 bp, and 25 bp. FIG. 5B. This is repeated for the requisite number of fragments that are then assembled and ligated at varying reaction temperatures. The assembly product is then used as the template for a subsequent PCR amplification. FIG. 5C.DNA agarose gel depicting the PCR product (1 pL loaded for all conditions) of a segment of the LuxABCDE cassette assembled with 5, 10, and 20-piece assemblies ligated at 16°C, 37°C, and 50°C for 25 bp overhangs. FIG. 5D. DNA agarose gel depicting the PCR product (1 pL loaded for all conditions) of a segment of the LuxABCDE cassette assembled with 5, 10, and 20-piece assemblies ligated at 75°C for 4 bp, 10 bp, and 25 bp overhangs.

[0048] FIGS. 6A-6B depict non-limiting exemplary schematics related to the mechanism and shortcomings of current USER cloning method. FIG. 6A. USER assembly generates overhangs by amplifying a target sequence with a terminal sequence of the form ANxT with a dU in place of the dT in the primers. The dU is targeted with USER enzyme to generate the 2WJ overhang for assembly. FIG. 6B. Unoptimized protocols can result in various byproducts which decrease the overall fidelity of assembly.

[0049] FIGS. 7A-7B depict non-limiting exemplary schematics and data related to how improvements to USER protocol are substantially burdened by secondary structure and crosstalk in overhangs. FIG. 7A. Schematic depicting the possible transformant genotypes after a 4-piece GFP assembly. The one permissible fragment encoding GFP does not correlate with cell survival while 3 required fragments encode for the backbone and are required for transformant survival on antibiotic selection. FIG. 7B. Plot depicting the transformation accuracy (y axis) vs yield (x axis) in colony forming units per pmol plasmid backbone transformed for the 4-piece GFP assembly conducted using overhangs not optimized for secondary structure at 22°C literature protocol under published conditions (grey), second digest site (blue), purification (green), and exonuclease digestion (yellow).

[0050] FIGS. 8A-8C depict data related to attBP and LoxP integrations occurring at expected recipient genomic location. FIG. 8A. PCR check with primers binding to genomic region flanking expected insert site near ^ / / / 'location in genome. Size of genomic insert increases from pre-integration 2.2 kB to post-integration 9.5 kB for attBP integration when checking 32 LUX+ colonies. FIG. 8B. Same genomic site comparing LoxP pre-integration 2.8 kB amplicon with post-integration 7.4 B amplicon for 32 LUX+ colonies. FIG. 8C. Reduction in colonies surviving when selecting against non-integrating plasmids. GFP+ chlor plasmids are transformed into LoxP pre-integration recipient strains containing the sacB negative selection gene. As the recipient genomic sacB locus is not lost with plasmid transformation, sucrose countersei ection can be used to reduce episomal transformants by over lOOx.

[0051] FIGS. 9A-9E depict non-limiting exemplary schematics and data related to how attBP and LoxP integration schemes can integrate with different orientations. FIG. 9A.Orientation of inserted genomic attP landing spot, in two different directions (square and triangle). Delivered constructs are either HighT 10-piece assemblies (blue), 19-piece assemblyamplicon ligated to backbone (pink), or PCR product from sequence verified template ligated to backbone (green). FIG. 9B. Integration efficiencies of the three constructs integrated into two different recipient cell orientations. FIG. 9C. Phenotypic spots of pre-integration attBP Recipients and post-integration cells with recipient landing sight oriented in two different directions. Resistance gened: +1 = Kanamycin, +2 = Carbenicillin, +3 = Chloramphenicol. FIG.9D. Cells with two different recipient LoxP / Lox2272 landing site orientations are transformed with products where the relative orientation of the distal LoxP = LI and Lox2272 = L2 are swapped. Delivered constructs are either HighT 10-piece assemblies (blue), 20-piece assembly amplicon (pink), or PCR product from sequence verified template (green) flanked by LI or L2 at distal ends. FIG. 9E. Integration efficiencies of the three different constructs sources, each tested independently with the two L1 / L2 or L2 / L1 orientations, and separately integrated in two different recipient cell orientations.

[0052] FIGS. 10A-10D depict non-limiting exemplary schematics related to how HighT can assemble precise isoforms using gDNA amplicons and Oligo Extension inputs. FIG.10A. Incorporation of Uracil nucleotides into gDNA amplicons can enable HighT fragment generation. FIG. 10B. Details of MAPT 14-exon location and 9 piece amplification scheme for MAPT isoform. FIG. 10C. PCR amplification of oligos used in Oligo Extension, USER digested and purified for HighT assembly. FIG. 10D. HighT assembly of 6 oligo extension products covers the entire VEGFA isoform coding sequence.

[0053] FIG. 11 depicts a non-limiting exemplary schematic related to how increased assembly fidelity enables more rapid in vitro assembly protocols. Sidewinder can enable a high fidelity for the proper connection that allows for highly pure in vitro intermediates to be generated and carried directly through for secondary rounds of heirarchical cloning. Alternative assembly techniques require an in vivo transformation in order to isolate correctly assembled clones prior to subsequent rounds of assembly.

[0054] FIGS. 12A-12C depict non-limiting exemplary schematics related to “Super USER” as a novel and improved method for DNA assembly. FIG. 12A. Classic USER design with 1 U in each primer and workflow. FIG. 12B. Intrinsic limitations of classic USER. FIG.12C. Super USER can solve the intrinsic limitations as a novel and improved DNA assembly method.

[0055] FIG. 13 depicts a non-limiting exemplary schematic related to introduction of an assembled product generated by the methods provided herein into an electrocompetent donor cell along with a conjugation plasmid for genome integration into a recipient cell.DETAILED DESCRIPTION

[0056] In the following detailed description, reference is made to the accompanyingdrawings, 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 may be 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.

[0057] 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

[0058] 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.

[0059] 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.

[0060] 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.”

[0061] 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 themolecule 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, IO’15M, or a number or a range between 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.

[0062] 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.

[0063] 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 can be 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.

[0064] 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.

[0065] 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, and those having inverted polarity wherein one or more intemucleotide linkages is a 3’ to 3’, a 5’ to 5’ or a 2’ to 2’ linkage.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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 withinthe 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.

[0070] 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).

[0071] As used herein, “melting temperature” or “Tm” of, e.g., a nucleic acid region (such as an overhang), 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 the intended reaction conditions.

[0072] 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.

[0073] 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 arenot 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.

[0074] 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.

[0075] 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.

[0076] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, 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.Super USER Cloning as a Novel Method of DNA Assembly

[0077] To generate longer DNA strands, multiple smaller DNA pieces need to be assembled in the correct order. Provided herein include novel DNA assembly techniques that can enable highly efficient, one-pot, sequence independent assembly of large numbers of DNA fragments.

[0078] The USER Cloning technique offers unique capabilities not found in any other DNA Assembly Technique. The USER Cloning technique relies on incorporating a deoxyuridine in the primers of assembly fragments in substitute for a deoxythymidine (FIG.12A i). Upon treatment with the USER enzyme mixture (Uracil DNA Glycosylase and a DNA glycosylase-lyase), a single nucleotide gap is generated in the double stranded DNA sequence, and a precise ssDNA overhang is generated (FIG. 12A ii,iii) Partnered assembly fragments can be subsequently joined by annealing with their ssDNA complement in solution (FIG. 12A iv,v).The USER cloning technique offers a unique capability over any other DNA assembly technique in that the start of the ssDNA overhang is precisely generated by the location of the deoxyuridine. Techniques that rely on imprecise overhang generation, such as Gibson Assembly, rely on non-specific degradation by an exonuclease to expose ssDNA overhangs of varying sequence and length. Alternatively, techniques that rely on precise overhang generation, such as Golden Gate Assembly, require restriction enzymes which put strict sequence limitations on the DNA fragments being assembled. As a result, the USER Cloning Technique is a favorable alternative to other assembly techniques due to its precise control overhang generation without any sequence restrictions in the DNA fragments being assembled. Unfortunately, the current USER Cloning technique is limited to 4-5 DNA fragments assembled with high efficiency, leaving the technique scarcely used.

[0079] The USER Cloning technique’s assembly accuracy can be limited by waste products. Although the sequence location of the ssDNA overhang generated with USER is precise, the overhang is not guaranteed to participate in assembly. With the current USER Cloning protocol, USER treatment results in the formation of a ssDNA waste product which may still bind its complement on the assembly fragment (FIG. 12B i). Additionally, even when ssDNA overhangs are properly exposed, they may have secondary structures (2° structures) which limit the reactivity of the exposed ssDNA overhang (FIG. 12B ii). The presence of either or both types of these unfavorable byproducts in the reaction mixture compete against the binding of the ssDNA overhang with the preferred assembly fragment, decreasing assembly accuracy and yield, thus limiting the number of fragments which can be assembled with the USER Cloning technique (FIG. 12B iii). The USER Cloning strategy can be optimized for improved performance by addressing these limitations and guaranteeing the presence of exposedssDNA overhangs.

[0080] Provided herein include methods, compositions, systems, and kits related an improved USER Cloning Technique (Super USER) which offers vastly improved ssDNA overhang generation using three novel innovations. First, Super USER can incorporate 2 (or more) deoxyuridine bases in the primers. By adding an additional deoxyuridine base in the primers, upon USER treatment to the sample, the waste fragment can be split in half. This can greatly decrease the affinity of the waste fragment for the overhang at higher temperatures (FIG.12C i). As a result, the assembly of DNA fragments with their proper partner can be heavily favored due to the greatly increased binding affinity between assembly fragments compared to their waste product. This can in principle be further expanded to multiple deoxyuridine bases in the primers following the same principle, while 2 deoxyuridine bases per primer is a good balance of ease of choosing overlapping regions and effects of improvements in some embodiments. Second, in some embodiments Super USER comprises the removal of waste fragments. In addition to reducing the binding affinity of the waste fragments, in some embodiments Super USER subsequently removes the waste fragments from the reaction mixture to further guarantee the presence of ssDNA overhangs that participate in assembly. This can be accomplished through purification and with enzymatic activity. After USER treatment, the DNA samples can passed through a size exclusion column such that the smaller waste fragments are separated from the larger assembly fragments (FIG. 12C ii). Alternatively, the DNA samples can be treated with a 5’ ssDNA exonuclease which will only act on free waste fragments in solution (FIG. 12C ii) Both these techniques for waste fragment removal eliminate the waste fragments from solution and work to guarantee the presence of exposed ssDNA overhangs, favoring the proper assembly of DNA fragments. Third, in some embodiments, Super USER optimizes the sequence of the generated ssDNA overhang. Since the USER cloning technique offers precise control over the length and the location of the ssDNA overhang being generated, these sequences can be optimized to allow greatly increased assembly efficiency. Thanks to the next generation of oligo confirmation prediction technology such as NUPACK, a user may optimize the ssDNA overhangs to minimize cross reactivity between non homologous ends and eliminate secondary structure in ssDNA overhangs (FIG. 12C iii).

[0081] Each of these strategies on their own can offer improvements to successful DNA assembly yield and accuracy on their own. When combined, they can offer substantial improvements over the current leading USER Cloning by favoring the assembly between proper DNA fragment partners, increasing assembly accuracy and yield (FIG. 12C iv). Super USER’S defined and optimized overhang sequences can allow for increased assembly accuracy for large number of fragments as with Golden-Gate, while maintaining the flexibility by having anoverabundance of potential assembly junctions in any given construct.

[0082] Provided herein, in some embodiments, are methods. The 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.

[0083] The non-canonical nucleotide(s) can comprise deoxyuridine, deoxyinosine, deoxy-7-methylguanosine, deoxy-5,6-dihydroxythymidine, deoxy-3 -methyladenosine, 5-methyl-deoxy cytidine, O-6-methyl-deoxy guanosine, 5-iodo-deoxyuridine, 8-oxy-deoxy guanine, 1,N6-ethenoadenine, 8-oxo-guanine (8oxoG), or any combination thereof. The non-canonical nucleotide(s) can comprise deoxyuridine. The cleavage agent(s) can comprise DNA glycosylase-lyase Endonuclease VIII, a DNA glycosylase, an AP cleaving agent, APE 1 (AP Endonuclease 1), Endo III (Endonuclease III), Endo IV (Endonuclease IV), Endo V (Endonuclease V), Endo VIII (Endonuclease VIII), Fpg (formamido-pyrimidine-DNA glycosylase), OGGI (8-oxoguanine DNA glycosylase 1), NEIL1 (Endonuclease Vlll-like 1), T7 Endo I (T7 Endonuclease I), T4 PDG (T4 pyrimidine dimer DNA glycosylase), UDG (uracil DNA glycosylase), SMUG1 (Single-strand selective monofunctional uracil DNA glycosylase), AAG (methylpurine DNA glycosylase), or any combination thereof. The contacting step can be performed in the presence of a 5’ ssDNA exonuclease configured to remove waste products derived from the 5’ terminal cleavage regions, optionally selected from the group comprising Red, RecJF, Exo VII, Dna2, T5 Exo, or any combination thereof. The 5’ terminal cleavage region can comprise 1, 2, 3, 4, or 5 non-canonical nucleotide(s). The step of providing m assembly precursors can comprise PCR amplification reaction(s) comprising precursor primer(s) having a 5’ terminal cleavage region(s) comprising said non-canonical nucleotide(s), optionally m sets of precursor primer(s). The cleavage agent(s) can comprise USER Enzyme, USER II Enzyme, and / or USER III Enzyme. Each set of precursor primer(s) can comprise a forward primer and a forward primer configured to amplify an assembly precursor, and the forward primer and / or reverse primer can have a 5’ terminal cleavage region(s) comprising said non-canonical nucleotide(s).

[0084] At least two of the m assembly precursors can be contacted with the one or more cleavage agents in separate reaction vessels. At least two of the m assembly precursors can be contacted with the one or more cleavage agents in a common reaction mixture. The method can comprise purifying the assembly precursors and / or assembly fragments, optionally one or more purification steps comprising: gel electrophoresis, optionally 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, optionally size exclusion column purification.

[0085] The 3’ overhang can comprise the sequence ANxWNyWNzT, wherein x, y, and z are positive integers. In some embodiments, the 3’ overhang exhibits no secondary structure (NSS), optionally at 30°C. The 3’ overhang can be optimized to minimize cross reactivity with non-complementary overhangs. The 3’ overhang can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or a number or a range between any two of these values, nucleotides in length. The 3’ overhang can comprise 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%, or a number or a range between any two of these values. The 3’ overhang can comprise a melting temperature (Tm) of about 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, 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, or a number or a range between any two of these values.

[0086] The contacting step can be performed at a first incubation temperature for a first period of time. The first incubation temperature can be about 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 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, or a number or a range between any two of these values, optionally 37°C. The first period of time can be about 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, optionally one hour.

[0087] One or more of the m assembly precursors can comprise an Uracil PrimerDimer Overhang Generation (UPDOG) product. Providing the m assembly precursors can comprise providing a first UPDOG primer and a second UPDOG primer capable of hybridizing each other, wherein the first UPDOG primer and second UPDOG primer each comprise 5’ terminal cleavage region(s) comprising two or more non-canonical nucleotide(s); and contacting the first UPDOG primer and the second UPDOG primer in the presence of a polymerase to generate the UPDOG product.

[0088] The method can comprise: providing n assembly fragments, wherein n is an integer greater than 1. At least two of the n assembly fragments can comprise or can be derived from the m assembly fragments generated by a method disclosed herein. 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, thereby generating an assembled product.

[0089] Provided herein, in some embodiments, are methods. The 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.

[0090] The method can comprise: generating one or more of the n assembly fragments according to a method disclosed herein. In some embodiments, contacting the m assembly precursors with the cleavage agent(s) and incubating the n assembly fragments in the presence of the ligase are performed simultaneously. The assembly fragments can comprise or can be derived from Golden Gate assembly fragments, optionally the method further can comprise contacting assembly precursors with Type-IIS and / or Type-IIP cleaving agents. 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. The method can comprise one or more Dpnl digestion steps.

[0091] The ligase can be capable of ligating nicks at junctions of hybridized assembly fragments to generate the assembled product. The ligase can be selected from the group comprising a thermostable ligase 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.

[0092] The assembly fragment can be at least 10, 25, 50, 75, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, or a number or a range between any two of these values, nucleotides in length. The assembly fragment can comprise 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%, or a number or a range between any two of these values. The assembly fragment can comprise a melting temperature (Tm) of about 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, 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, or a number or a range between any two of these values.

[0093] The incubating step can be performed at second incubation temperature for a second period of time. The second incubation temperature can be about 8°C, 9°C, 10°C, 11 °C, 12°C, 13°C, 14°C, 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, 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, 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, or a number or a range between any two of these values, optionally 50°C. The second period of time can be about 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, or a number or a range between any two of these values, optionally 16 hr. The second incubation temperature can be at least about 0.5°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 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, or a number or a range between any two of these values, higher than the median 3’ overhang Tm.

[0094] The integers n or m 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.

[0095] In some embodiments, n is 3 or more, and the assembled product is linear. The first assembly fragment of the n assembly fragments can comprise a second 3’ overhang. Each (z)th assembly fragment of the n assembly fragments can comprise a first 3’ overhang and a second 3’ overhang, wherein 1 < z < n. The (zz)th assembly of the n assembly fragments fragment can comprise a first 3’ overhang. The first 3’ overhang of each (z)th assembly fragment can be complementary to the second 3’ overhang of the (z-l)th assembly fragment. The second 3’ overhang of each (z)th assembly fragment can be complementary to the first 3’ overhang of the (z+1 )th assembly fragment.

[0096] In some embodiments, n is 3 or more, and the assembled product is circular. Each assembly fragment of the n assembly fragments can comprise a first 3’ overhang and a second 3’ overhang. The first 3’ overhang of the first assembly fragment of the n assembly fragments can be complementary to the second 3’ overhang of the (zz)th assembly fragment of the n assembly fragments.

[0097] In some embodiments, for each (z)th assembly fragment, wherein 1 < z < n the first 3’ overhang of the (z)th assembly fragment is complementary to the second 3’ overhang of the (z-l)th assembly fragment, and the second 3’ overhang of the (z)th assembly fragment is complementary to the first 3’ overhang of the (z+l)th assembly fragment. The first assembly fragment of the n assembly fragments can comprise a first terminal region, optionally a 5’ terminal region. The (zz)th assembly fragment of the n assembly fragments can comprise a second terminal region, optionally a 3’ terminal region. In some embodiments, one or more of the n assembly fragments comprise an internal segment, wherein the internal segment does not comprise the 3’ overhang(s), and wherein the internal segment is double-stranded.

[0098] In some embodiments, the assembly precursors and / or the assembly fragments comprise or are derived from synthetic oligonucleotides. In some embodiments, the assembly precursors and / or the assembly fragments comprise or are derived from Sidewinder products, TADA products, rolling circle amplification products, restriction enzyme digestion products, reverse transcription products, CRISPR-excised products, PCR amplification products, template-independent polymerase products, recombinase-generated products, phage-derived products, or any combination thereof.

[0099] In some embodiments, the method further comprises PCR amplification of the assembled product, or a product thereof, to generate an amplified product. PCRamplification 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. PCR amplification can comprise PCR primer(s) having an overhang, and wherein the final synthetic sequence can comprise the sequence of said overhang.

[0100] The method can comprise purification of the assembled product, the amplified product, or products thereof. In some embodiments, said purification step compromises: 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.

[0101] The assembled product, the amplified product, or products thereof, can comprise a final synthetic sequence, and the final synthetic sequence can be at least about 500 bp, 750 bp, 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 final synthetic sequence can comprise 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. The final synthetic sequence can comprise 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. The final synthetic sequence can comprise 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. The final synthetic sequence can comprise 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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 ofcytokines 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).

[0106] 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.

[0107] 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 group comprising 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, areceptor 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.

[0108] 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, 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. The one or more payload proteins can comprise components of a synthetic proteincircuit, 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.

[0109] In some embodiments, a payload gene encodes an isoform of a payload protein comprising two or more exons, optionally a therapeutically relevant isoform. In some embodiments, providing n assembly fragments comprises: PCR amplification of one or more one exons from genomic DNA, optionally patient-derived genomic DNA. At least one of the two or more exons can be encoded in the 5’ end of an amplification primer. At least one assembly fragment of the n assembly fragments can be an UPDOG product comprising two or more exons.

[0110] In some embodiments, one or more of the payload gene(s) comprise: (a) a 5’UTR and / or a 3’UTR; (b) 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 (c) 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.[OHl] At least one of the payload genes can be operably connected to a promoter selected from the group comprising: (a) an RNA pol I promoter; (b) a pol II promoter, optionally CMV, SV40 early region or adenovirus major late promoter; (c) pol III promoter, optionally a U6 or Hl promoter; (d) a minimal promoter, optionally TATA, miniCMV, and / or miniPromo; (e) a bacteriophage promoter, optionally a bacteriophage T3 promoter, a bacteriophage T7 promoter, a bacteriophage SP6 promoter, or a combination thereof; (f) a tissue-specific promoter and / or a lineage-specific promoter; (g) 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-shockpromoter; (h) 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.

[0112] The method can comprise replication of the assembled product, the amplified product, or products thereof, in a cell, optionally episomal replicons. The method can comprise delivery of the assembled product, the amplified product, or products thereof, to a cell. Delivery can comprise one or more of transformation, transfection, transduction, conjugation, electroporation, lipid-mediated transfection, liposome-mediated delivery, nanoparticle-mediated delivery, viral vector-mediated delivery, microinjection, biolistic delivery, calcium phosphate-mediated transfection, PEG-mediated transformation or transfection. The method can comprise conjugative transfer from a donor cell to a recipient cell.

[0113] The final synthetic sequence can be or can comprise all or a portion of a vector, The vector can comprise a viral vector, a plasmid, a transposable element, a naked DNA vector, or any combination thereof. The vector 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.

[0114] 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 syntheticsequence 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.

[0115] The method can comprise integration of the final synthetic sequence, or a portion thereof, into the genome of a cell, optionally via a site-specific recombinase. The sitespecific recombinase can be a tyrosine recombinase, optionally selected from the group comprising HK022 recombinase, Cre, lambda phage, phage 186, phi80, P21, Flp, XerC / D, XerA, lambda integrase (Int), P2 integrase, FimB, FimE, HbiF, Rci, or any combination thereof. The site-specific recombinase can be a serine recombinase, optionally selected from the group comprising Bxbl, Tn3, y6 resolvase, Gin, Hin, TP901-1, C31, TGI, Rvl, C.IS607-like serine transposases, or any combination thereof. The method can comprise integration without episomal replicon intermediate(s).

[0116] 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 can comprise 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 HIE 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.

[0117] The assembled product, the amplified product, or product thereof, can comprise a circular DNA molecule comprising: a recombination site, optionally an attB recombination site; terminator(s); and / or a transcriptionally inactive first selection marker gene configured to be transcriptionally active upon correct integration. The method can comprise introducing the circular DNA molecule into a recipient cell comprising: (a) a recombinase protein, optionally a tyrosine recombinase; and (b) an integration site comprising a complementary recombination site, optionally an attP recombination site. The integration site can comprise an upstream constitutive promoter and / or a transcriptionally active second selection marker gene. In some embodiments, the terminator(s) render the second selection marker gene transcriptionally inactive upon correct integration. The introduction into the recipient cell can comprise conjugative transfer from a donor cell.

[0118] The assembled product, the amplified product, or product thereof, can comprise a linear DNA molecule comprising: (a) a first recombination site and a second recombination site, optionally situated on the 5’ and 3’ terminal ends, respectively, further optionally LoxP (LI) and Lox2272 (L2) recombination sites; and / or (b) a first selection marker gene. The method can comprise introducing the linear DNA molecule into a recipient cell comprising: (a) a recombinase protein, optionally a serine recombinase; and (b) an integration site comprising the first recombination site and the second recombination site, optionally LoxP (LI) and Lox2272 (L2) recombination sites. The integration site can comprise one or more selection marker genes situated between the first recombination site and the second recombination site, The introduction into the recipient cell can comprise conjugative transfer from a donor cell.

[0119] The first assembly fragment can be an invariant fragment, wherein all instances of the invariant first assembly fragment are identical. The first assembly fragment can be a variant fragment, wherein two or more instances of the variant first assembly fragment differ with respect to the sequence of the internal segment. At least one (z)th assembly fragment can be an invariant fragment, wherein all instances of the invariant (z)th assembly fragment are identical. At least one (z)th assembly fragment can be a variant fragment, wherein two or more instances of the variant (z)th assembly fragment differ with respect to the sequence of the internal segment. The (zz)th assembly fragment can be an invariant fragment, wherein all instances of the invariant (zz)th assembly fragment are identical. The (zz)th assembly fragment can be a variant fragment, wherein two or more instances of the variant (zz)th assembly fragment differ with respect to the sequence of the internal segment.

[0120] Variant fragments can comprise predefined codon variations, optionally codons variations configured to achieve modified and / or improved protein function(s). At least one of the n assembly 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, 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, 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%, or 0.01%, from the theoretical proportion of occurrence for that codon.

[0121] 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 assembly 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 assembly 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 assembly fragments or can comprise a mis-assembled junction. The mis-ligation rate at a junction 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. 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, 1000-fold, or a number or a range between any two of these values, greater than the yield of a polynucleotide assembly method not comprising a ligation temperature higher than the median melting temperature of the 3’ overhangs, 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 assembly fragments become a component of an assembled product.

[0122] Provided herein, in some embodiments, are compositions. The composition can 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 nucleic acid comprising the final synthetic sequence, or a portion thereof. The composition can comprise a cell comprising the final synthetic sequence, or a portion thereof.

[0123] 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: (a) 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; (b) 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; (c) an infectious agent, an anti-infectious agent, or a mixture thereof; (d) a cytotoxic agent, optionally a chemotherapeutic agent, a biologic agent, a toxin, a radioactive isotope, or any combination thereof; and / or (e) 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. 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 sequence verification and subsequently eukaryotic cells for expression, optionally mammalian, yeast, insect, plant, or fungal cells.

[0124] Provided herein, in some embodiments, are system for synthesizing nucleic acids. Provided herein, in some embodiments, are kits. The system or kit can comprise: one or more of the m assembly precursors, m sets of precursor primer(s), and n assembly fragments disclosed herein. The system or kit can comprise: one or more cleavage agent(s), optionally DNA glycosylase-lyase Endonuclease VIII, a DNA glycosylase, an AP cleaving agent, APE 1 (AP Endonuclease 1), Endo III (Endonuclease III), Endo IV (Endonuclease IV), Endo V (Endonuclease V), Endo VIII (Endonuclease VIII), Fpg (formamido-pyrimidine-DNA glycosylase), OGGI (8-oxoguanine DNA glycosylase 1), NEIL1 (Endonuclease Vlll-like 1), T7 Endo I (T7 Endonuclease I), T4 PDG (T4 pyrimidine dimer DNA glycosylase), UDG (uracil DNA glycosylase), SMUG1 (Single-strand selective monofunctional uracil DNA glycosylase), AAG (methylpurine DNA glycosylase), or any combination thereof. The system or kit can comprise: a non-thermostable ligase, a thermostable ligase, 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); 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). 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 kit or 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.TADA

[0125] The systems, methods, compositions, and kits provided herein can, in some embodiments, be employed in concert with the systems, methods, compositions, and kits for TADA-based DNA assembly described in U.S. Provisional Patent Application Ser. No.63 / 777,905, entitled, “Toehold Assisted DNA Assembly (TADA) as a Novel Method of DNA Assembly,” filed March 26, 2025, and in a PCT Application entitled, “Toehold Assisted DNA Assembly (TADA) as a Novel Method of DNA Assembly,” filed March 25, 2026, the contents of which are incorporated herein by reference in their entireties. The assembly precursors and / or the assembly fragments can comprise or can be derived from TADA products. In some embodiments the assembled products, or products thereof, generated by methods disclosed herein can be used as inputs for further assembly using TADA-based DNA assembly methods.Linear TADA

[0126] 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 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. 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 (< / )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 maskingoligonucleotide is complementary to the masking toehold of the (z / )th downstream masking oligonucleotide.

[0127] 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

[0128] 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 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, 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 n 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 (zy+ljth 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.

[0129] 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.TADA Methods

[0130] 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, 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.

[0131] 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 thefragment 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.Sidewinder

[0132] 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 JUNCTION DNA ASSEMBLY,” filed January 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. The assembly precursors and / or the assembly fragments can comprise or can be derived from Sidewinder products. In some embodiments the assembled products, or products thereof, generated by methods disclosed herein can be used as inputs for further assembly using Sidewinder-based DNA assembly methods.

[0133] 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 firstterminal 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 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 methods, compositions, systems, and kits provided herein can comprise the generation of a linear product.

[0134] 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.

[0135] 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 (Zr)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.

[0136] 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 linkage between 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 notcomprise 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 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.EXAMPLES

[0137] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure.Example 1High Temperature Ligation Enables in vitro Assembly of Eukaryotic Genes

[0138] The ability to efficiently construct synthetic DNA products is essential to studying and engineering biology. There is an abundance of widely adopted DNA assembly technologies suitable for generating constructs from diverse DNA inputs, but these techniques suffer from low specificity of connection during assembly, which decreases the accuracy, efficiency, and throughput of constructing synthetic DNA. Herein is provided a new paradigm for DNA assembly where ligation of single strand DNA complementary overhangs at reaction temperatures significantly higher than the melting temperature of the cohesive sticky ends results in lower mis-assembly rates. Herein is provided High Temperature (HighT) assembly that when adapted to conventional assembly technologies for plasmid assembly, improves accuracy by 40 percentage points and increases correct transformants by 4 orders of magnitude. HighT assembly principles were applied to develop cloning pipelines for the development of efficient genomic assembly-to-integration protocols, the construction of specific low-abundance isoforms of medically relevant proteins from oligos and human gDNA, and the production of a 10-kb GC-rich genomic region from the almost extinct Northern White Rhino created entirely de novo and in vitro.Introduction

[0139] The contemporary study of biology is shaped by our understanding of the relationship between DNA molecules and their corresponding functions. Synthetic DNA iscrucial to these investigations as it enables us to precisely control the information encoded in engineered constructs to more systematically probe the correlation between genotype and phenotype. Long synthetic DNA molecules are constructed by assembling smaller DNA fragments derived from chemically or enzymatically synthesized oligos, or sourced from preexisting DNA templates such as bacterial plasmids, genomic DNA (gDNA), or complementary DNA (cDNA). Through a combination of these diverse inputs, a user can cover the theoretical sequence space required for bioengineering of diverse systems through the efficient construction of DNA molecules.

[0140] The ability to construct DNA polymers from any source is intrinsically limited by the misconnection rate between DNA fragments during assembly - a problem compounded by an increasing number of assembly junctions. A novel DNA assembly technique, Sidewinder, was previously developed to address this misconnection issue and enable the assembly of large numbers of fragments into complex DNA constructs. Sidewinder assembly is directed by highly optimized barcode sequences not present in the final construct via a 3 -Way Junction (3WJ) intermediate, enabling unprecedented specificity of 1 in 1,000,000 misconnection rate.

[0141] Expanding beyond reducing misconnection rates via the 3WJ, provided herein include methods and compositions to improve specificity for 2-Way Junction (2WJ) approaches. The 2WJ suffers from higher misconnection rates since the junction information which directs assembly is constrained to the sequence space of the final synthetic construct. This leads to unoptimized assemblies where exposed single strand DNA (ssDNA) overhangs can form secondary structures, mis-associate, and mis-ligate (FIG. 1A). To increase reaction specificity while operating within 2WJ sequence constraints, it was observed that previous 2WJ techniques share a common feature where annealing and ligation reactions typically occur at temperatures at or below the melting temperatures (Tm) of the fragment overhangs. Contrasting with this existing paradigm, provided herein is High Temperature (HighT) ligation, defined by ligation at temperatures significantly higher than the melting temperature of assembly overhangs. While this was previously perceived to be an unfavorable condition, it was hypothesized HighT may increase the stringency of 2WJ connection rate (FIG. 1A) to enable larger and more robust assemblies with reduced incorrect byproducts.

[0142] First it was validated that HighT ligations enable more robust 2WJ assembly. Then the 2WJ design and construction framework was systematically optimized. This was accomplished with the nucleic acid design software NUPACK to computationally design ssDNA overhangs to reduce self-reactive secondary structure formation and reduced cross-reactivity with all other ssDNA overhangs present in the HighT reaction. Using a range of input DNAincluding plasmids, gDNA, and oligo assemblies, enzymatic processing and DNA purification conditions were then optimized to synergize with HighT assembly. Furthermore, the improved assembly conditions enable direct PCR amplification from HighT reactions that can be used in subsequent HighT rounds for in vitro hieratically assembly of larger constructs without the need for in vivo intermediates.

[0143] This Example demonstrates that the increased specificity afforded by HighT can improve conventional assembly technology accuracy and enables over 10,000* increase correct transformants for multi-fragment assemblies. The improvement in accuracy with HighT enables rapid one-step assembly-to-integration protocols, demonstrated by 10-piece HighT assemblies and HighT amplicons being directly cloned, transformed, and recombined into bacterial genomes through two different integration mechanisms. Next, this Example demonstrates a new paradigm enabled by HighT to precisely construct specific low-abundance isoforms of eukaryotic genes from gDNA templates as opposed to cloning from cDNA or de novo synthesis. Lastly, 3WJ Sidewinder assembly was combine with the 2WJ HighT pipeline to construct 10-kb, GC-rich linear dsDNA encoding the genomic region from the nearly extinct Northern White Rhino created entirely de novo and in vitro.ResultsHighT enables high fidelity ligations

[0144] For ligation-based assembly methods, the conventional consideration has been of the balance between enzyme activity and the stability of the interaction between the overhangs being ligated. These two factors seemingly conflict as a higher reaction temperature increases ligase activity while destabilizing interactions between overhangs whereas a lower reaction temperature decreases ligase activity while stabilizing interactions between overhangs. Prior literature demonstrations have generally decided in favor of lower temperature ligations, prioritizing overhang stability at the cost of decreased enzyme activity. However, in addition to stabilizing intended overhang interactions, lower temperatures will also stabilize all the unintended DNA interactions previously described (FIG. 1A).

[0145] It was hypothesized that high ligation temperatures significantly above overhang Tm, in addition to increasing enzyme activity, may increase the specificity of assembly such that assembly conditions should favor high temperature ligations for more accurate and robust assemblies (FIG. 1A). There is literature that suggests that ligation significantly above overhang Tmmay be possible even with particularly short overhang sequences. Provided herein include methods comprising High Temperature (HighT) assembly to increase specificity of overhang interactions and increase ligase activity for more robust 2WJ assembly.

[0146] To rapidly and systematically test multiple ligation temperatures, an oligo-based assembly (FIGS. 5A-5B) was performed to separate the ligation from any digestion required for exposing ssDNA overhangs, allowing a user to more directly test the effect of temperature on ligation independent from the prior overhang processing steps. Assemblies composing a segment of the LuxABCDE bioluminescence cassette were conducted at ligation temperatures 16°C, 37°C, 50°C, and 75°C. This was done for overhangs of 4 bases (analogous to Golden Gate assembly), 10 bases (analogous to USER or SLIC assembly), and 25 bases (analogous to Gibson assembly) for increasing reaction sizes of 5-pieces, 10-pieces, and 20-pieces. A PCR amplification of the final assembly product is used as a qualitative read out of assembly accuracy and yield (FIG. IB, FIG. 5B).

[0147] At 16°C, evidence was seen of mis-assembly via the absence of the target band or presence of multiple shorter products for each 4 bp, 10 bp, and 25 bp overhang length, particularly for the larger multi-fragment assemblies (FIG. 1C, FIG. 5C). Improved quality of assembly was observed when conducting the assembly using the same fragments at 37°C and 50°C for all overhang lengths (FIG. 1C, FIG. 5C). By plotting the distribution of the predicted overhang Tmit was seen that HighT ligation enables efficient multi -fragment assembly even when the ligation temperature is significantly above the median overhang T m (FIG. ID) Even at exceedingly high 75°C ligation, successful assembly up to 5-pieces for 4 bp overhang and 10-pieces for 10 bp and 25 bp overhangs was seen (FIG. 5D). These data suggest that 50°C may be a more optimal ligation temperature to increase assembly accuracy without sacrificing product yield.

[0148] Although improved assembly specificity was qualitatively observed assessed by the intensity of the clean agarose gel band, it is possible that the final PCR step conducted here may have boosted the yield of the reaction beyond what may be seen with direct cloning, particularly for the shorter overhangs. In order to challenge this possibility, a 4-piece USER assembly was designed for a green fluorescent protein (GFP) plasmid where assembly accuracy can be determined by the presence of the green-fluorescence (GFP+) phenotype (FIGS. 6A-6B, FIG. 7A). The assembly fragments were obtained via PCR and they were processed according to a published protocol for USER assembly which utilizes -8-12 base overhangs for room temperature (22°C) ligation (FIGS. 6A-6B). Next assembly conditions with HighT and a series of additional modifications to the assembly protocol were then systematically tested in order to improve assembly accuracy and yield (FIG. IE).

[0149] The standard literature protocol with 22°C ligation results in an average accuracy of 50.0% GFP+ colonies with a yield of 2.01 x 105GFP+ colonies / pmol plasmid transformed. This was then compared to HighT ligation at 50°C which results in comparable accuracy of 51.2% GFP+ clones but notably, results in an order of magnitude increase in yield to2.79 x 106GFP+ colonies / pmol (FIG. IF a).

[0150] These data support the HighT hypothesis by demonstrating that increased ligation temperature above the overhang Tmnot only yields significant product but also offers improved assembly for both oligo-based and PCR-based assembly methods.Optimization of 2WJ PCR-based assembly

[0151] It was hypothesized that continued improvements to 2WJ techniques can occur by further increasing the specificity of overhang interactions and decreasing the likelihood of DNA fragments associating with an unintended assembly partner. Towards this end, a series of improvements was developed and applied to the 4-piece GFP HighT assembly protocol. These improvements all work to bias the overhangs generated for the assembly to be exposed, mutually exclusive sequences free from secondary structure to improve intended assembly partner ligation (FIG. IE). This includes: (i) optimizing overhang sequences to be free from secondary structures or cross reactivity, (ii) additional digestion sites for exposing overhangs, (iii) purification of digested fragments, (iv) enzymatic digestion of waste, in addition to (v) HighT ligation.

[0152] First, the disclosed 4-piece assembly was redesigned to ensure that the USER overhang sequences were free from secondary structure at 30°C (i.e. the average Tmof the overhangs) or above by using the NUPACK web browser (Methods). NUPACK was also used to assess all exposed ssDNA overhangs participating in the reaction to test for crosstalk between non-partnered assembly fragments. With the redesigned no secondary structure (NSS) overhangs there was an ability to increase the average accuracy of the assembly from 50.0% to 91.8% and increase efficiency from 2.01 x io5GFP+ colonies / pmol to 1.82 x 106GFP+ colonies / pmol at 22°C (FIG. IF P). It was found that the NSS overhangs substantially enhanced the improvements to accuracy and yield afforded by the subsequent protocol modifications compared to when secondary structure is present (FIG. 7B). This suggests that the optimization of overhang sequences is an essential factor to consider for successful USER assembly.

[0153] The subsequent modifications to the protocol were all shown using the disclosed improved NSS overhangs, as each offers additional advantages over this new established baseline by ensuring more exposed ssDNA overhang participate in the assembly process. These modifications and their resultant improvements were: including two digestion sites (FIG. IF y 88.3% fluorescent with 5.61 x 106GFP + colonies / pmol), purification of digested product (FIG. IF 8, 96.7% fluorescence with 3.14 x 106GFP + colonies / pmol), exonuclease digestion of residual waste (FIG. IF e, 88.7% fluorescence with 4.47 x io7GFP + colonies / pmol), and lastly HighT assembly (FIG. IF , 86.5% fluorescence with 1.03 x 108GFP + colonies / pmol).

[0154] Next an additional assembly condition was tested that combines all modifications into a single reaction, which when conducted at 30°C ligation resulted in an accuracy of 89.9% and an efficiency of 7.41 x 108GFP + colonies / pmol (FIG. IF T|) and at 50°C ligation resulted in an accuracy of 82.7% and efficiency of 1.27 x 109GFP + colonies / pmol (FIG. IF 0). Overall, the combination of these optimizations can result in up to a 40-percentage point increase in accuracy compared to standard USER, while also resulting in nearly 10,000x increased yield of correct clones. These reaction conditions also enable a user to approach the theoretical maximum accuracy and yield for this construct, as demonstrated by the positive control of a nicked miniprep plasmid (FIG. IF).

[0155] In addition to direct cloning, it was hypothesized that the improvements to 2WJ assembly may enable a PCR to be conducted using the assembly reaction as template, resulting in an “Assembly Amplicon” (FIG. IB). Due to PCR’s bias towards amplification of smaller products it was hypothesized other 2WJ protocols do not implement PCR of the assembled product since any mis-ligations resulting in truncated product may be preferentially amplified, preventing amplification of the full-length target product. However, the improvements put forward to 2WJ assembly may substantially reduce the presence of truncated products in the assembly reaction, enabling preferential amplification of full-length assembly product in some embodiments. PCR of an Assembly Amplicon can allow for entirely in vitro selection of the properly assembled final product, increasing the yield and enabling the possibility of in vitro hierarchical cloning with multiple rounds of assembly without the need for cellular intermediates.

[0156] To demonstrate Assembly Amplicons enabled by HighT, a 20-piece assembly was designed with one required fragment containing the origin of replication and the antibiotic resistance gene while the 6.1 kb LuxABCDE cassette was split into 19 permissible fragments. Using the combined improvements to the 2WJ protocol, ligations were conducted at 22°C, 30°C, and 50°C and PCR amplified the Lux cassette. Only the 50°C protocol produces a single strong band at the correct size corresponding to the Assembly Amplicon of the 19 permissible PCR-based fragments of the Lux cassette (FIG. 1G). The assemblies conducted at lower ligation temperatures produce amplicons of the incorrect size (FIG. 1G).

[0157] Nanopore sequencing of the Assembly Amplicons allows insight into the identities of the final products. When aligned to the target 6.1kb Lux sequence, the 50°C Assembly Amplicon has consistent coverage spanning the entire Lux cassette with each fragment in the correct order and correct orientation (FIG. 1H). In contrast, the 22°C and 30°C have inconsistent coverage, limited full length final product, and precipitous drops in coverage consistent with mis-assemblies between fragments 1 & 9 at 22°C and mis-assemblies betweenfragments 2 & 12 and 1 & 15 at 30°C (FIG. 1H). The sequencing data was then checked for all possible fragment connections to check for correctly or incorrectly ligated assemblies. The 22°C and 30°C Assembly Amplicons had a junction mis-connection rate of approximately 1 in 11 (91.1% accurate) and 1 in 15 (93.1% accurate) while 50°C had a reduced misconnection rate to approximately 1 in 140 (99.3% accurate) (FIG. II).

[0158] The 20-piece Lux assembly was also transformed under different assembly conditions to compare yield and accuracy both with and without HighT and Assembly Amplicon. With a published ligation protocol at 22°C (room temperature) in addition to disclosed 2WJ modifications, it was shown one is able to successfully produce a full 20-piece assembly with an average accuracy of 9.8% while retaining a high efficiency with 2.17 x 105Lux+ colonies per pmol of assembly transformed (FIG. 1 J). Next, HighT 50°C ligation was applied to the 20-piece USER assembly and greatly improved accuracy to 76.4% Lux+ clones was observed while maintaining a comparable yield of 2.14 x 105Lux+ colonies / pmol (FIG.1 J). This corresponds to a junction connection rate of 98.7% for this assembly under these conditions. The literature characterizations of USER demonstrate relatively small assemblies, usually around 5 DNA fragments assembled simultaneously with a decrease in accuracy and efficiency as the number of fragments increases indicating that the 2WJ modifications described enable larger multi-fragment assemblies to be conducted. When cloning the HighT Assembly Amplicon of the Lux cassette from 19 individually processed fragments, a slight decrease in proportion of Lux+ clones at 66.4% was observed but gain nearly two orders of magnitude increased yield of 1.18 x 107Lux+ colonies / pmol (FIG. 1 J).One-step assembly and integration

[0159] In addition to carrying DNA on episomal replicons, genomic integration of synthetic DNA is often deployed for improved construct expression and stability. A variety of mechanisms have been used to integrate large pieces of DNA into genomes, including naturally occurring bacteriophage integrases as well as synthetic constructs such as Cas-transposons. In particular, site-specific recombinases have been widely explored due to their well-defined recognition sequences that can be used to direct insertions, excisions, inversions and translocations of DNA. These site-specific recombinases largely fall into two families named after the amino acid residue that catalyzes the protein-DNA covalent bond, either tyrosine recombinases or serine recombinases.

[0160] We theorized that the high yield and low mis-assembly rate afforded by HighT could enable rapid DNA assembly, transformation, and integration of multipiece synthetic inserts into recipient genomes without episomal replicon intermediates. To accomplish this, two independent integration schemes were developed, the first of which uses a tyrosinerecombinase to integrate HighT circularized DNA and the second uses a serine recombinase to integrate HighT linear DNA.

[0161] For circular integration, the HK022 tyrosine recombinase was used to direct integration. This recombinase could theoretically work interchangeably with similar recombinases (lambda phage, phage 186, phi80, and P21). These systems rely upon three major components: a recombinase, an attP recombination site, and its complementary attB recombination site. Using these three components, a mechanism was created to integrate HighT synthetic DNA into a user defined location within recipient genomes (FIG. 2A). The system was designed such that successful integration at a defined location would result in the true gain of a transcriptionally active antibiotic marker.

[0162] First, cargo DNA and a selection cassette DNA were assembled via HighT (FIG. 2A i, ii) to construct an integration plasmid (FIG. 2A iii) that is transformed into recipient cells (FIG. 2A iv). The recipient contains the recombinase protein and a pre-engineered user defined integration site containing a transcriptionally active fluorescent marker. Upon on target recombination (FIG. 2A v), a transcriptionally inactive antibiotic selection marker gains an upstream constitutive promoter to enable selection (FIG. 2A vi). This transcriptional activation of the antibiotic marker is directly coupled to transcriptional inactivation of the fluorescent marker, which functions as an additional verification of correct integrants (FIG. 2B).

[0163] Using this attBP integration system, the ability to directly integrate a 10-piece HighT assembly containing the Lux operon was demonstrated to yield transformants of 19.06% Lux+ clones with 1.36 x 103Lux+ colonies / pmol DNA. (FIG. 2C). When implementing the HighT Assembly Amplicon, a 19-piece assembly of the Lux cassette was successfully amplify resulting in higher yield and accuracy with 30.62% LUX+ clones with 1.43 x 104LUX+ colonies / pmol DNA (FIG. 2C). This increase in accuracy and yield is achieved despite the 2x increase in number of assembly fragments. PCRs flanking the genomic location of the insertion confirmed all Lux positive cells had insertions at the correct location (FIG. 8A) with no observed off-target integrations.

[0164] For the second integration scheme, a system was designed that uses serine recombinases to integrate linear dsDNA (FIG. 2D). Input DNA containing flanking recombination sites are derived from HighT ligations (FIG. 2D i) or from HighT Assembly Amlicons (FIG. 2D ii) and are transformed (FIG. 2D iii) into a cell with a recombinase and preintegrated recombination site (FIG. 2D iv). Integration of HighT assemblies is accomplished through two independent crossing over events between distally located recombination sites, one at each end of the assembly (FIG. 2D v). It was chosen to leverage the well-studied Cre-LoxP serine integrase system, where one Cre recombinase protein can independently act on twodifferent non-cross reactive orthogonal 34-bp recognition sites, LoxP and Lox2272, allowing separate recombination events to occur from the expression of a single recombinase protein.

[0165] The gain of an antibiotic marker in the assembly product is directly coupled to the replacement of the genomic region (FIG. 2D vi), resulting in a simultaneous loss of a genomically integrated negative selection marker for additional assurance of correct (FIG. 2E).While all clones genotyped showed integration at the correct location (FIG. 8B), the ability to use the negative selection marker to counterselection against non-integrating plasmids was still demonstrated (FIG. 8C).

[0166] Using the disclosed LoxP system, integration of both 10-piece HighT direct assembly (58.58% LUX+ with 1.22 x 103LUX+ colonies / pmol DNA) and and 20-piece HighT Assembly Amplicon (76.62% LUX+ with 3.18 x 103LUX+ colonies / pmol DNA) was demonstrated (FIG. 2F). To demonstrate reproducibility of HighT integrations and demonstrate the ability of both the attBP and LoxP systems to control insert DNA orientation, 10-piece assemblies, Assembly Amplicons, and control amplicons were integrated into separate recipient cells with mirrored oriented landing sites (FIGS. 9A-9E).

[0167] These data demonstrate that the improvements afforded by the improved 2WJ assembly protocols enable robust integration of assembly product directly into host genomes with no requirement for stable episomal replicon intermediates.Cloning eukaryotic coding sequences

[0168] HighT can be used to construct definitive DNA sequences to encode medically relevant protein isoforms. One single human gene can produce multiple isoforms through alternative splicing of mRNA with exon compositions differing between tissue type and disease state. Three proteins were selected whose NCBI references indicate alternative splicing patterns: the MAPT gene which creates the tau protein implicated in Alzheimer’s disease, the VEGFA protein which regulates blood vessel formation and whose upregulation is often found in growing tumors, and the BRCA1 tumor suppressor gene whose mutation is linked to breast cancer.

[0169] While dominant variants of small isoforms can be readily isolated from cDNA amplicons for cloning, low abundance isoforms or longer isoforms which are amplified less preferentially can be more difficult or impractical to isolate. An additional fundamental limitation of amplification from cDNA is the abundance of single nucleotide point mutations (SNPs) in the construct resulting from the high error rate of transcription and subsequent reverse transcription processes (FIG. 3A). The error rate of transcription is reported to be 10’5-10’6per bp, reverse transcription to be 10’4-10’5per bp, and the error rate of eukaryotic genomic DNA replication with proofreading and error correction at 1 O'9- 1 O'10per bp. As such, using patientderived genomic DNA as a low SNP-rate template for PCR amplification of specific exons which are subsequently combined via high fidelity HighT ligation offers an inherent advantage in both aspects over conventional methodologies. This Example next illustrates that both template DNA in the form of human gDNA as well as synthetically produced oligos can be used in HighT cloning to construct definitive isoforms of human proteins regardless of natural abundance and length (FIG. 3A).

[0170] In order to identify long mRNA isoforms from the medically relevant MAPT, VEGFA, and BRCA1 genes, an amplicon library was generated from commercially available reverse transcribed mRNA converted to cDNA from healthy human heart, brain, spleen, skeletal muscle, and testis tissue, with heart yielding most consistent expression for all target genes. The heart cDNA reads were mapped onto to the genomic chromosomal reference sequence (sample MAPT depiction in FIG. 3B) and combined that information with NCBI mRNA reads to identify exon locations (or alternative splicing variant regions) and determine one of the longest naturally occurring specific isoforms for each MAPT, VEGFA, and BRCA1.

[0171] For MAPT, to construct the target isoform of 14 exons, a 9-piece assembly was designed from amplicons targeting MAPT gDNA exons, taking advantage longer 5’ ends of amplification primers to encode for some small exons (FIG. 3B and FIGS. 10A-10B). The low abundance of this long isoform in heart cDNA amplicons resulted in just 0.001223% of reads being assigned to this isoform. In contrast, the disclosed gDNA targeted assembly of yielded >95% accurate assemblies assigned to this isoform (FIG. 3C). VEGFA was also constructed using gDNA as template with a 4-piece assembly to compose 9 identified exons, yielding an assembly with over 90% the target isoform when constructed with HighT ligation compared to 0 reads in the cDNA amplicon Nanopore sequencing (FIG. 3D).

[0172] While some exons can be easily incorporated in the 5’ end of the gDNA amplification primer, sometimes long stretches of multiple short exons cannot be encoded in amplification primers, making gDNA amplification impractical in these instances. To address this challenge while still leveraging HighT and the advancements to 2WJ assembly, an oligobased assembly method termed Uracil Primer Dimer Overhang Generation (UPDOG) was designed. In isolated PCR reactions, overlapping primers are extended to make short, synthetic 2WJ assembly fragments. Then by processing with the disclosed improved 2WJ protocol and assembly with HighT, UPDOG is able to generate an entirely synthetic product (FIG. 10C).VEGFA constructed entirely with UPDOG yielded -70% correct constructs from the scripted DNA design with no template employed (FIG. 3D).

[0173] Combining both gDNA and UPDOG assembly in a hybrid manner can utilize the advantages of both techniques. Employing gDNA amplification for longer exons andsynthetic UPDOG cloning for shorter exons enables a streamlined protocol for uniformly processing fragments to construct low abundance isoforms. To synthesize the target isoform of BRCA1, a 13-fragment assembly was designed to construct a 25-exon splicing BRCA1 variant. This hybrid assembly was able to yield over 75% correct constructs compared with 0% abundance of this longest isoform from cDNA amplification (FIG. 3E). Relative to cDNA amplifications, HighT has qualitatively cleaner DNA products for the construction of all 3 target isoforms of MAPT, VEGFA, and BRCA1 while enabling control over exactly which isoform is derived (FIG. 3F).In vitro assembly of large and complex DNA

[0174] De novo construction of large DNA products remains a premium commodity that is not commonly utilized in routine research applications. It was hypothesized this perceived barrier limits the scope of research which requires large synthetic DNA. De novo construction of DNA sequences has been historically limited by the accuracy and reliability of the assembly techniques used to stitch the chemically synthesized synthetic DNA oligos together. Beyond this, construction of large DNA products usually requires an in vivo intermediate step where shorter DNA assemblies are conducted and subcloned into bacteria (FIG. 11), ultimately increasing costs and time due to limited throughput and lack of automation. Further, there are instances, particularly in pharmaceutical applications, where cell-free DNA is required due to concerns regarding byproducts that persist after an in vivo subcloning steps. As a result, it would be advantageous for there to be an assembly technique or combination of techniques which were reliable enough to produce large DNA products (10 kb and beyond) de novo and entirely in vitro.

[0175] Towards this end, the 3WJ assembly technique, Sidewinder, was combined with the disclosed 2WJ HighT assembly for the high-fidelity construction of a linear, 10 kb, dsDNA product. To produce 10 kb entirely de novo and cell-free, Sidewinder was first used to produce ten 1 kb DNA products starting from synthetic oligos. The 3WJ assembly intermediates are then amplified with dU containing primers, simultaneously removing the 3WJ and incorporating a dU residue into the assembled products. These products are then processed as described and assembled with HighT ligation to a final size of 10 kb. Finally, with a PCR amplification, a user can select for the final target 10 kb sequence and generate a 10 kb Assembly Amplicon, all in vitro and cell-free (FIG. 4A).

[0176] The target for the de novo cell-free construction was the genomic sequence and upstream context for the Sonic Hedgehog (SHH) gene of the northern white rhino. The northern white rhino is a functionally extinct species with only two females remaining and SHH plays an important role in embryonic development. In addition to the large genomic gene size, italso is a very complex sequence for DNA construction as it has regions which are incredibly GC rich, with multiple 1 kb regions with over 60% GC content, 50 bp regions with as high as 94% GC content, as well as independent stretches of 11 and 15 consecutive guanine bases (FIG. 4B).

[0177] Sidewinder was used to construct each of the 1 kb intermediates and successfully produces a single, strong, target amplicon of the expected size despite the high GC content (FIG. 4C). Due to the low misconnection rate of Sidewinder, these in vitro intermediates can then be directly processed for a second round of assembly according to the disclosed HighT protocol and subsequently amplified for a 10 kb Assembly Amplicon. The extreme size of this assembly as well as the high GC content makes this a difficult amplification even if PCR amplifying from a perfect template. Despite these extreme conditions, the PCR of the final 10 kb assembly shows a strong target amplicon of the expected size (FIG. 4D) resulting in >90% of amplicons sequenced directly align to the full 10-kB expected reference (FIG. 4E).

[0178] To obtain a sequence perfect clone, the 1-kB fragments were also subassembled onto a pSClOl backbone for sequencing (Table 2), and SNP-free sequences were confirmed with whole plasmid Nanopore and for homopolymeric regions additional pair-end Sanger Sequencing. Error free 1-kB assemblies were HighT assembled onto a final BAC backbone (Table 2), again with whole plasmid Nanopore and Sanger for homopolymeric regions to obtain a completely SNP free 10-kB clone.Discussion

[0179] In this Example, a novel paradigm for the assembly of DNA molecules was conceptualized and demonstrated with 2WJ techniques. Specifically, HighT ligation is provided herein which offers a new paradigm over conventional protocols through the demonstration of accurate and robust assemblies under reaction temperatures significantly above the Tmof the ssDNA overhangs directing assembly. Further improvements to the design and processing the assembly greatly enhances assembly accuracy and efficiently to allow for more reliable and robust construction of DNA molecules.

[0180] The disclosed HighT 2WJ paradigm was applied under a variety of assembly contexts including assemblies from oligos with short 4 bp overhangs (analogous to Golden Gate), 10 bp overhangs (analogous to SLIC) 25 bp overhangs (analogous to Gibson assembly), and in the novel approach Uracil Primer Dimer Overhang Generation (UPDOG), each time generating highly precise assembly products. When HighT ligation applied to PCR-based multifragment assemblies, a user is able to break new barriers for USER assembly reaction sizes of up to 20 fragments assembled simultaneously, showing a 40 point increase in accuracy and over four order of magnitude increase in yield. This greatly enhanced fidelity increases assembly accuracy substantially such that PCR can be conducted on the assembly product, enablingAssembly Amplicon generation for in vitro hierarchical assembly.

[0181] HighT was applied in a variety of contexts that offer demonstrations of new workflows for ease of studying and engineering biological systems. Through two independent integration schemes, it was shown that the improvements afforded by the improved 2WJ assembly protocols enable robust integration of assembly product directly into host bacterial genomes with no requirement for stable episomal replicon intermediates. This can streamline genome engineering by saving on the time and costs associated with sequencing plasmid intermediates prior to genomic integration, enabling more efficient protocol automation.

[0182] Reliable HighT assembly also facilitates the generation of important eukaryotic genes and specific isoforms. Long, rare mRNA isoforms from the medically relevant MAPT, VEGFA, and BRCA1 genes were identified which are impractical to isolate from cDNA amplification. Through the combined approaches of gDNA amplification and synthetic construction a user may more reliably isolate isoforms of interest via HighT construction.

[0183] Finally, HighT advancements paired with the state-of-the-art assembly technique from oligos, Sidewinder, enables the de novo and cell-free construction of a complex 10 kb target sequence. The interfacing of the two techniques enables a demonstration of entirely in vitro hierarchical assembly, where a user is able to generate a large synthetic construct rapidly and entirely in vitro. Historically, many biological tools that have been crucial to conducting research and making therapeutics have been sourced from existing genetic components found from living organisms such as CRISPR / Cas9 technologies, transposons, and therapeutics like biologies to name a few. With the capacity for producing large genomic regions de novo the disclosed compositions and methods enable a user to expand the repertoire of sequences to include historic genes that are no longer accessible by PCR amplification and leverage these sequences to find new and useful tools and therapeutics. Additionally, improvements to Al will expand the user sequence space even further to include DNA sequences which have never existed, adding to the need for rapid de novo construction.

[0184] These results offer a distinct set of advantages that can be applied to 2WJ assembly techniques that can be leveraged for the more efficient engineering of biological systems.Materials and MethodsOligo Purchasing

[0185] Amplification primers and assembly primers containing dU residues were ordered from Integrated DNA Technologies and Millipore-Sigma with standard desalt purification and at the lowest mass allowed, usually 25 nmol. Oligos for generating the oligobased assembly fragments were purchased from Millipore-Sigma with Standard DNA Synthesisfor DNA Oligos in Tubes which has a max oligo length of 120 bases, ordered with standard desalt purification at 50 nmol. The 10 kb SHH Sidewinder oligos which were shipped in water at 50 pM. All other oligos were shipped dry. All oligos are listed in Table 1.Table 1: OligonucleotidesSEQ Method Construct Use FIG. Oligo ID IDNO:HTP-1 1 O-B A LuxABC 4bp Assembly 1cHTP-2 2 O-B A LuxABC 4bp Assembly 1cHTP-3 3 O-B A LuxABC 4bp Assembly 1cHTP-4 4 O-B A LuxABC 4bp Assembly 1cHTP-5 5 O-B A LuxABC 4bp Assembly 1cHTP-6 6 O-B A LuxABC 4bp Assembly 1cHTP-7 7 O-B A LuxABC 4bp Assembly 1cHTP-8 8 O-B A LuxABC 4bp Assembly 1cHTP-9 9 O-B A LuxABC 4bp Assembly 1cHTP-10 10 O-B A LuxABC 4bp Assembly 1cHTP-11 11 O-B A LuxABC 4bp Assembly 1cHTP-12 12 O-B A LuxABC 4bp Assembly 1cHTP-13 13 O-B A LuxABC 4bp Assembly 1cHTP-14 14 O-B A LuxABC 4bp Assembly 1cHTP-15 15 O-B A LuxABC 4bp Assembly 1cHTP-16 16 O-B A LuxABC 4bp Assembly 1cHTP-17 17 O-B A LuxABC 4bp Assembly 1cHTP-18 18 O-B A LuxABC 4bp Assembly 1cHTP-19 19 O-B A LuxABC 4bp Assembly 1cHTP-20 20 O-B A LuxABC 4bp Assembly 1cHTP-21 21 O-B A LuxABC 4bp Assembly 1cHTP-22 22 O-B A LuxABC 4bp Assembly 1cHTP-23 23 O-B A LuxABC 4bp Assembly 1cHTP-24 24 O-B A LuxABC 4bp Assembly 1cHTP-25 25 O-B A LuxABC 4bp Assembly 1cHTP-26 26 O-B A LuxABC 4bp Assembly 1cHTP-27 27 O-B A LuxABC 4bp Assembly 1cHTP-28 28 O-B A LuxABC 4bp Assembly 1cHTP-29 29 O-B A LuxABC 4bp Assembly 1cHTP-30 30 O-B A LuxABC 4bp Assembly 1cHTP-31 31 O-B A LuxABC 4bp Assembly 1cHTP-32 32 O-B A LuxABC 4bp Assembly 1cHTP-33 33 O-B A LuxABC 4bp Assembly 1cHTP-34 34 O-B A LuxABC 4bp Assembly 1cHTP-35 35 O-B A LuxABC 4bp Assembly 1cHTP-36 36 O-B A LuxABC 4bp Assembly 1cHTP-37 37 O-B A LuxABC 4bp Assembly 1cHTP-38 38 O-B A LuxABC 4bp Assembly 1cHTP-39 39 O-B A LuxABC 4bp Assembly 1cHTP-40 40 O-B A LuxABC 4bp Assembly 1cHTP-41 41 O-B A LuxABC 4bp Assembly 1cHTP-42 42 O-B A LuxABC lObp Assembly 1cHTP-43 43 O-B A LuxABC lObp Assembly 1cHTP-44 44 O-B A LuxABC lObp Assembly 1cHTP-45 45 O-B A LuxABC lObp Assembly 1cHTP-46 46 O-B A LuxABC lObp Assembly 1cHTP-47 47 O-B A LuxABC lObp Assembly 1cHTP-48 48 O-B A LuxABC lObp Assembly 1cHTP-49 49 O-B A LuxABC lObp Assembly 1cHTP-50 50 O-B A LuxABC lObp Assembly 1cHTP-51 51 O-B A Lux ABC lObp Assembly 1c HTP-52 52 O-B A Lux ABC lObp Assembly 1c HTP-53 53 O-B A Lux ABC lObp Assembly 1c HTP-54 54 O-B A Lux ABC lObp Assembly 1c HTP-55 55 O-B A Lux ABC lObp Assembly 1c HTP-56 56 O-B A Lux ABC lObp Assembly 1c HTP-57 57 O-B A Lux ABC lObp Assembly 1c HTP-58 58 O-B A Lux ABC lObp Assembly 1c HTP-59 59 O-B A Lux ABC lObp Assembly 1c HTP-60 60 O-B A Lux ABC lObp Assembly 1c HTP-61 61 O-B A Lux ABC lObp Assembly 1c HTP-62 62 O-B A Lux ABC lObp Assembly 1c HTP-63 63 O-B A Lux ABC lObp Assembly 1c HTP-64 64 O-B A Lux ABC lObp Assembly 1c HTP-65 65 O-B A Lux ABC lObp Assembly 1c HTP-66 66 O-B A Lux ABC lObp Assembly 1c HTP-67 67 O-B A Lux ABC lObp Assembly 1c HTP-68 68 O-B A Lux ABC lObp Assembly 1c HTP-69 69 O-B A Lux ABC lObp Assembly 1c HTP-70 70 O-B A Lux ABC lObp Assembly 1c HTP-71 71 O-B A Lux ABC lObp Assembly 1c HTP-72 72 O-B A Lux ABC lObp Assembly 1c HTP-73 73 O-B A Lux ABC lObp Assembly 1c HTP-74 74 O-B A Lux ABC lObp Assembly 1c HTP-75 75 O-B A Lux ABC lObp Assembly 1c HTP-76 76 O-B A Lux ABC lObp Assembly 1c HTP-77 77 O-B A Lux ABC lObp Assembly 1c HTP-78 78 O-B A Lux ABC lObp Assembly 1c HTP-79 79 O-B A Lux ABC lObp Assembly 1c HTP-80 80 O-B A Lux ABC lObp Assembly 1c HTP-81 81 O-B A Lux ABC lObp Assembly 1c HTP-82 82 O-B A Lux ABC 25bp Assembly 1c HTP-83 83 O-B A Lux ABC 25bp Assembly 1c HTP-84 84 O-B A Lux ABC 25bp Assembly 1c HTP-85 85 O-B A Lux ABC 25bp Assembly 1c HTP-86 86 O-B A Lux ABC 25bp Assembly 1c HTP-87 87 O-B A Lux ABC 25bp Assembly 1c HTP-88 88 O-B A Lux ABC 25bp Assembly 1c HTP-89 89 O-B A Lux ABC 25bp Assembly 1c HTP-90 90 O-B A Lux ABC 25bp Assembly 1c HTP-91 91 O-B A Lux ABC 25bp Assembly 1c HTP-92 92 O-B A Lux ABC 25bp Assembly 1c HTP-93 93 O-B A Lux ABC 25bp Assembly 1c HTP-94 94 O-B A Lux ABC 25bp Assembly 1c HTP-95 95 O-B A Lux ABC 25bp Assembly 1c HTP-96 96 O-B A Lux ABC 25bp Assembly 1c HTP-97 97 O-B A Lux ABC 25bp Assembly 1c HTP-98 98 O-B A Lux ABC 25bp Assembly 1c HTP-99 99 O-B A Lux ABC 25bp Assembly 1c HTP-100 100 O-B A Lux ABC 25bp Assembly 1c HTP-101 101 O-B A Lux ABC 25bp Assembly 1c HTP-102 102 O-B A Lux ABC 25bp Assembly 1c HTP-103 103 O-B A Lux ABC 25bp Assembly 1c HTP-104 104 O-B A Lux ABC 25bp Assembly 1c HTP-105 105 O-B A Lux ABC 25bp Assembly 1c HTP-106 106 O-B A Lux ABC 25bp Assembly 1c HTP-107 107 O-B A Lux ABC 25bp Assembly 1c HTP-108 108 O-B A Lux ABC 25bp Assembly 1c HTP-109 109 O-B A Lux ABC 25bp Assembly 1cHTP-110 110 O-B A Lux ABC 25bp Assembly 1cHTP-111 111 O-B A Lux ABC 25bp Assembly 1cHTP-112 112 O-B A Lux ABC 25bp Assembly 1cHTP-113 113 O-B A Lux ABC 25bp Assembly 1cHTP-114 114 O-B A Lux ABC 25bp Assembly 1cHTP-115 115 O-B A Lux ABC 25bp Assembly 1cHTP-116 116 O-B A Lux ABC 25bp Assembly 1cHTP-117 117 O-B A Lux ABC 25bp Assembly 1cHTP-118 118 O-B A Lux ABC 25bp Assembly 1cHTP-119 119 O-B A Lux ABC 25bp Assembly 1cHTP-120 120 O-B A Lux ABC 25bp Assembly 1cHTP-121 121 O-B A Lux ABC 25bp Assembly 1cHTP-122 122 O-B A LuxABC 4bp, lObp, 25 bp Amplification 1cHTP-123 123 O-B A LuxABC 4bp, lObp, 25 bp Amplification 1cHTP-124 124 O-B A LuxABC 4bp, lObp, 25 bp Amplification 1cHTP-125 125 O-B A LuxABC 4bp, lObp, 25 bp Amplification 1cHTP-126 126 USER 4-piece GFP, control (has Amplification / Cloning If -control, a, secondary structure) 7bHTP-127 127 USER 4-piece GFP, control (has Amplification / Cloning If -control, a, secondary structure) 7bHTP-128 128 USER 4-piece GFP, control (has Amplification / Cloning If -control, a, secondary structure) 7bHTP-129 129 USER 4-piece GFP, control (has Amplification / Cloning If -control, a, secondary structure) 7bHTP-130 130 USER 4-piece GFP, control (has Amplification / Cloning If -control, a, secondary structure) 7bHTP-131 131 USER 4-piece GFP, control (has Amplification / Cloning If -control, a, secondary structure) 7bHTP-132 132 USER 4-piece GFP, control (has Amplification / Cloning If -control, a, secondary structure) 7bHTP-133 133 USER 4-piece GFP, control (has Amplification / Cloning If -control, a, secondary structure) 7bHTP-134 134 USER 4-piece GFP, 2dU control (has Amplification / Cloning 7b secondary structure)HTP-135 135 USER 4-piece GFP, 2dU control (has Amplification / Cloning 7b secondary structure)HTP-136 136 USER 4-piece GFP, 2dU control (has Amplification / Cloning 7b secondary structure)HTP-137 137 USER 4-piece GFP, 2dU control (has Amplification / Cloning 7b secondary structure)HTP-138 138 USER 4-piece GFP, 2dU control (has Amplification / Cloning 7b secondary structure)HTP-139 139 USER 4-piece GFP, 2dU control (has Amplification / Cloning 7b secondary structure)HTP-140 140 USER 4-piece GFP, 2dU control (has Amplification / Cloning 7b secondary structure)HTP-141 141 USER 4-piece GFP, 2dU control (has Amplification / Cloning 7b secondary structure)HTP-142 142 USER 4-piece GFP, No Secondary Amplification / Cloning If P, 5, e , < n,Structure 0HTP-143 143 USER 4-piece GFP, No Secondary Amplification / Cloning If P, 5, e , < n,Structure 0HTP-144 144 USER 4-piece GFP, No Secondary Amplification / Cloning If P, 5, e , < n,Structure 0HTP-145 145 USER 4-piece GFP, No Secondary Amplification / Cloning If P, 5, e , < n,Structure 0HTP-146 146 USER 4-piece GFP, No Secondary Amplification / Cloning If P, 5, e , < n,Structure 0HTP-147 147 USER 4-piece GFP, No Secondary Amplification / Cloning If P, 5, e , < n,Structure 0HTP-148 148 USER 4-piece GFP, No Secondary Amplification / Cloning If P, 5, e , < n,Structure 0HTP-149 149 USER 4-piece GFP, No Secondary Amplification / Cloning If P, 5, e , < n,Structure 0HTP-150 150 USER 4-piece GFP, 2dU No Secondary Amplification / Cloning if Y StructureHTP-151 151 USER 4-piece GFP, 2dU No Secondary Amplification / Cloning if Y StructureHTP-152 152 USER 4-piece GFP, 2dU No Secondary Amplification / Cloning If Y StructureHTP-153 153 USER 4-piece GFP, 2dU No Secondary Amplification / Cloning If Y StructureHTP-154 154 USER 4-piece GFP, 2dU No Secondary Amplification / Cloning if Y StructureHTP-155 155 USER 4-piece GFP, 2dU No Secondary Amplification / Cloning if Y StructureHTP-156 156 USER 4-piece GFP, 2dU No Secondary Amplification / Cloning if Y StructureHTP-157 157 USER 4-piece GFP, 2dU No Secondary Amplification / Cloning if Y StructureHTP-158 158 USER 10,20 LUX ABCDE Amplification / Cloning lg, ij HTP-159 159 USER 10,20 LUX ABCDE Amplification / Cloning 1g, ij HTP-160 160 USER 10,20 LUX ABCDE Amplification / Cloning 1g, ij HTP-161 161 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-162 162 USER 10,20 LUX ABCDE Amplification / Cloning 1g, Ij, 2c, 2f, 9 HTP-163 163 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-164 164 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-165 165 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-166 166 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-167 167 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-168 168 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-169 169 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-170 170 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-171 171 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-172 172 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-173 173 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-174 174 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij, 2c, 2f, 9 HTP-175 175 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij HTP-176 176 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-177 177 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-178 178 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-179 179 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-180 180 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-181 181 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-182 182 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-183 183 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-184 184 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-185 185 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-186 186 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-187 187 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-188 188 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-189 189 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-190 190 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-191 191 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-192 192 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-193 193 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-194 194 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-195 195 USER 20 Lux ABCDE Amplification / Cloning lg, Ij HTP-196 196 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij HTP-197 197 USER 10,20 LUX ABCDE Amplification / Cloning lg, Ij HTP-198 198 USER 10 Lux AttB, 10 Lux LoxP Amplification / Cloning 2c, 2f, 9HTP-199 199 USER 10 Lux AttB, 10 Lux LoxP Amplification / Cloning 2c, 2f, 9HTP-200 200 USER 10 Lux AttB, 10 Lux LoxP Amplification / Cloning 2c, 2f, 9 HTP-201 201 USER 10 Lux AttB Amplification / Cloning 2c, 9 HTP-202 202 USER 10 Lux AttB Amplification / Cloning 2c, 9 HTP-203 203 USER 10 Lux LoxP Amplification / Cloning 2f, 9 HTP-204 204 USER 10 Lux LoxP Amplification / Cloning 2f, 9 HTP-205 205 USER 10 Lux LoxP Amplification / Cloning 2f, 9 HTP-206 206 Genome Dif Site Genome Check LoxP Dif Site Genome Check 8 Check AttB Integration LoxP AttB Integration HTP-207 207 Genome Dif Site Genome Check LoxP Dif Site Genome Check 8 Check AttB Integration LoxP AttB Integration HTP-208 208 USER 10 Lux LoxP Amplification / Cloning 9 HTP-209 209 USER 10 Lux LoxP Amplification / Cloning 9 HTP-210 210 cDNA and MAPT cDNA Amplification Sequencing 3c USER HTP-211 211 cDNA and MAPT cDNA Amplification Sequencing 3c USER HTP-212 212 cDNA VEGFA cDNA Amplification Sequencing 3d HTP-213 213 cDNA VEGFA cDNA Amplification Sequencing 3d HTP-214 214 USER VEGFA Assembly Amplification Amplification / Cloning 3d and VEGFA gDNA HighTHTP-215 215 USER VEGFA Assembly Amplification Amplification / Cloning 3d and VEGFA gDNA HighTHTP-216 216 cDNA and BRCA1 cDNA Amplification + Sequencing, 3e UPDOg Assembly Ampification Amplification, and CloningHTP-217 217 cDNA and BRCA1 cDNA Amplification + Sequencing, 3e UPDOg Assembly Ampification Amplification, and CloningHTP-218 218 USER MAPT gDNA HighT Assembly 3c HTP-219 219 USER MAPT gDNA HighT Assembly 3c HTP-220 220 USER MAPT gDNA HighT Assembly 3c HTP-221 221 USER MAPT gDNA HighT Assembly 3c HTP-222 222 USER MAPT gDNA HighT Assembly 3c HTP-223 223 USER MAPT gDNA HighT Assembly 3c HTP-224 224 USER MAPT gDNA HighT Assembly 3c HTP-225 225 USER MAPT gDNA HighT Assembly 3c HTP-226 226 USER MAPT gDNA HighT Assembly 3c HTP-227 227 USER MAPT gDNA HighT Assembly 3c HTP-228 228 USER MAPT gDNA HighT Assembly 3c HTP-229 229 USER MAPT gDNA HighT Assembly 3c HTP-230 230 USER MAPT gDNA HighT Assembly 3c HTP-231 231 USER MAPT gDNA HighT Assembly 3c HTP-232 232 USER MAPT gDNA HighT Assembly 3c HTP-233 233 USER MAPT gDNA HighT Assembly 3c HTP-234 234 USER MAPT gDNA HighT Assembly 3c HTP-235 235 USER MAPT gDNA HighT Assembly 3c HTP-236 236 UPDOG VEGFA UPDOG Assembly 3c HTP-237 237 UPDOG VEGFA UPDOG Assembly 3c HTP-238 238 UPDOG VEGFA UPDOG Assembly 3c HTP-239 239 UPDOG VEGFA UPDOG Assembly 3c HTP-240 240 UPDOG VEGFA UPDOG Assembly 3c HTP-241 241 UPDOG VEGFA UPDOG Assembly 3c HTP-242 242 UPDOG VEGFA UPDOG Assembly 3c HTP-243 243 UPDOG VEGFA UPDOG Assembly 3c HTP-244 244 UPDOG VEGFA UPDOG Assembly 3c HTP-245 245 UPDOG VEGFA UPDOG Assembly 3c HTP-246 246 UPDOG VEGFA UPDOG Assembly 3c HTP-247 247 UPDOG VEGFA UPDOG Assembly 3c HTP-248 248 USER VEGFA gDNA HighT Assembly 3cHTP-249 249 USER VEGFA gDNA HighT Assembly 3cHTP-250 250 USER VEGFA gDNA HighT Assembly 3c HTP-251 251 USER VEGFA gDNA HighT Assembly 3c HTP-252 252 USER VEGFA gDNA HighT Assembly 3c HTP-253 253 USER VEGFA gDNA HighT Assembly 3c HTP-254 254 USER VEGFA gDNA HighT Assembly 3c HTP-255 255 USER VEGFA gDNA HighT Assembly 3c HTP-256 256 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-257 257 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-258 258 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-259 259 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-260 260 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-261 261 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-262 262 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-263 263 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-264 264 USER BRCA1 HighT+UPDOG Assembly 3e HTP-265 265 USER BRCA1 HighT+UPDOG Assembly 3e HTP-266 266 USER BRCA1 HighT+UPDOG Assembly 3e HTP-267 267 USER BRCA1 HighT+UPDOG Assembly 3e HTP-268 268 USER BRCA1 HighT+UPDOG Assembly 3e HTP-269 269 USER BRCA1 HighT+UPDOG Assembly 3e HTP-270 270 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-271 271 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-272 272 USER BRCA1 HighT+UPDOG Assembly 3e HTP-273 273 USER BRCA1 HighT+UPDOG Assembly 3e HTP-274 274 USER BRCA1 HighT+UPDOG Assembly 3e HTP-275 275 USER BRCA1 HighT+UPDOG Assembly 3e HTP-276 276 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-277 277 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-278 278 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-279 279 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-280 280 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-281 281 UPDOG BRCA1 HighT+UPDOG Assembly 3e HTP-282 282 Sidewinder NWR-SSH Assembly 4 HTP-283 283 Sidewinder NWR-SSH Assembly 4 HTP-284 284 Sidewinder NWR-SSH Assembly 4 HTP-285 285 Sidewinder NWR-SSH Assembly 4 HTP-286 286 Sidewinder NWR-SSH Assembly 4 HTP-287 287 Sidewinder NWR-SSH Assembly 4 HTP-288 288 Sidewinder NWR-SSH Assembly 4 HTP-289 289 Sidewinder NWR-SSH Assembly 4 HTP-290 290 Sidewinder NWR-SSH Assembly 4 HTP-291 291 Sidewinder NWR-SSH Assembly 4 HTP-292 292 Sidewinder NWR-SSH Assembly 4 HTP-293 293 Sidewinder NWR-SSH Assembly 4 HTP-294 294 Sidewinder NWR-SSH Assembly 4 HTP-295 295 Sidewinder NWR-SSH Assembly 4 HTP-296 296 Sidewinder NWR-SSH Assembly 4 HTP-297 297 Sidewinder NWR-SSH Assembly 4 HTP-298 298 Sidewinder NWR-SSH Assembly 4 HTP-299 299 Sidewinder NWR-SSH Assembly 4 HTP-300 300 Sidewinder NWR-SSH Assembly 4 HTP-301 301 Sidewinder NWR-SSH Assembly 4 HTP-302 302 Sidewinder NWR-SSH Assembly 4 HTP-303 303 Sidewinder NWR-SSH Assembly 4 HTP-304 304 Sidewinder NWR-SSH Assembly 4 HTP-305 305 Sidewinder NWR-SSH Assembly 4 HTP-306 306 Sidewinder NWR-SSH Assembly 4 HTP-307 307 Sidewinder NWR-SSH Assembly 4 HTP-308 308 Sidewinder NWR-SSH Assembly 4HTP-309 309 Sidewinder NWR-SSH Assembly 4HTP-310 310 Sidewinder NWR-SSH Assembly 4 HTP-311 311 Sidewinder NWR-SSH Assembly 4 HTP-312 312 Sidewinder NWR-SSH Assembly 4 HTP-313 313 Sidewinder NWR-SSH Assembly 4 HTP-314 314 Sidewinder NWR-SSH Assembly 4 HTP-315 315 Sidewinder NWR-SSH Assembly 4 HTP-316 316 Sidewinder NWR-SSH Assembly 4 HTP-317 317 Sidewinder NWR-SSH Assembly 4 HTP-318 318 Sidewinder NWR-SSH Assembly 4 HTP-319 319 Sidewinder NWR-SSH Assembly 4 HTP-320 320 Sidewinder NWR-SSH Assembly 4 HTP-321 321 Sidewinder NWR-SSH Assembly 4 HTP-322 322 Sidewinder NWR-SSH Assembly 4 HTP-323 323 Sidewinder NWR-SSH Assembly 4 HTP-324 324 Sidewinder NWR-SSH Assembly 4 HTP-325 325 Sidewinder NWR-SSH Assembly 4 HTP-326 326 Sidewinder NWR-SSH Assembly 4 HTP-327 327 Sidewinder NWR-SSH Assembly 4 HTP-328 328 Sidewinder NWR-SSH Assembly 4 HTP-329 329 Sidewinder NWR-SSH Assembly 4 HTP-330 330 Sidewinder NWR-SSH Assembly 4 HTP-331 331 Sidewinder NWR-SSH Assembly 4 HTP-332 332 Sidewinder NWR-SSH Assembly 4 HTP-333 333 Sidewinder NWR-SSH Assembly 4 HTP-334 334 Sidewinder NWR-SSH Assembly 4 HTP-335 335 Sidewinder NWR-SSH Assembly 4 HTP-336 336 Sidewinder NWR-SSH Assembly 4 HTP-337 337 Sidewinder NWR-SSH Assembly 4 HTP-338 338 Sidewinder NWR-SSH Assembly 4 HTP-339 339 Sidewinder NWR-SSH Assembly 4 HTP-340 340 Sidewinder NWR-SSH Assembly 4 HTP-341 341 Sidewinder NWR-SSH Assembly 4 HTP-342 342 Sidewinder NWR-SSH Assembly 4 HTP-343 343 Sidewinder NWR-SSH Assembly 4 HTP-344 344 Sidewinder NWR-SSH Assembly 4 HTP-345 345 Sidewinder NWR-SSH Assembly 4 HTP-346 346 Sidewinder NWR-SSH Assembly 4 HTP-347 347 Sidewinder NWR-SSH Assembly 4 HTP-348 348 Sidewinder NWR-SSH Assembly 4 HTP-349 349 Sidewinder NWR-SSH Assembly 4 HTP-350 350 Sidewinder NWR-SSH Assembly 4 HTP-351 351 Sidewinder NWR-SSH Assembly 4 HTP-352 352 Sidewinder NWR-SSH Assembly 4 HTP-353 353 Sidewinder NWR-SSH Assembly 4 HTP-354 354 Sidewinder NWR-SSH Assembly 4 HTP-355 355 Sidewinder NWR-SSH Assembly 4 HTP-356 356 Sidewinder NWR-SSH Assembly 4 HTP-357 357 Sidewinder NWR-SSH Assembly 4 HTP-358 358 Sidewinder NWR-SSH Assembly 4 HTP-359 359 Sidewinder NWR-SSH Assembly 4 HTP-360 360 Sidewinder NWR-SSH Assembly 4 HTP-361 361 Sidewinder NWR-SSH Assembly 4 HTP-362 362 Sidewinder NWR-SSH Assembly 4 HTP-363 363 Sidewinder NWR-SSH Assembly 4 HTP-364 364 Sidewinder NWR-SSH Assembly 4 HTP-365 365 Sidewinder NWR-SSH Assembly 4 HTP-366 366 Sidewinder NWR-SSH Assembly 4 HTP-367 367 Sidewinder NWR-SSH Assembly 4 HTP-368 368 Sidewinder NWR-SSH Assembly 4HTP-369 369 Sidewinder NWR-SSH Assembly 4HTP-370 370 Sidewinder NWR-SSH Assembly 4 HTP-371 371 Sidewinder NWR-SSH Assembly 4 HTP-372 372 Sidewinder NWR-SSH Assembly 4 HTP-373 373 Sidewinder NWR-SSH Assembly 4 HTP-374 374 Sidewinder NWR-SSH Assembly 4 HTP-375 375 Sidewinder NWR-SSH Assembly 4 HTP-376 376 Sidewinder NWR-SSH Assembly 4 HTP-377 377 Sidewinder NWR-SSH Assembly 4 HTP-378 378 Sidewinder NWR-SSH Assembly 4 HTP-379 379 Sidewinder NWR-SSH Assembly 4 HTP-380 380 Sidewinder NWR-SSH Assembly 4 HTP-381 381 Sidewinder NWR-SSH Assembly 4 HTP-382 382 Sidewinder NWR-SSH Assembly 4 HTP-383 383 Sidewinder NWR-SSH Assembly 4 HTP-384 384 Sidewinder NWR-SSH Assembly 4 HTP-385 385 Sidewinder NWR-SSH Assembly 4 HTP-386 386 Sidewinder NWR-SSH Assembly 4 HTP-387 387 Sidewinder NWR-SSH Assembly 4 HTP-388 388 Sidewinder NWR-SSH Assembly 4 HTP-389 389 Sidewinder NWR-SSH Assembly 4 HTP-390 390 Sidewinder NWR-SSH Assembly 4 HTP-391 391 Sidewinder NWR-SSH Assembly 4 HTP-392 392 Sidewinder NWR-SSH Assembly 4 HTP-393 393 Sidewinder NWR-SSH Assembly 4 HTP-394 394 Sidewinder NWR-SSH Assembly 4 HTP-395 395 Sidewinder NWR-SSH Assembly 4 HTP-396 396 Sidewinder NWR-SSH Assembly 4 HTP-397 397 Sidewinder NWR-SSH Assembly 4 HTP-398 398 Sidewinder NWR-SSH Assembly 4 HTP-399 399 Sidewinder NWR-SSH Assembly 4 HTP-400 400 Sidewinder NWR-SSH Assembly 4 HTP-401 401 Sidewinder NWR-SSH Assembly 4 HTP-402 402 Sidewinder NWR-SSH Assembly 4 HTP-403 403 Sidewinder NWR-SSH Assembly 4 HTP-404 404 Sidewinder NWR-SSH Assembly 4 HTP-405 405 Sidewinder NWR-SSH Assembly 4 HTP-406 406 Sidewinder NWR-SSH Assembly 4 HTP-407 407 Sidewinder NWR-SSH Assembly 4 HTP-408 408 Sidewinder NWR-SSH Assembly 4 HTP-409 409 Sidewinder NWR-SSH Assembly 4 HTP-410 410 Sidewinder NWR-SSH Assembly 4 HTP-411 411 Sidewinder NWR-SSH Assembly 4 HTP-412 412 Sidewinder NWR-SSH Assembly 4 HTP-413 413 Sidewinder NWR-SSH Assembly 4 HTP-414 414 Sidewinder NWR-SSH Assembly 4 HTP-415 415 Sidewinder NWR-SSH Assembly 4 HTP-416 416 Sidewinder NWR-SSH Assembly 4 HTP-417 417 Sidewinder NWR-SSH Assembly 4 HTP-418 418 Sidewinder NWR-SSH Assembly 4 HTP-419 419 Sidewinder NWR-SSH Assembly 4 HTP-420 420 Sidewinder NWR-SSH Assembly 4 HTP-421 421 Sidewinder NWR-SSH Assembly 4 HTP-422 422 Sidewinder NWR-SSH Assembly 4 HTP-423 423 Sidewinder NWR-SSH Assembly 4 HTP-424 424 Sidewinder NWR-SSH Assembly 4 HTP-425 425 Sidewinder NWR-SSH Assembly 4 HTP-426 426 Sidewinder NWR-SSH Assembly 4 HTP-427 427 Sidewinder NWR-SSH Assembly 4 HTP-428 428 Sidewinder NWR-SSH Assembly 4HTP-429 429 Sidewinder NWR-SSH Assembly 4HTP-430 430 Sidewinder NWR-SSH Assembly 4 HTP-431 431 Sidewinder NWR-SSH Assembly 4 HTP-432 432 Sidewinder NWR-SSH Assembly 4 HTP-433 433 Sidewinder NWR-SSH Assembly 4 HTP-434 434 Sidewinder NWR-SSH Assembly 4 HTP-435 435 Sidewinder NWR-SSH Assembly 4 HTP-436 436 Sidewinder NWR-SSH Assembly 4 HTP-437 437 Sidewinder NWR-SSH Assembly 4 HTP-438 438 Sidewinder NWR-SSH Assembly 4 HTP-439 439 Sidewinder NWR-SSH Assembly 4 HTP-440 440 Sidewinder NWR-SSH Assembly 4 HTP-441 441 Sidewinder NWR-SSH Assembly 4 HTP-442 442 Sidewinder NWR-SSH Assembly 4 HTP-443 443 Sidewinder NWR-SSH Assembly 4 HTP-444 444 Sidewinder NWR-SSH Assembly 4 HTP-445 445 Sidewinder NWR-SSH Assembly 4 HTP-446 446 Sidewinder NWR-SSH Assembly 4 HTP-447 447 Sidewinder NWR-SSH Assembly 4 HTP-448 448 Sidewinder NWR-SSH Assembly 4 HTP-449 449 Sidewinder NWR-SSH Assembly 4 HTP-450 450 Sidewinder NWR-SSH Assembly 4 HTP-451 451 Sidewinder NWR-SSH Assembly 4 HTP-452 452 Sidewinder NWR-SSH Assembly 4 HTP-453 453 Sidewinder NWR-SSH Assembly 4 HTP-454 454 Sidewinder NWR-SSH Assembly 4 HTP-455 455 Sidewinder NWR-SSH Assembly 4 HTP-456 456 Sidewinder NWR-SSH Assembly 4 HTP-457 457 Sidewinder NWR-SSH Assembly 4 HTP-458 458 Sidewinder NWR-SSH Assembly 4 HTP-459 459 Sidewinder NWR-SSH Assembly 4 HTP-460 460 Sidewinder NWR-SSH Assembly 4 HTP-461 461 Sidewinder NWR-SSH Assembly 4 HTP-462 462 Sidewinder NWR-SSH Assembly 4 HTP-463 463 Sidewinder NWR-SSH Assembly 4 HTP-464 464 Sidewinder NWR-SSH Assembly 4 HTP-465 465 Sidewinder NWR-SSH Assembly 4 HTP-466 466 Sidewinder NWR-SSH Assembly 4 HTP-467 467 Sidewinder NWR-SSH Assembly 4 HTP-468 468 Sidewinder NWR-SSH Assembly 4 HTP-469 469 Sidewinder NWR-SSH Assembly 4 HTP-470 470 Sidewinder NWR-SSH Assembly 4 HTP-471 471 Sidewinder NWR-SSH Assembly 4 HTP-472 472 Sidewinder NWR-SSH Assembly 4 HTP-473 473 Sidewinder NWR-SSH Assembly 4 HTP-474 474 Sidewinder NWR-SSH Assembly 4 HTP-475 475 Sidewinder NWR-SSH Assembly 4 HTP-476 476 Sidewinder NWR-SSH Assembly 4 HTP-477 477 Sidewinder NWR-SSH Assembly 4 HTP-478 478 Sidewinder NWR-SSH Assembly 4 HTP-479 479 Sidewinder NWR-SSH Assembly 4 HTP-480 480 Sidewinder NWR-SSH Assembly 4 HTP-481 481 Sidewinder NWR-SSH Assembly 4 HTP-482 482 Sidewinder NWR-SSH Assembly 4 HTP-483 483 Sidewinder NWR-SSH Assembly 4 HTP-484 484 Sidewinder NWR-SSH Assembly 4 HTP-485 485 Sidewinder NWR-SSH Assembly 4 HTP-486 486 Sidewinder NWR-SSH Assembly 4 HTP-487 487 Sidewinder NWR-SSH Assembly 4 HTP-488 488 Sidewinder NWR-SSH Assembly 4HTP-489 489 Sidewinder NWR-SSH Assembly 4HTP-490 490 Sidewinder NWR-SSH Assembly 4 HTP-491 491 Sidewinder NWR-SSH Assembly 4 HTP-492 492 Sidewinder NWR-SSH Assembly 4 HTP-493 493 Sidewinder NWR-SSH Assembly 4 HTP-494 494 Sidewinder NWR-SSH Assembly 4 HTP-495 495 Sidewinder NWR-SSH Assembly 4 HTP-496 496 Sidewinder NWR-SSH Assembly 4 HTP-497 497 Sidewinder NWR-SSH Assembly 4 HTP-498 498 Sidewinder NWR-SSH Assembly 4 HTP-499 499 Sidewinder NWR-SSH Assembly 4 HTP-500 500 Sidewinder NWR-SSH Assembly 4 HTP-501 501 Sidewinder NWR-SSH Assembly 4 HTP-502 502 Sidewinder NWR-SSH Assembly 4 HTP-503 503 Sidewinder NWR-SSH Assembly 4 HTP-504 504 Sidewinder NWR-SSH Assembly 4 HTP-505 505 Sidewinder NWR-SSH Assembly 4 HTP-506 506 Sidewinder NWR-SSH Assembly 4 HTP-507 507 Sidewinder NWR-SSH Assembly 4 HTP-508 508 Sidewinder NWR-SSH Assembly 4 HTP-509 509 Sidewinder NWR-SSH Assembly 4 HTP-510 510 Sidewinder NWR-SSH Assembly 4 HTP-511 511 Sidewinder NWR-SSH Assembly 4 HTP-512 512 Sidewinder NWR-SSH Assembly 4 HTP-513 513 Sidewinder NWR-SSH Assembly 4 HTP-514 514 Sidewinder NWR-SSH Assembly 4 HTP-515 515 Sidewinder NWR-SSH Assembly 4 HTP-516 516 Sidewinder NWR-SSH Assembly 4 HTP-517 517 Sidewinder NWR-SSH Assembly 4 HTP-518 518 Sidewinder NWR-SSH Assembly 4 HTP-519 519 Sidewinder NWR-SSH Assembly 4 HTP-520 520 Sidewinder NWR-SSH Assembly 4 HTP-521 521 Sidewinder NWR-SSH Assembly 4 HTP-522 522 Sidewinder NWR-SSH Assembly 4 HTP-523 523 Sidewinder NWR-SSH Assembly 4 HTP-524 524 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-525 525 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-526 526 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-527 527 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-528 528 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-529 529 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-530 530 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-531 531 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-532 532 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-533 533 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-534 534 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-535 535 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-536 536 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-537 537 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-538 538 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-539 539 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-540 540 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-541 541 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-542 542 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-543 543 Sidewinder NWR-SSH Amplification / Cloning 4 HTP-544 544 USER NWR-SSH, lOkb Amplification 4 HTP-545 545 USER NWR-SSH, lOkb Amplification 4 All oligos purity=DesaltNWR-SSH=Northem White Rhino SSHO-B A=Oligo-Based AssemblyFragment Design

[0186] The 4-piece GFP assemblies in FIGS. 1A-1J were designed systematically as to reduce bias that may come from specifically chosen overhangs.

[0187] For the negative control, the first overhang selected was the first valid sequence of the form ANxWNyWNzT (where one W is an A and the other a T) with a Tmof 30°C at the end of the GFP coding sequence. This ensured that the overhang could be compatible with a two dU condition and that the entire GFP gene could be a single permissible fragment. The plasmid was then divided into four equal length fragments. The location for the next overhang was chosen to be the first valid overhang of the form ANxWNyWNzT with a melting temperature at 30°C after moving an equidistant 917 bases from the terminal T of the previous overhang. This was repeated until all 4 overhangs were chosen. Through this process, the ReplOl-GFP junction has substantial crosstalk with itself and the GFP-KanR junction has a looped secondary structure as determined by the NUPACK web application at 30°C, IpM (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).

[0188] For the no secondary structure (NSS) assembly, all overhangs were chosen to be of the form ANxWNyWNzT with a Tmof 30°C and were ensured to have no secondary structure and minimal cross reactivity (less than 13% concentration defect at 1 pM) at 30°C. The first junction was created after the coding sequence of GFP using pre-generated sequences because the junction flanking the GFP gene is not present in a coding sequence. Subsequent junctions were again chosen by moving an equidistance from the previous overhang and choosing the first valid junction that satisfied the NSS conditions. All secondary structure and cross reactivity of chosen overhangs were checked using NUPACK web application.

[0189] For the two dU condition, the position of the second dU was chosen which most evenly reduces the melting temperature of both resultant waste strands as to maximize the difference in Tmbetween the waste strands and the Tmof the intended assembly partner.

[0190] The PCR-based multi-fragment Lux assemblies were designed by splitting the Lux cassette into the requisite number of fragments, counting the backbone as one fragment. The ANxWNyWNzT sequence chosen was ensured to have no secondary structure and minimal cross reactivity when considering complexes of size two at 30°C (less than 35.3% concentration defect at 1 pM for the 20-piece assembly at 30°C). All secondary structure and cross reactivity of chosen overhangs were checked using NUPACK web-browser. Junctions flanking the Lux cassette were designed using synthetic overhangs were designed using pre-generated sequences.

[0191] All primers used to amplify DNA fragments are listed in Table 1. All final constructs and templates can be found in FASTA format in Table 2.Table 2: Sequences Used and ConstructedSEQ ID NO:KanR GFP pSClOl Standard-USER (3666 bp) 546 KanR GFP pSClOl NSS-USER (3682 bp) 547 LuxABCDE pl5a CmR(7869bp) 548 LuxABC oligo-based (470 bp) 549 Northern White Rhino Genomic SSH (10000 bp) 550 CmR pl5a mGL SacB Template (4157 bp) 551 KanR pSClOl SacB Template (4297 bp) 552 Genomically Integrated attB dif landing site orientation 1 (2063 bp) 553 Genomically Integrated attB dif landing site orientation2 (2062 bp) 554 LUX attB Assembled Plasmid for Integration (7299 bp) 555 Genomically Integrated 2LoxP dif landing site orientation 1 (2666 bp) 556 Genomically Integrated 2LoxP dif landing site orientation2 (2666 bp) 557 LUX 2LoxP Assembled Linear for Integration Orientation 1 (7093 bp) 558 LUX 2LoxP Assembled Linear for Integration Orientation2 (7093 bp) 559 MAPT Assembled Isoform 560 MAPT Exonl 561 MAPT Exon2 562 MAPT Exon3 563 MAPT Exon4 564 MAPT Exon5 565 MAPT Exon6 566 MAPT Exon7 567 MAPT Exon8 568 MAPT Exon9 569 MAPT Exon 10 570 MAPT Exon 11 571 MAPT Exon 12 572 MAPT Exon 13 573 MAPT Exon 14 574 VEGFA Assembled Isoform 575 VEGFA Exonl 576 VEGFA Exon2 577 VEGFA Exon3 578 VEGFA Exon4 579 VEGFA Exon5 580 VEGFA Exon6 581 VEGFA Exon7 582 VEGFA Exon8 583 VEGFA Exon9 584 BRCA1 Assembled Isoform 585 BRCA1 Exonl 586 BRCA1 Exon2 587 BRCA1 Exon3 588 BRCA1 Exon4 589 BRCA1 Exon5 590 BRCA1 Exon6 591 BRCA1 Exon7 592 BRCA1 Exon 8 593BRCA1 Exon9 594BRCA1 Exon 10 595 BRCA1 Exon 11 596BRCA1 Exon 12 597BRCA1 Exon 13 598BRCA1 Exon 14 599BRCA1 Exon 15 600BRCA1 Exon 16 601BRCA1 Exon 17 602BRCA1 Exon 18 603BRCA1 Exon 19 604BRCA1 Exon20 605BRCA1 Exon21 606BRCA1 Exon22 607BRCA1 Exon23 608BRCA1 Exon24 609BRCA1 Exon25 610Northern White Rhino Sonic Hedgehog gDNA 611 Replication backbone pSClOl for IkB NWR in-vivo sub-assemblies 612Replication backbone BAC for lOkB NWR in-vivo full-assembly 613PCR amplification and purification

[0192] The polymerase used for amplification of input PCR fragments or final amplification of Assembly Amplicons are listed in Tables 3-5. PCRs are conducted in 50pL reactions.

[0193] The backbone fragments for the GFP construct assemblies were amplified from 2.5 ng of a KanR, pSClOl, SacB template. The GFP gene was amplified from 2.5ng of a CmR, pl 5a, mGL, SacB template. SacB was utilized in the templates such that sucrose selection could be used to select against any residual template in the final transformation. The backbone fragment for the Lux construct assemblies was amplified from 2.5 ng of a CmR, pl5a, mGL, SacB template such that GFP could be used to identify, and sucrose selection could be used to select against, any residual template in the final transformation. The fragments of the Lux cassette were amplified from 2.5 ng of a TetR, BAC, LuxABCDE template. Sequences for the templates are listed in Table 2.

[0194] Post PCR amplification of assembly fragments, purification was done using a QIAquick PCR Purification Kit (Qiagen). Multiple 50uL PCR reactions can be passed simultaneously through the same purification column to increase the final concentration of the purified amplicon. Purified products were eluted in 10% elution buffer.Fragment processing

[0195] The conditions used to process and assemble each construct demonstrated in this paper are listed in Tables 3-5.Table 3: Fragment ProcessingFIG Construct Assembly Polymerase USER RecJF Dpnl Fragment Fragment Source Fragment Purification Amplification1c Lux 5-pc, 4bp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 16C1c Lux 10-pc, 4bp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 16C1c Lux 20-pc, 4bp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 16C1c Lux 5-pc, 4bp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 37C1c Lux 10-pc, 4bp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 37C1c Lux 20-pc, 4bp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 37C1c Lux 5-pc, 4bp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 50C1c Lux 10-pc, 4bp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 50C1c Lux 20-pc, 4bp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 50C1c Lux 5-pc, lObp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 16C1c Lux 10-pc, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction lObp, 16C1c Lux 20-pc, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction lObp, 16C1c Lux 5-pc, lObp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 37C1c Lux 10-pc, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction lObp, 37C1c Lux 20-pc, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction lObp, 37C1c Lux 5-pc, lObp, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction 50C1c Lux 10-pc, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction lObp, 50C1c Lux 20-pc, oligos (synthetic) N / A N / A N / A N / A PAGE Extraction lObp, 50CIf 4-piece GFP, - PCR (2.5ng Q5u (NEB) 1 pL per 40 N / A N / A N / A control template) pmol dU, duringassemblyIf 4-piece GFP, a PCR (2.5ng Q5u (NEB) 1 pL per 40 N / A N / A N / A template) pmol dU, duringassemblyIf 4-piece GFP, 0 PCR (2.5ng Q5u (NEB) 1 pL per 40 N / A N / A N / A template) pmol dU, duringassemblyIf 4-piece GFP, Y PCR (2.5ng Q5u (NEB) 1 pL per 40 N / A N / A N / A template) pmol dU, duringassemblyIf 4-piece GFP, 5 PCR (2.5ng Q5u (NEB) 1 pL per 40 N / A N / A Kleen Spin template) pmol dU, during (BioRad)assemblyIf 4-piece GFP, e PCR (2.5ng Q5u (NEB) 1 pL per 40 IpL N / A N / A template) pmol dU, duringassemblyIf 4-piece GFP, PCR (2.5ng Q5u (NEB) 1 pL per 40 N / A N / A N / A template) pmol dU, duringassemblyIf 4-piece GFP, n PCR (2.5ng Q5u (NEB) 1 pL per 40 IpL N / A Kleen Spin template) pmol dU, during (BioRad) assemblyIf 4-piece GFP, 0 PCR (2.5ng Q5u (NEB) 1 pL per 40 IpL N / A Kleen Spin template) pmol dU, during (BioRad) assembly1g 19-piece Lux PCR (2.5ng Q5u (NEB) 1 pL per 40 4 pL 4 pL Kleen Spin Assembly template) pmol dU, prior (BioRad) Amplicon 22C to assembly1g 19-piece Lux PCR (2.5ng Q5u (NEB) 1 pL per 40 4 pL 4 pL Kleen Spin Assembly template) pmol dU, prior (BioRad) Amplicon 30C to assembly1g 19-piece Lux PCR (2.5ng Q5u (NEB) 1 pL per 40 4 pL 4 pL Kleen Spin Assembly template) pmol dU, prior (BioRad) Amplicon 50C to assemblyIj 20-piece Lux PCR (2.5ng Q5u (NEB) 1 pL per 40 4 pL 4 pL Kleen Spin 22C template) pmol dU, prior (BioRad) to assemblyIj 20-piece Lux PCR (2.5ng Q5u (NEB) 1 pL per 40 4 pL 4 pL Kleen Spin 50C template) pmol dU, prior (BioRad) to assemblyIj 20-piece Lux PCR (lpL lg 50C QR2xT 1 pL per 40 N / A N / A Monarch DNA 50C Assembly assembly pmol dU, prior gel extraction amplicon amplicon) to assembly2c 9 piece Lux PCR (2.5ng QR2xT 2.5 pL per 30pL 4 pL 4 pL Qiagen PCR assembly + attB template) purified PCR Purification backbones2c 19 piece PCR (lpL lg 50C QR2xT 2.5 pL per 30pL 4 pL 4 pL Monarch DNA assembly assembly purified PCR gel extraction + amplicon + attB amplicon, 2.5ng Qiagen PCR backbone backbone) Purification 9 Control PCR PCR (2.5ng QR2xT 2 pL per 30pL N / A 0.5 pL Qiagen PCR from plasmid + template) purified PCR Purification attB backbone2f 10 piece LoxP PCR (2.5ng QR2xT 2.5 pL per 30pL 4 pL 4 pL Qiagen PCR assembly with template) purified PCR Purification antibiotic2f 19 piece LoxP PCR (lpL lg 50C QR2xT N / A N / A N / A Monarch DNA Assembly assembly gel extraction + Amplicon amplicon) Qiagen PCR Purification 9 Control PCR PCR (2.5ng QR2xT N / A N / A N / A Monarch DNA LoxP from template) gel extraction plasmid3c MAPT gDNA PCR (IpL Human QR2xT 2 pL per 50 pL N / A 0.5 pL Qiagen PCR Assembled heart gDNA as Purification Isoforms template)3d VEGFA gDNA PCR (IpL Human QR2xT 1 pL per 50pL N / A 0.5 pL Monarch DNA Assembled heart gDNA as purified PCR gel extraction + Isoforms template) Qiagen PCR Purification 3d VEGFA oligos (synthetic) QR2xT 1 pL per 50pL N / A 0.5 pL Monarch DNA UPDOG purified PCR gel extraction + Isoforms Qiagen PCR Purification 3e BRCA1 gDNA PCR (IpL Human QR2xT 1 pL per 50pL N / A 0.5 pL Monarch DNA Assembled heart gDNA as purified PCR gel extraction + Isoforms template) + oligos Qiagen PCR (synthetic) PurificationQR2xT: Quanta Repliqa 2x Toughmix (QuantaBio)Table 4: Assembly ConditionsFIG Construct Buffer Ligase pmol per fragment in Volume Thermocycler Polymerase assembly assembly condition Assembly Amplicon 1c Lux 5-pc, 4bp, 16C T4LB IpL 0.00045 pmol 50pL 1 PGXL T4L1c Lux 10-pc, 4bp, 16C T4LB IpL 0.00045 pmol 50pL 1 PGXL T4L1c Lux 20-pc, 4bp, 16C T4LB IpL 0.00045 pmol 50pL 1 PGXL T4L1c Lux 5-pc, 4bp, 37C HTLB 2pL 0.00045 pmol 50pL 4 PGXL TaqL1c Lux 10-pc, 4bp, 37C HTLB 2pL 0.00045 pmol 50pL 4 PGXL TaqL1c Lux 20-pc, 4bp, 37C HTLB 2pL 0.00045 pmol 50pL 4 PGXL TaqL1c Lux 5-pc, 4bp, 50C HTLB 2pL 0.00045 pmol 50pL 5 PGXL TaqL1c Lux 10-pc, 4bp, 50C HTLB 2pL 0.00045 pmol 50pL 5 PGXL TaqL1c Lux 20-pc, 4bp, 50C HTLB 2pL 0.00045 pmol 50pL 5 PGXL TaqL1c Lux 5-pc, lObp, 16C T4LB IpL 0.00045 pmol 50pL 1 PGXL T4L1c Lux 10-pc, lObp, 16C T4LB IpL 0.00045 pmol 50pL 1 PGXL T4L1c Lux 20-pc, lObp, 16C T4LB IpL 0.00045 pmol 50pL 1 PGXL T4L1c Lux 5-pc, lObp, 37C HTLB 2pL 0.00045 pmol 50pL 4 PGXL TaqL1c Lux 10-pc, lObp, 37C HTLB 2pL 0.00045 pmol 50pL 4 PGXL TaqL1c Lux 20-pc, lObp, 37C HTLB 2pL 0.00045 pmol 50pL 4 PGXL TaqL1c Lux 5-pc, lObp, 50C HTLB 2pL 0.00045 pmol 50pL 5 PGXL TaqL1c Lux 10-pc, lObp, 50C HTLB 2pL 0.00045 pmol 50pL 5 PGXL TaqL1c Lux 20-pc, lObp, 50C HTLB 2pL 0.00045 pmol 50pL 5 PGXL TaqLIf 4-piece GFP, -control T4LB 2.5pL 0.075pmol Required, 20pL 2 N / A T4L 0.225pmol permissibleIf 4-piece GFP, a T4LB 2.5pL 0.075pmol Required, 20pL 2,5 N / A Hi-T4L 0.225pmol permissibleIf 4-piece GFP, 0 T4LB 2.5pL 0.075pmol Required, 20pL 2 N / A T4L 0.225pmol permissibleIf 4-piece GFP, Y T4LB 2.5pL 0.075pmol Required, 20pL 2 N / A T4L 0.225pmol permissibleIf 4-piece GFP, 5 T4LB 2.5pL 0.075pmol Required, 20pL 2 N / A T4L 0.225pmol permissibleIf 4-piece GFP, e Cutsmart 2.5pL 0.075pmol Required, 20pL 2 N / A T4L 0.225pmol permissibleIf 4-piece GFP, T4LB 2.5pL 0.075pmol Required, 20pL 3 N / A T4L 0.225pmol permissibleIf 4-piece GFP, n T4LB 2.5pL 0.075pmol Required, 20pL 3 N / A T4L 0.225pmol permissibleIf 4-piece GFP, 0 HTLB IpL 0.075pmol Required, 20pL 5 N / ATaqL 0.225pmol permissible1g 19-piece Lux T4LB 2.5pL 0.1284pmol 50pL 2 QR2xT Assembly Amplicon T4L permissible22C1g 19-piece Lux T4LB 2.5pL 0.1284pmol 50pL 3 QR2xT Assembly Amplicon T4L permissible30C1g 19-piece Lux HTLB 2pL 0.1284pmol 50pL 5 QR2xT Assembly Amplicon TaqL permissible50CIj 20-piece Lux 22C T4LB 2.5pL 0.0428pmol Required, 50pL 2 N / A T4L 0.1284pmolpermissibleIj 20-piece Lux 50C HTLB 2pL 0.0428pmol Required, 50pL 5 N / A TaqL 0.1284pmolpermissibleIj 20-piece Lux 50C HTLB 2pL 0.0428pmol Required, 50pL 5 QR2xT Assembly amplicon TaqL 0.1284pmolpermissible2c 9-piece Lux assembly T4LB IpL 0.3pmol fragment, 30pL 7 N / A + attB backbones T4L 0. Ipmol backbon2c 19-piece assembly T4LB IpL 0. Ipmol Assembly 30pL 7 QR2xT amplicon + attB T4L Ampicon, 0.0333pmolbackbone backbon9 Control PCR from T4LB IpL 0.03333 pmol 30pL 7 N / A plasmid + attB T4L backbone, 0.1 pMolbackbone insert2f 10-piece LoxP T4LB IpL Ipmol 30pL 7 N / A assembly with T4Lantibiotic2f 19-piece LoxP N / A N / A N / A N / A N / A QR2xT Assembly Amplicon9 Control PCR LoxP N / A N / A N / A N / A N / A N / A from plasmid3c MAPT gDNA HTLB IpL Ipmol 30pL 5 QR2xT Assembled Isoforms TaqL3d VEGFA gDNA HTLB IpL 0.25 pmol 50pL 5 QR2xT Assembled Isoforms TaqL3d VEGFA UPDOG HTLB IpL 0.25 pmol 50pL 5 QR2xT Isoforms TaqL3e BRCA1 gDNA HTLB IpL 0.025 pmol 50pL 5 QR2xT Assembled Isoforms TaqLT4LB: T4 Ligase Buffer (NEB)HTLB: Hifi Taq Ligase Buffer (NEB)TaqL: Taq Ligase (NEB)T4L: T4 Ligase (NEB)QR2xT: Quanta Repliqa 2x Toughmix (QuantaBio)PGXL: Primestar GXL (Takara Bio)Table 5: Notes Regarding MethodsFIG Construct Notes1c Lux 5-pc, 4bp, 16C IpL of assembly s used as template for Assembly Amplicon PCR1c Lux 10-pc, 4bp, 16C IpL of assembly s used as template for Assembly Amplicon PCR1c Lux 20-pc, 4bp, 16C IpL of assembly s used as template for Assembly Amplicon PCR1c Lux 5-pc, 4bp, 37C IpL of assembly s used as template for Assembly Amplicon PCR1c Lux 10-pc, 4bp, 37C IpL of assembly s used as template for Assembly Amplicon PCR1c Lux 20-pc, 4bp, 37C IpL of assembly s used as template for Assembly Amplicon PCR1c Lux 5-pc, 4bp, 50C IpL of assembly s used as template for Assembly Amplicon PCR1c Lux 10-pc, 4bp, 50C IpL of assembly s used as template for Assembly Amplicon PCR1c Lux 20-pc, 4bp, 50C IpL of assembly s used as template for Assembly Amplicon PCR1c Lux 5-pc, lObp, 16C IpL of assembly s used as template for Assembly Amplicon PCR1c Lux 10-pc, lObp, 16C IpL of assembly is used as template for Assembly Amplicon PCR 1c Lux 20-pc, lObp, 16C IpL of assembly is used as template for Assembly Amplicon PCR1c Lux 5-pc, lObp, 37C IpL of assembly is used as template for Assembly Amplicon PCR1c Lux 10-pc, lObp, 37C IpL of assembly is used as template for Assembly Amplicon PCR1c Lux 20-pc, lObp, 37C IpL of assembly is used as template for Assembly Amplicon PCR1c Lux 5-pc, lObp, 50C IpL of assembly is used as template for Assembly Amplicon PCR1c Lux 10-pc, lObp, 50C IpL of assembly is used as template for Assembly Amplicon PCR1c Lux 20-pc, lObp, 50C IpL of assembly is used as template for Assembly Amplicon PCRIf 4-piece GFP, -control DPN1 digestion occurs prior to assembly. USER digestion occurs during assemblyIf 4-piece GFP, a DPN1 digestion occurs prior to assembly. USER is added and protocol 2 is run for digestion. Instead of 22°C incubation consistent with protocol 2, a 50°C 16hr ligation is doneIf 4-piece GFP, f DPN1 digestion occurs prior to assembly. USER digestion occurs during assemblyIf 4-piece GFP, Y DPN1 digestion occurs prior to assembly. USER digestion occurs during assemblyIf 4-piece GFP, 5 DPN1 digestion and USER digestion occurs prior to assemblyIf 4-piece GFP, e DPN1 digestion occurs prior to assembly. USER digestion and RecJF digestion occurs during assembly. 2pL ATP added to Cutsmart buffer to fascilitate ligation If 4-piece GFP, DPN1 digestion occurs prior to assembly. USER digestion occurs during assembly. Reaction was purified after assembly with a QIAquick PCR Purification Kit (Qiagen). Concentrations were further measured by Qubit lx dsDNA High Sensitivity Assay Kit (Invitrogen, ThermoFisher Scientific) prior to transformation.If 4-piece GFP, n DPN1 digestion and USER digestion occurs prior to assembly. Reaction was purified after assembly with a QIAquick PCR Purification Kit (Qiagen).Concentrations were further measured by Qubit lx dsDNA High Sensitivity Assay Kit (Invitrogen, ThermoFisher Scientific) prior to transformation.If 4-piece GFP, 0 DPN1 digestion and USER digestion occurs prior to assembly. Reaction was purified after assembly with a QIAquick PCR Purification Kit (Qiagen).Concentrations were further measured by Qubit lx dsDNA High Sensitivity Assay Kit (Invitrogen, ThermoFisher Scientific) prior to transformation.1g 19-piece Lux Assembly IpL of assembly is used as template for Assembly Amplicon PCR Amplicon 22C1g 19-piece Lux Assembly IpL of assembly is used as template for Assembly Amplicon PCR Amplicon 30C1g 19-piece Lux Assembly IpL of assembly is used as template for Assembly Amplicon PCR Amplicon 50CM 20-piece Lux 22C DPN1 digestion, USER digestion, and RecJF digestion occurs prior to assembly Ij 20-piece Lux 50C Reaction was purified after assembly with a QIAquick PCR Purification Kit (Qiagen). Concentrations were further measured by Qubit lx dsDNA High Sensitivity Assay Kit (Invitrogen, ThermoFisher Scientific) prior to transformation.Ij 20-piece Lux 50C 50C Assembly Amplicon from 1g used as single permissible fragment processed Assembly amplicon as described here2f 19-piece LoxP Direct PCR off of Assembly AmpliconAssembly Amplicon3d VEGFA gDNA IpL of assembly is used as template for Assembly Amplicon PCR Assembled Isoforms3d VEGFA UPDOG IpL of assembly is used as template for Assembly Amplicon PCRIsoforms3e BRCA1 gDNA IpL of assembly is used as template for Assembly Amplicon PCRAssembled Isoforms

[0196] All assembly fragments from plasmid templates were treated with Dpnl (New England Biolabs) for Ihr at 37°C with a 20 min heat inactivation at 80°C in a 50 pL reaction of lx CutSmart using 1 pL Dpnl per 50 pL of initial PCR reaction. This digestion occurs after PCR purification of the initial fragments and prior to assembly. Conditions which perform USERdigestion prior to assembly had the USER enzyme mix added during this step. An additional purification was conducted after this digestion using either QIAquick PCR Purification Kit (Qiagen), Monarch DNA Gel Extraction Kit (New England Biolabs), or Quantum Prep PCR Kleen Spin Column (Biorad) (Tables 3-5). Kleen column purification had the temperature of the sample raised to 50°C immediately after digestion prior to passing through the column and was the method used for the FIG. 15.

[0197] All USER assemblies were treated with IpL USER enzyme mix (New England Biolabs) per 40pmol dU (or minimally IpL if total moles less than 40 pmol).

[0198] Purified products were then measured by Qubit lx dsDNA High Sensitivity Assay Kit, (Invitrogen, ThermoFisher Scientific) and stored at -20°C until assembly.

[0199] Oligos-based assembly fragments from FIG. 1 had oligos suspended by hand in lx TE buffer at pH 8.0 (Corning, ThermoFisher Scientific) to a final concentration of lOOuM based on manufacturers reported weight. To ensure adequate resuspension of the dried oligos, if the volume required to for a final concentration of lOOuM was less than 50uL of TE buffer according to the manufacturer’s reported weight, oligos would be resuspended in a volume of 50uL of buffer resulting in a lower final concertation. The concentration of all oligos were additionally measured using the Qubit ssDNA Assay Kit (Invitrogen, ThermoFisher Scientific) and subsequent calculations were based upon these measurements. Assembly fragments were generated from resuspended stock oligos by annealing top and bottom oligos to form a heteroduplex. This was done at luM in a 50uL reaction in lx T4 ligase buffer (New England Biolabs). The mixture would then be phosphorylated using luL of T4PNK (New England Biolabs) at 37°C for Ihr.

[0200] Sidewinder fragments were shipped at 50 pM in water and processed based on this reported concentration. The amount of coding oligo from the stock resuspension required for a 2uM final concentration in 25uL would be individually phosphorylated in a 22.5uL reaction using luL of T4PNK (New England Biolabs) in lxT4 ligase buffer at 37°C for Ihr, followed by an enzyme deactivation at 80°C for 10 minutes. The corresponding volume of stock barcode oligo needed for luM in 25uL was then added to the mix for a total volume of 25 pL.

[0201] All heteroduplexes from both experiments were annealed in PCR tubes on a thermocycler with an initial denaturation of 98°C for 10 minutes, followed by a gradual decrease in temperature down to 25°C at -1°C per minute. Once fragments are annealed, they were kept at 4°C until use. PAGE gel extraction of annealed heteroduplexes was performed on all fragments using 8% TBE gel (Invitrogen, ThermoFisher Scientific) and run at 200v for 35 minutes. Gel extraction was done according to published DNA nanotechnology protocol.Fragment assembly

[0202] Processed fragments were assembled under one of the following thermocycler conditions (Tables 3-5).

[0203] (1) 16°C Protocol: (a) 16°C get to temperature (i. addition of ligase); (b) 16°C for 16 hr; (c) 4°C hold.

[0204] (2) 22°C (literature protocol): (a) 37°C for 15 min; (b) 30°C for 15 min; (c) 10°C for 10 (i. addition of ligase); (d) room temperature (22°C) for 15 min.

[0205] (3) 30°C Protocol: (a) 37°C for 15 min; (b) 30°C for 30 min (i. addition of ligase); (c) 30°C for 125 min; (d) 65°C for 10.

[0206] (4) 37°C Protocol: (a) 37°C get to temperature (i. addition of ligase); (b) 37°C for 16 hr; (c) 4°C hold.

[0207] (5) 50°C Protocol: (a) 50°C get to temperature (i. addition of ligase); (b) 50°C for 16 hr; (c) 4°C hold.

[0208] (6) 75°C Protocol: (a) 75°C get to temperature (i. addition of ligase); (b) 75°C for 16 hr; (c) 4°C hold.

[0209] (7) Anneal Dropdown: (a) 45°C for 15 min; (b) 37°C for 15 min; (c) 30°C for 15 min; (d) 10°C for 10 min (i. addition of ligase); (e) 30°C for 1 hour.Human cDNA and gDNA assembling

[0210] Amplicon libraries were generated by select amplification of MAPT, VEGFA, and BRCA1 using designed primers (Table 1). PCR Ready Frist Strand cDNA from healthy human tissues were purchased from BioChain. cDNA from human heart, brain, spleen, skeletal muscle, and testis tissue were tested, with heart providing most consistent expression with isoform diversity across all 3 gene targets.

[0211] Exon fragments for MAPT, VEGFA, and BRCA1 were amplified similarly but from BioChain Human Heart gDNA (Table 1).Sidewinder 1 kb sub-assemblies

[0212] The Sidewinder assemblies for 1 kb SHH sub-assemblies were conducted at between 1-3 nM in 50 pL lx Hifi Taq Buffer (New England Biolabs). Two thermocycler conditions were utilized for assembly of the Sidewinder 1 kb sub-assemblies. Assemblies were conducted in 50uL reactions in lx HiFi Taq buffer (New England Biolabs). For sub-assemblies SI, S2, S3, S4, S5, S6, S9, and S10, a thermocycler protocol was used with 85°C for 5 minutes followed by the addition of 2uL of Taq ligase (New England Biolabs), then a slow decrease of -0.1°C per 6s down to 37°C, incubate at 37°C for 20 minutes, and repeat the thermocycling 85°C-37°C three additional times. For sub-assemblies S7, and S8, the “cycling” protocol of 85°C for 5 minutes, followed by the addition of 2uL of Taq ligase, then the reaction then cycles between85°C for 1 minute and 50°C for 2 minutes for 100 cycles. These cycles are then followed by 50°C for 1 hr.

[0213] PCR is then conducted on the final assembly as described.SHH 10 kb assemblies

[0214] For in vitro assembly, 1 kb Sidewinder sub-assemblies S3, S4, S7, S8, S9, and S10 were amplified from Sidewinder product amplicons, and SI, S2, S5, and S6 were amplified directly from Sidewinder product using RepliQa HiFi ToughMix (Quantabio) as described in PCR amplification and purification. Amplicons were purified using a QIAquick PCR Purification Kit (Qiagen), eluted in 40 pL of 10% Elution Buffer, and digested in a 50 pL reaction containing 1 pL USER enzyme (New England Biolabs), 5 pL 10x CutSmart buffer, and 5 pL FEO. Reactions were incubated at 37 °C for 1 hour followed by 80 °C for 20 minutes. Fragments were then gel extracted using the Monarch DNA Gel Extraction Kit (New England Biolabs). Concentrations of each sub-assembly were measured by Qubit lx dsDNA High Sensitivity Assay Kit (Invitrogen, ThermoFisher Scientific). Assemblies were set up with 0.4 pmol of each fragment in 50 pL lx Hifi Taq Buffer (New England Biolabs) using the 50 °C Super USER assembly described earlier. The resulting 10 kb SHH assembly product were amplified and sequenced by PacBio.

[0215] For in vivo assembly, all 1 kb Sidewinder sub-assemblies were amplified from Sidewinder product amplicons. Sub-assemblies were purified using QIAquick, digested with USER enzyme as described for the in vitro assembly, purified again with QIAquick, and quantified by Qubit. Each fragment was assembled into the pSClOl backbone using the 50 °C Super USER assembly and transformed as previously described.

[0216] All in vivo assemblies were validated by Nanopore sequencing through the GENEWIZ service. Sub-assemblies SI, S2, S4, and S5, which contain homopolymers >8 bp, were also sent for Sanger sequencing to confirm these regions. Sequence-validated fragments were amplified from colony resuspension, purified, digested, gel extracted, and assembled with 0.4 pmol of each construct as described for the in vitro assembly. The final 10 kb SHH product was assembled into the BAC backbone, transformed, and sequenced to identify SNP-free clones.Sequencing Analysis

[0217] The coverage plots of 22°C, 30°C and 50°C assembly amplicon were generated by aligning Oxford Nanopore reads (FASTQ) to the 6.1 kb LuxABCDE casette reference sequence using minimap2 (v2.30) with the map-ont preset, and alignments were streamed directly to samtools (vl.6) for coordinate sorting and indexing. Per-base coverage was calculated with samtools depth -aa to include zero-coverage positions and coverage values were parsed into contig-length-matched arrays. Coverage profiles were concatenated across contigsand plotted as read depth versus reference position in Python using NumPy and Matplotlib.

[0218] The fragment connection analysis was done by generating a list of all valid connections between all fragments accounting for 5’ and 3’ orientation. Using these -650 base references, Oxford Nanopore reads (FASTQ) were aligned using minimap2 (-x map-ont, -k 15, -w 10, -N 150, — secondary=yes, -p 0.6, 8 threads) and alignments were filtered by requiring coverage of >0.75* the specific reference length (absolute floor 250 bp) and >0.82 identity (MAPQ not thresholded). To prevent strand-mirroring double counts, passing hits were deduplicated per read by collapsing alignments with the same junction assignment and similar read coordinates using 25 bp-binned query start / end intervals. Each retained hit was collapsed into one of three connection classes where the left (L) and right (R) end of two fragments are connected in sense-sense orientation (RL) sense-antisense orientation (RR*) or sense-antisense orientation (LL*).

[0219] Oxford Nanopore Sequencing was used for sequence verification and generation of the coverage plots for the 19-piece Assembly Amplicons, amplicons from human cDNA, HighT isoform assemblies, and NWR SHH HighT Assembly Amplicons were analyzed with Nanopore sequencing.

[0220] Version2: Nanopore sequencing reads of cDNA PCR products were aligned to gDNA reference sequences using minimap2 and further processed with SAMtools to generate a coverage plot showing the exons to be selected. For PCR products of the assembly reaction of the selected exons, plasmidsaurus Premium sequencing service was used for analysis. Fastq reads that are shorter than 10% of the desired isoform or that do not contain both PCR primers were discarded since those are likely sequencing artifacts. Remaining reads were aligned against the desired isoform sequence using the blastn-short program of Blastn. Alignments with more than 70% of query coverage and more than 70% of percent identity were considered valid hits. Constructed isoforms were identified based on the order of the fragments in the alignment. Isoforms identified were present at >1% of filtered reads, with all reads <1% binned into Other.

[0221] Homopolymers in NWR assemblies validated through pair end Sanger sequencing using GENEWIZ Sanger sequencing service.Agarose sei imaging

[0222] All gel images are 1-2% agarose gels stained with Sybr Safe (Invitrogen, ThermoFisher Scientific) run at 135V for 25 minutes in 0.5x TBE buffer. The 1 kb+ ladder (New England Biolabs). Oligo-based assembly gels depict 1 pL of final PCR loaded onto the gel. 19-piece LuxABCDE assembly gel depicts 1 pL of final PCR loaded onto the gel. Sidewinder assembly gel depicts 50ng of sample loaded onto the gel. The 10 kb sample depicts 5 pL final PCR loaded onto gel.Transformation

[0223] Outside of attBP and LoxP tranformations (below section), all transformations were done using NEB 10-beta electrocompetent E. coli cells (New England Biolabs). Assemblies were processed as described. 2 pL purified products were added to 25 pL electrocompetent cells and transferred to an electroporation cuvette and pulsed at 2500V. Then 2 mL NEB 10-beta Stable Outgrowth Media (New England Biolabs) is added to the cuvette and entire volume is transferred to a 15 mL tube for a 1-hour recovery at 37°C with shaking.

[0224] A dilution series is then established assuming 2 mL final volume by adding 22.2 pL of the n-1 to 200 pL 10% Luria-Bertani (LB) to make the nth dilution. All 200 pL were spread on an LB-agar plate using glass beads. Antibiotic concentrations and negative marker concentrations used in this study are: Carbenicillin 100 pg / mL, Chloramphenicol 20 pg / mL, Kanamycin 50 pg / mL, Spectinomycin 60 pg / mL, Streptomycin 100 pg / mL, Tetracycline 10 pg / mL, Sucrose 7.5% w / v. Plates were then incubated for 24-48 hours until all clones could be clearly distinguished.Strain Engineering and Electrocompetency

[0225] For LambdaRed recombination, Cre-LoxP recombination, or attBP recombination, electrocompetent cells were prepared similarly. Cells containing recombination machinery were grown overnight in 10 mL of LB plus respective antibiotics. OD was measured at 600 nm using NanoDrop Onec. The next day cells were diluted to OD = 0.05 in 500-1000 mL of LB and antibiotics and incubated at 37C with 170 rpm shaking. Cultures were grown until OD = 0.2, where Arabinose was added to final 0.5% concentration to induce recombination machinery. At OD = 0.5, cells were placed on ice for 30 minutes. Cultures were spun down at 4C for 10 minutes at 4000 rpm and washed with pre-chilled water, then again with pre-chilled 15% glycerol. Cells were spun down and resuspended in 750 pL of 15% glycerol before flash frozen in liquid nitrogen and stored at 80C.

[0226] For the attBP and LoxP recipient cells, MDS42 was used as the parental strain and LambdaRed recombination was first used to knockout RecA and the native HK022 attB sites by integrating a chlor-sacB selection cassette before patching out with a 120 bp primer to generate a scarless deletion (DNA in Table 2). A PCR amplicon of the attP or 2LoxP landing site was recombined in at the dif site. Strains were cured of the LambdaRed plasmid before having the pSC101-HK022 (AmpR) or pSClOl-Cre (TetR) plasmids introduced. These final strains were made induced and electrocompetent as described above for integration experiments.Transformation and Plating attBP and LoxP Ligations

[0227] Fifty- 100 ng of purified ligations were transformed into electrocompetent cells and recovered for 4 hours before plating. attBP transformants were serially diluted onplates containing chloramphenicol, Kanamycin, and carbenicillin. LoxP transformants were plated on just chloramphenicol. Transformations were performed in triplicate, from independent ligations reactions. Cells were restreaked on same selection plates before genotyping (FIGS.8A-8C).Pheno typing and Imaging

[0228] For phenotypic spots, colonies were grown overnight, OD normalized to 0.2, then 5 pL of cells were spotted on agar plates containing selective media.

[0229] Imaging of colorimetric, luminescence and GFP was performed using BioRad ChemiDoc Imaging System with following settings: Chemiluminescent Blot 30s (white tray), Colorimetric Blot 0.5s (white tray), GFP on SYBR Green 0.5s (blue tray).Statistics

[0230] All reported assemblies in FIGS. IF, 1J, 2C, 2F were done with three biological replicates. Each sample was an independent assembly reaction that was transformed a single time to get respective colony counts. Data presentation is composed of the mean and standard deviation of the biological replicates.

[0231] 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 art that 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.

[0232] 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.

[0233] 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, suchan 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.

[0234] 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.

[0235] 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.

[0236] 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 method, comprising:providing m assembly precursors, wherein m is a positive integer, wherein the assembly precursors are double-stranded DNA molecules comprising one or two 5’ overhang-forming regions, wherein each 5’ overhang-forming region comprises two or more 5’ terminal cleavage regions, wherein each of the 5’ terminal cleavage regions comprises one or more non-canonical nucleotide(s); andcontacting the m assembly precursors with cleavage agent(s) configured to remove the non-canonical nucleotide(s) to generate m assembly fragments, wherein each of the m assembly fragments comprises one or two 3’ overhang(s) exposed by removal of the corresponding 5' terminal cleavage region(s) of the 5’ overhang-forming region, wherein each 3’ overhang begins at a position corresponding to the 3’ most non- canonical nucleotide of the corresponding 5’ terminal cleavage region.

2. The method of claim 1, wherein the non-canonical nucleotide(s) comprises deoxyuridine, deoxyinosine, deoxy-7-m ethylguanosine, deoxy-5,6-dihydroxythymidine, deoxy-3 -methyladenosine, 5-methyl-deoxycytidine, O-6-methyl-deoxyguanosine, 5-iodo-deoxyuridine, 8-oxy-deoxy guanine, l,N6-ethenoadenine, 8-oxo-guanine (8oxoG), or any combination thereof.

3. The method of claim 2, wherein the non-canonical nucleotide(s) comprise deoxyuridine.

4. The method of any one of claims 1-3, wherein the cleavage agent(s) comprise DNA glycosylase-lyase Endonuclease VIII, a DNA glycosylase, an AP cleaving agent, APE 1 (AP Endonuclease 1), Endo III (Endonuclease III), Endo IV (Endonuclease IV), Endo V (Endonuclease V), Endo VIII (Endonuclease VIII), Fpg (formamido-pyrimidine-DNA glycosylase), OGGI (8-oxoguanine DNA glycosylase 1), NEIL1 (Endonuclease VUI-like 1), T7 Endo I (T7 Endonuclease I), T4 PDG (T4 pyrimidine dimer DNA glycosylase), UDG (uracil DNA glycosylase), SMUG1 (Single-strand selective monofunctional uracil DNA glycosylase), AAG (methylpurine DNA glycosylase), or any combination thereof.

5. The method of any one of claims 1-4, wherein the contacting step is performed in the presence of a 5’ ssDNA exonuclease configured to remove waste products derived from the 5’ terminal cleavage regions, optionally selected from the group comprising Red, RecJF, Exo VII, Dna2, T5 Exo, or any combination thereof.

6. The method of any one of claims 1-5, wherein the 5’ terminal cleavage region comprises 1, 2, 3, 4, or 5 non-canonical nucleotide(s), optionally the step of providing m assembly precursors comprises PCR amplification reaction(s) comprising precursor primer(s) having a 5’ terminal cleavage region(s) comprising said non-canonical nucleotide(s), optionallym sets of precursor primer(s).

7. The method of any one of claims 1-6, wherein at least two of the m assembly precursors are contacted with the one or more cleavage agents in separate reaction vessels.

8. The method of any one of claims 1-7, wherein at least two of the m assembly precursors are contacted with the one or more cleavage agents in a common reaction mixture.

9. The method of any one of claims 1-8, wherein the method comprises purifying the assembly precursors and / or assembly fragments, optionally one or more purification steps comprising:gel electrophoresis, optionally 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, optionally size exclusion column purification.

10. The method of any one of claims 1-9, wherein the 3’ overhang:comprises the sequence ANxWNyWNzT, wherein x, y, and z are positive integers; exhibits no secondary structure (NSS), optionally at 30°C;is optimized to minimize cross reactivity with non-complementary overhangs; is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, 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%, or 100%; and / or comprises a melting temperature (Tm) of about 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, 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, or 75°C.

11. The method of any one of claims 1-10, wherein the contacting step is performed at a first incubation temperature for a first period of time, optionally:the first incubation temperature is about 8°C, 9°C, 10°C, 11°C, 12°C, 13°C,14°C, 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, or 40°C, optionally 37°C; andthe first period of time is about 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, optionally one hour.

12. The method of any one of claims 1-11, wherein one or more of the m assembly precursors comprises an Uracil Primer Dimer Overhang Generation (UPDOG) product, and wherein providing the m assembly precursors comprises:providing a first UPDOG primer and a second UPDOG primer capable of hybridizing each other, wherein the first UPDOG primer and second UPDOG primer each comprise 5’ terminal cleavage region(s) comprising two or more non-canonical nucleotide(s); andcontacting the first UPDOG primer and the second UPDOG primer in the presence of a polymerase to generate the UPDOG product.

13. The method of any one of claims 1-12, wherein the method comprises:providing n assembly fragments, wherein at least two of the n assembly fragments comprise or are derived from the m assembly fragments generated by the method of any one of claims 1-12, wherein the n assembly fragments are doublestranded DNA molecules comprising one or two 3’ overhang(s) configured to hybridize to a complementary 3’ overhang of another of the n assembly fragments; and incubating the n assembly fragments in the presence of a ligase, thereby generating an assembled product.

14. A method, comprising:providing n assembly fragments, wherein n is an integer greater than 1, wherein the n assembly fragments are 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; andincubating 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.

15. The method of claim 14, wherein the method comprises generating one or more of the n assembly fragments according to the method of any one of claims 1-13.

16. The method of claim 15, wherein contacting the m assembly precursors with the cleavage agent(s) and incubating the n assembly fragments in the presence of the ligase are performed simultaneously.

17. The method of any one of claims 13-16, wherein the assembly fragments comprise or are derived from Golden Gate assembly fragments, optionally the method further comprises contacting assembly precursors with Type-IIS and / or Type-IIP cleaving agents.

18. The method of any one of claims 13-17, wherein the ligase is capable of ligating nicks at junctions of hybridized assembly fragments to generate the assembled product.

19. The method of any one of claims 13-18, wherein the ligase is selected from the group comprising a thermostable ligase 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.

20. The method of any one of claims 1-19, wherein the assembly fragment:is at least 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, or 5000, 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%; and / or comprises a melting temperature (Tm) of about 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, 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, or 75°C.

21. The method of any one of claims 1-20, wherein the incubating step is performed at second incubation temperature for a second period of time, optionally:the second incubation temperature is about 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 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, 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, 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, or 75°C, optionally 50°C; andthe second period of time is about 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, optionally 16 hr.

22. The method of any one of claims 1-21, wherein the second incubation temperature is at least about 0.5°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 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, or 50°C, higher than the median 3’ overhang Tm.

23. The method of any one of claims 1-22, wherein n or m 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.

24. The method of any one of claims 13-23, wherein n is 3 or more, wherein the assembled product is linear,wherein the first assembly fragment of the n assembly fragments comprises a second 3 ’ overhang,wherein each (z)th assembly fragment of the n assembly fragments comprises a first 3 ’ overhang and a second 3 ’ overhang, wherein 1 < z < «,wherein the (zz)th assembly of the n assembly fragments fragment comprises a first 3 ’ overhang,wherein the first 3’ overhang of each (z)th assembly fragment is complementary to the second 3’ overhang of the (z-l)th assembly fragment, andwherein the second 3’ overhang of each (z)th assembly fragment is complementary to the first 3’ overhang of the (z+l)th assembly fragment.

25. The method of any one of claims 13-24, wherein n is 3 or more, wherein the assembled product is circular,wherein each assembly fragment of the n assembly fragments comprises a first 3’ overhang and a second 3 ’ overhang,wherein the first 3’ overhang of the first assembly fragment of the n assembly fragments is complementary to the second 3’ overhang of the (zz)th assembly fragment of the n assembly fragments, andwherein for each (z)th assembly fragment, wherein 1 < z < nthe first 3’ overhang of the (z)th assembly fragment is complementary to the second 3’ overhang of the (z-l)th assembly fragment, andthe second 3’ overhang of the (z)th assembly fragment is complementary to the first 3’ overhang of the (z+l)th assembly fragment.

26. The method of any one of claims 13-25, wherein:the first assembly fragment of the n assembly fragments comprises a first terminal region, optionally a 5’ terminal region; and / orthe (zz)th assembly fragment of the n assembly fragments comprises a second terminal region, optionally a 3’ terminal region.

27. The method of any one of claims 13-26, wherein one or more of the n assembly fragments comprise an internal segment, wherein the internal segment does not comprise the 3’ overhang(s), and wherein the internal segment is double-stranded.

28. The method of any one of claims 1-27, wherein the assembly precursors and / or the assembly fragments:comprise or are derived from synthetic oligonucleotides; and / orcomprise or are derived from Sidewinder products, TADA products, rolling circle amplification products, restriction enzyme digestion products, reverse transcription products, CRISPR-excised products, PCR amplification products, template-independent polymerase products, recombinase-generated products, phage-derived products, or any combination thereof.

29. The method of any one of claims 13-28, 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; and / orPCR amplification comprises PCR primer(s) having an overhang, and wherein the final synthetic sequence comprises the sequence of said overhang.

30. The method of any one of claims 13-29, wherein the method comprises purification of the assembled product, the amplified product, or products thereof, optionally said purification step compromises: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.

31. The method of any one of claims 13-30, wherein the assembled product, the amplified product, or products thereof, comprises a final synthetic sequence, and wherein the final synthetic sequence is at least about 500 bp, 750 bp, 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.

32. The method of claim 31, wherein the final synthetic sequence: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.

33. The method of claim 31 or 32, 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).

34. The method of any one of claims 1-33, 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.

35. The method of any one of claims 1-34, 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 anycombination 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 any combination 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 partiallydifferentiated 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-APOBEC1 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 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.

36. The method of any one of claims 1-35, 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.

37. The method of any one of claims 1-36, 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.

38. The method of any one of claims 1-37, 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 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.

39. The method of any one of claims 1-38, 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 ofa 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.

40. The method of any one of claims 1-39, 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 gate, AND logic gate, NOR logic gate, NAND logic gate, IMPLY logic gate, NIMPLY logic gate, XOR logic gate, and an XNOR logic gate.

41. The method of any one of claims 1-40, 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.

42. The method of any one of claims 1-41, 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.

43. The method of any one of claims 1-42, wherein a payload gene encodes an isoform of a payload protein comprising two or more exons, optionally a therapeutically relevant isoform.

44. The method of any one of claims 1-43, wherein providing n assembly fragments comprises:PCR amplification of one or more one exons from genomic DNA, optionally patient-derived genomic DNA,optionally at least one of the two or more exons is encoded in the 5’ end of an amplification primer.

45. The method of any one of claims 1-44, wherein at least one assembly fragment of the n assembly fragments is an UPDOG product comprising two or more exons.

46. The method of any one of claims 1-45, 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 elementcomprises 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.

47. The method of any one of claims 1-46, 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 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.

48. The method of any one of claims 13-47, wherein the method comprises replication of the assembled product, the amplified product, or products thereof, in a cell,optionally episomal replicons.

49. The method of any one of claims 13-48, wherein the method comprises delivery of the assembled product, the amplified product, or products thereof, to a cell, optionally delivery comprises one or more of transformation, transfection, transduction, conjugation, electroporation, lipid-mediated transfection, liposome-mediated delivery, nanoparticle-mediated delivery, viral vector-mediated delivery, microinjection, biolistic delivery, calcium phosphate-mediated transfection, PEG-mediated transformation or transfection, further optionally the method comprises conjugative transfer from a donor cell to a recipient cell.

50. The method of any one of claims 31-49, 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.

51. The method of any one of claims 31-50, 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.

52. The method of any one of claims 31-51, wherein the method comprisesintegration of the final synthetic sequence, or a portion thereof, into the genome of a cell, optionally via a site-specific recombinase, further optionally:the site-specific recombinase is a tyrosine recombinase, optionally selected from the group comprising HK022 recombinase, Cre, lambda phage, phage 186, phi80, P21, Flp, XerC / D, XerA, lambda integrase (Int), P2 integrase, FimB, FimE, HbiF, Rci, or any combination thereof;the site-specific recombinase is a serine recombinase, optionally selected from the group comprising Bxbl, Tn3, y6 resolvase, Gin, Hin, TP901-1, C31, TGI, Rvl, C.IS607-like serine transposases, or any combination thereof; and / orthe method comprises integration without episomal replicon intermediate(s).

53. The method of any one of claims 31-52, 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.

54. The method of any one of claims 13-53, wherein the assembled product, the amplified product, or product thereof, comprises a circular DNA molecule comprising:a recombination site, optionally an attB recombination site;terminator(s); and / ora transcriptionally inactive first selection marker gene configured to be transcriptionally active upon correct integration.

55. The method of any one of claims 1-54, wherein the method comprises introducing the circular DNA molecule into a recipient cell comprising:a recombinase protein, optionally a tyrosine recombinase; andan integration site comprising a complementary recombination site, optionally an attP recombination site,optionally the integration site comprises an upstream constitutive promoter and / or a transcriptionally active second selection marker gene,optionally the terminator(s) render the second selection marker gene transcriptionally inactive upon correct integration,optionally the introduction into the recipient cell comprises conjugative transfer from a donor cell.

56. The method of any one of claims 13-55, wherein the assembled product, the amplified product, or product thereof, comprises a linear DNA molecule comprising:a first recombination site and a second recombination site, optionally situated on the 5’ and 3’ terminal ends, respectively, further optionally LoxP (LI) and Lox2272 (L2) recombination sites; and / ora first selection marker gene.

57. The method of any one of claims 1-56, wherein the method comprises introducing the linear DNA molecule into a recipient cell comprising:a recombinase protein, optionally a serine recombinase; andan integration site comprising the first recombination site and the second recombination site, optionally LoxP (LI) and Lox2272 (L2) recombination sites, optionally the integration site comprises one or more selection marker genes situated between the first recombination site and the second recombination site, optionally the introduction into the recipient cell comprises conjugative transfer from a donor cell.

58. The method of any one of claims 1-57,(a) wherein the first assembly fragment:is an invariant fragment, wherein all instances of the invariant first assembly fragment are identical; oris a variant fragment, wherein two or more instances of the variant first assembly fragment differ with respect to the sequence of the internal segment; (b) wherein at least one (z')th assembly fragment is an invariant fragment, wherein all instances of the invariant (z)th assembly fragment are identical;(c) wherein at least one (z')th assembly fragment is a variant fragment, wherein two or more instances of the variant (z)th assembly fragment differ with respect to the sequence of the internal segment; and / or(d) wherein the (zz)th assembly fragment:is an invariant fragment, wherein all instances of the invariant (zz)th assembly fragment are identical; oris a variant fragment, wherein two or more instances of the variant (zz)th assembly fragment differ with respect to the sequence of the internal segment.

59. The method of claim 58, wherein variant fragments comprise predefined codon variations, optionally codons variations configured to achieve modified and / or improved protein function(s).

60. The method of any one of claims 58-59, wherein at least one of the n assembly 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.

61. The method of any one of claims 1-60,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.

62. The method of any one of claims 13-61,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 assembly 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 assembly 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 assembly fragments or comprise a mis-assembled junction; and / orwherein the mis-ligation rate at a junction is less than 1 in 1000, 1 in 10000, 1 in 100000, 1 in 1000000, 1 in 10000000, or 1 in 100000000.

63. The method of any one of claims 1-62, 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 a ligation temperature higher than the median melting temperature of the 3’ overhangs, optionally Polymerase Cycling Assembly (PCA), Gibson assembly, USER, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly.

64. The method of any one of claims 1-63, 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 assembly fragments become a component of an assembled product.

65. A composition, comprising:assembled products, or products thereof, generated by the method of any one of claims 13-64.

66. The composition of claim 65, wherein the composition comprises a plurality of cells comprising the assembled products, or products thereof.

67. A method, comprising:providing the combinatorial library of any one of claims 60-61, or a product thereof;expressing the one or more payload genes in cell(s); andscreening for a property of interest.

68. The method of claim 67, wherein screening comprises fluorescence-activated cell sorting (FACS), cell viability assay, ELISA, co-immunoprecipitation, a bead-based immunoassay, or any combination thereof.

69. The method of any one of claims 67-68, 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.

70. The method of any one of claims 67-69, 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, 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 / 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.

71. The method of any one of claims 67-70, 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.

72. The method of any one of claims 67-71, 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.

73. A kit, comprising:one or more of the m assembly precursors, m sets of precursor primer(s), and n assembly fragments of any one of claims 1-64,one or more cleavage agent(s), optionally DNA glycosylase-lyase Endonuclease VIII, a DNA glycosylase, an AP cleaving agent, APE 1 (AP Endonuclease 1), Endo III (Endonuclease III), Endo IV (Endonuclease IV), Endo V (Endonuclease V), Endo VIII (Endonuclease VIII), Fpg (formamido-pyrimidine-DNA glycosylase), OGGI (8-oxoguanine DNA glycosylase 1), NEIL1 (Endonuclease Vlll-like 1), T7 Endo I (T7 Endonuclease I), T4 PDG (T4 pyrimidine dimer DNA glycosylase), UDG (uracil DNA glycosylase), SMUG1 (Single-strand selective monofunctional uracil DNA glycosylase), AAG (methylpurine DNA glycosylase), or any combination thereof.a non-thermostable ligase, a thermostable ligase, 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 / ora ligation buffer, optionally comprising:a reaction buffer configured to support annealing, ligation, and / or amplification;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 / orone 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.

74. A system for synthesizing nucleic acids, comprising:one or more of the m assembly precursors, m sets of precursor primer(s), and n assembly fragments of any one of claims 1-64,one or more cleavage agent(s), optionally DNA glycosylase-lyase Endonuclease VIII, a DNA glycosylase, an AP cleaving agent, APE 1 (AP Endonuclease 1), Endo III (Endonuclease III), Endo IV (Endonuclease IV), Endo V (Endonuclease V), Endo VIII (Endonuclease VIII), Fpg (formamido-pyrimidine-DNA glycosylase), OGGI (8-oxoguanine DNA glycosylase 1), NEIL1 (Endonuclease Vlll-like 1), T7 Endo I (T7 Endonuclease I), T4 PDG (T4 pyrimidine dimer DNA glycosylase), UDG (uracil DNA glycosylase), SMUG1 (Single-strand selective monofunctional uracil DNA glycosylase), AAG (methylpurine DNA glycosylase), or any combination thereof.a non-thermostable ligase, a thermostable ligase, 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 / ora ligation buffer, optionally comprising:a reaction buffer configured to support annealing, ligation, and / or amplification;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 / orone 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 / orcolumn purification reagent(s),optionally the system does not comprise one or more reagents employed with Polymerase Cycling Assembly (PCA), Gibson assembly, Yeast Assembly, Homologous Recombination, and / or Golden Gate assembly.

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