Abstract:
This invention provides processes to assemble many (greater than 20) partially overlapping single stranded DNA molecules (fragments) having preselected sequences, followed by extension of those strands that hybridize at terminal overlap regions, and ligation of the extend products, creating a double-stranded DNA assembly. These processes use non-standard nucleotides carrying heterocyclic nucleobase analogs that implement non-standard hydrogen bonding patterns; these allow controlled annealing of the single stranded fragments via Watson-Crick rules, with less or no interference from a range of non-Watson Crick interactions, hairpin formations, or off-target hybridization displayed by standard nucleobases. This process includes an optional conversion step that replaces non-standard nucleobase pairs with standard nucleobase pairs, generating large synthetic DNA (LS-DNA) molecules containing only natural nucleotides. As useful application, this invention allows the assembly of genes encoding whole proteins (typically 1000-3000 nucleotide pairs) from a collection of single stranded DNA fragments at reduced cost and effort.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a nonprovisional application that derives from, and claims priority to, U.S. nonprovisional application Ser. No. 13/936,309 filed Jul. 9, 2013 which derived from and claimed priority to provisional patent application 61/669,295, which was filed Jul. 9, 2012, for “Inexpensive Autonomous Assembly of Ultra-Large (UL) DNA Constructs”. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with government support under a contract awarded by the United States Defense Advanced Research Project Agency (HR0011-12-C-0064). The government has certain rights in the invention. 
    
    
     THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT 
     Not applicable 
     INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC 
     None 
     BACKGROUND OF THE INVENTION 
     (1) Field of the Invention 
     This invention is in the field of nucleic acid chemistry, more specifically the synthesis of nucleic acids, more specifically to the synthesis of large DNA molecules, and more specifically to processes and procedures that create large (more than 1000 base pairs) double-stranded DNA via the assembly of large numbers of short single stranded DNA molecules having preselected sequences. 
     (2) Description of Related Art 
     Synthetic biology needs processes to enable low-cost and rapid assembly of many synthetic DNA fragments into large DNA assemblies. For example, in 2012, DARPA issued a small business grant solicitation seeking technology to assemble single-stranded synthetic fragments to give 20,000 bp ML-DNA constructs. A short while earlier, the Army Research Office issued a small business grant solicitation seeking companies to design software to allow 30,000 base pairs of single stranded DNA self-assemble to form nanostructures. 
     Unfortunately, the realities behind the biophysics of DNA make these goals fanciful, if the attempt is made with standard DNA. With just four nucleotides, the information density of standard DNA is too low to allow (without exquisite design) more than ca. a dozen single strands to self-assemble upon simple mixing. With more fragments containing only natural nucleotides, the vagaries of “strong” and “weak” G:C and A:T pairs, hairpins, off-target Watson-Crick hybridization, and non-Watson Crick interactions (e.g wobble and major groove binding) defeat self-assembly. These can be illustrated by mentioning the following problems: 
     Problem (A). 
     Different DNA base pairs do not contribute uniformly to duplex stability. The largest source of this non-uniformity in strand hybridization is a feature of standard DNA that joins A:T pairs by just two hydrogen bonds and G:C pairs by three. Thus, A:T pairs contribute to duplex stability consistently less than G:C pairs. This makes it challenging to design DNA fragments with different nucleotide compositions that hybridize to their complements with the same affinity. 
     Problem (B). 
     DNA strands can interact in ways outside of those specified by the canonical Watson-Crick pair. In addition to wobble pairing (e.g. G:T pairs), DNA can form major groove interactions (e.g. G-quartets). These, illustrated in  FIG. 1  and  FIG. 2 , can (in appropriate contexts) be stronger than Watson-Crick pairing and can defeat pairing between large numbers of single stranded DNA molecules designed solely by applying Watson-Crick rules. 
     Problem (C). 
     Intra-strand folding can defeat desired inter-strand interactions needed for hybridization, primer extension, and ligation. Hairpin structures formed by a single strand, for example, can easily disrupt inter-strand hybridization that intended for a multi-strand assembly ( FIG. 3 ). The easy accessibility of hairpins can be illustrated by some simple mathematics. The 5′-nucleotide of a standard DNA molecule must be G, A, T, or C. Whatever it is, it can find a complementary C, T, A, or G (respectively) with a one-in-four probability at each base farther into the sequence. Within a random sequence 64 nucleotides in length, the final one, two, and three nucleotides will find perfect complements 16 times, 4 times, and once within that sequence, on average. These will form hairpins with stems that are joined by one, two, and three perfect base pairs respectively. Stems with four or five pairs and loops of 2-5 nucleotides are adequate to disrupt hybridization. Therefore, loops must be avoided by design, and this design becomes difficult to manage as the number of fragments increase. 
     Problem (D): 
     Even if DNA had access only to Watson-Crickery, even if all nucleobase pairs contributed equally to duplex stability, and even if single strands never folded by themselves, the autonomous self-assembly problem would still not be trivial. With only four nucleotide letters to encode information, the information density of natural DNA is low. For a bacterial sized genome having a random sequence, all 10mers are present once. Overlapping 10mers are more than adequate to support ligation, even if they include one or two mismatches, at temperatures when typical ligases operate. This low information density makes it essentially impossible to do reliable self-assembly from any more than a dozen or so fragments. Each complement is present at low concentrations, making the rates at which they find each other low a priori. The rate of hybridization is slowed as GACT DNA fragments find “off target” GACT fragments, bind to them, and dwell for a time before dissociating to seek their “on target” fragments. 
     Given these realities of the chemical structure of natural DNA, it is hardly surprising that Nature rarely does what synthetic biologists want to do: Large-scale assembly by way of the hybridization of multiple single stranded fragments. Non-uniformity in the binding of sequences of natural nucleotides make it essentially impossible to assemble by autonomous hybridization of thousands (or more) nucleobase pairs, even if the primary products have no errors at all. Therefore, in natural biology, large-scale DNA assemblies are carried forward carefully from generation to generation, with strand displacement at the core of polymerization and specifically targeted ligation events that do not allow the DNA to wander into multiple single strands. 
     Thus, most large synthetic DNA (LS-DNA) molecules today are obtained via the “Gibson method” [Gibson 2011], rather than the spontaneous self-assembly of many single DNA strands prepared by synthesis. The Gibson method reproduces in vitro the natural Szostak process for recombination in vivo [Szostak et al. 1983]. It starts with pre-annealed duplexes, cuts them back with a 3′-exonuclease to generate sticky ends (without cutting back so far as to disrupt the duplex) and then uses the resulting sticky ends to assemble the duplexes with overhangs. Expert intervention is required at many steps in the process, creating costs. 
     While [Gibson 2011] speaks of single strand assembly, including single stranded assembly in yeast cells [Gibson 2009], they teach that to “ensure that error-free molecules are obtained at a reasonable efficiency, only eight to twelve 60-base oligos are assembled at one time” [Gibson 2011]. This teaching, we presume, reflects the problems listed above, which are deeply embedded in the molecular structure of natural DNA. These drive the need for inventive processes to allow LS-DNA assembly from multiple single stranded synthetic DNA fragments. 
     BRIEF SUMMARY OF THE INVENTION 
     This invention provides processes to assemble more than 12, and preferably at least 20, strands of single stranded DNA fragments, preferably 50-100 nucleotides in length, and more preferably 50-80 nucleotides in length, to give large synthetic DNA (LS-DNA) constructs. Those fragments intended to be ligated must have free 5′-phosphate groups at the ends to be ligated and/or free 3′-hydroxyl groups at the ends to be ligated. Those fragments intended to be extended must have free 3′-hydroxyl groups at the ends to be extended. Further, only those 3′-ends annealed to another of the DNA fragments with a 3′-underhang will be extended ( FIGS. 5-10 ). As useful applications, this invention therefore allows the assembly, from a collection of single stranded DNA fragments, of genes encoding whole proteins (typically 1000-3000 nucleotide pairs), plasmid-sized constructs (5000-10000 base pairs), artificial chromosomes (such as BACs having 10,000 to 300,000 nucleotide pairs), or even chromosomes (having a million or more nucleotide pairs). 
    
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) 
         FIG. 1 . Illustration of Problem (A). Wobble pairing can cause single stranded oligonucleotides to not interact as predicted by Watson-Crick rules, especially in an annealing attempt that involves more than 12 single stranded oligonucleotides (and certainly 20 or more). As AEGIS pairs are all joined by three hydrogen bonds, AEGIS pairs are stronger than A:T pairs, allowing correct pairs to dominate over wobble, which involve two hydrogen bonds [Benner et al. 2010]. 
         FIG. 2 . Further illustration of Problem (A). Major groove binding interactions can cause single stranded oligonucleotides to not anneal as predicted by Watson-Crick pairing, especially if the annealing attempt involves more than 12 single stranded oligonucleotides (and certainly 20 or more). Various of the AEGIS purines cannot form these major groove interactions, if they are implemented in a form that lacks a nitrogen atom at what would be formally called “position 7” on the analogous purine. These include P and 7-deazaisoguanosine. 
         FIG. 3 . Illustration of Problem (B). Hairpins prevent certain single stranded oligonucleotides from interacting with their target as predicted by Watson-Crick pairing. Hairpins that involving a folding back of the 3′-end also can be extended by polymerases to give undesired products. While hairpins can be avoided by careful design if only a few oligonucleotides are being assembled, they are difficult to avoid by design if the assembly attempt involves more than 12 single stranded oligonucleotides (and certainly for 20 or more). Hairpins cannot form if the ends contain AEGIS components (shown in cartoon form). 
         FIG. 4 . Watson-Crick pairing follow two complementarity rules: (a) size (large purines pair with small pyrimidines) and (b) hydrogen bonding (on purine pu and pyrimidine py ring analogs, hydrogen bond acceptors, A, pair with donors D). Rearranging D and A groups on the nucleobases creates artificially expanded genetic information systems (AEGIS). A central teaching of this application is that various heterocyclic systems can implement the same hydrogen bonding interaction. For example, isoguanine and 7-deazaisoguanine both implement the puDDA hydrogen bonding pattern. 
         FIG. 5 . (a) Proposed chemical mechanisms for the overall conversion of Z:P pairs to C:G pairs, completed with two copying steps. (b) Copying steps are shown, where the first polymerase extension cycle creates a natural strand by placing C opposite P and G opposite B. Alternatively, if dZTP or dPTP is present in small amounts, the conversion can be completed in three or more cycles, or in the products of PCR amplification. 
         FIG. 6 . (a) Proposed chemical mechanisms for the overall conversion of S:B pairs to T:A pairs, completed with three copying steps, where the minor enol tautomer of isoG pairs with T. (b) Copying steps shown involve three cycles, where a small amount of dBTP is incorporated to allow the dS in the ligated product to first be copied with a dB. 
         FIG. 7 . Cartoon illustrating the annealing and extension parts of the anneal-extend-ligate process using AEGIS components and a hypothetical cyclic target. The AEGIS components shown are Z and P; analogous cartoons can be drawn using the AEGIS components S and B. The DNA strands are preselected to have regions that “partially overlap”, meaning that the 3′-end of one or more of the fragments anneals to another fragment so that its 3′-end can be extended using its annealed partner as a template. The overlapping annealed segments preferably have two or more AEGIS components, although a single AEGIS component is not excluded. The extension must be done by a polymerase that is not “strand displacing”; an example is Phusion DNA polymerase. Shown are not actual sequences, but rather generic sequences to illustrate the concept; therefore sequence listing entries are not required by statute, regulations, or USPTO procedures. In practice, the paired regions are longer (presently preferred 12-20 nucleotides, more preferably 15-18, with similar melting temperatures), and the single stranded regions to be copied are longer. The presently preferred total length of the fragments is 40-100 nucleotides, more preferably 50-80 nucleotides, and most preferably 50-60 nucleotides, with the preferred length dependent on the level of error in the synthetic DNA fragments themselves. 
         FIG. 8 . Cartoon illustrating the extended products ready for the ligation part of the anneal-extend-ligate process using AEGIS components and a hypothetical cyclic target. N&#39;s in italics are those added by the polymerase against the N&#39;s in the preselected sequence. These are not actual sequences, but rather generic sequences to illustrate the concept; therefore sequence listing entries are not required by statute, regulations, or USPTO procedures. In practice, the paired regions are longer (presently preferred 12-20 nucleotides, more preferably 15-18, with similar melting temperatures), and the single stranded regions to be copied are longer. The presently preferred total length of the fragments is 40-100 nucleotides, more preferably 50-80 nucleotides, and most preferably 50-60 nucleotides, with the preferred length dependent on the level of error in the synthetic DNA fragments themselves. 
         FIG. 9 . Cartoon illustrating the product after ligation of the anneal-extend-ligate process using AEGIS components and a hypothetical cyclic target. These are not actual sequences, but rather generic sequences to illustrate the concept; therefore sequence listing entries are not required by statute, regulations, or USPTO procedures. In practice, the paired regions are longer (presently preferred 12-20 nucleotides, more preferably 15-18, with similar melting temperatures), and the single stranded regions to be copied are longer. The presently preferred total length of the fragments is 40-100 nucleotides, more preferably 50-80 nucleotides, and most preferably 50-60 nucleotides, with the preferred length dependent on the level of error in the synthetic DNA fragments themselves. 
         FIG. 10 . Cartoon illustrating the conversion step following the anneal-extend-ligate process using AEGIS components and a hypothetical cyclic target. Lower case nucleotides are those that arose from conversion. These are not actual sequences, but rather generic sequences to illustrate the concept; therefore sequence listing entries are not required by statute, regulations, or USPTO procedures. This is illustrated with Z:P conversion; see Example 1 for S:B conversion. In practice, the paired regions are longer (presently preferred 12-20 nucleotides, more preferably 15-18, with similar melting temperatures), and the single stranded regions to be copied are longer. The presently preferred total length of the fragments are 40-100 nucleotides, more preferably 50-80 nucleotides, and most preferably 50-60 nucleotides, with the preferred length dependent on the level of error in the synthetic DNA fragments themselves. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     (1) Use of Non-Standard Nucleotides to Address the Problems in Multi-Fragment Assembly 
     The instant invention add nucleotides to the “alphabet” of standard DNA, specifically, components of an artificially expanded genetic information systems (AEGIS) ( FIG. 4 ). By shuffling hydrogen bond donor and acceptor groups, AEGIS adds up to eight nucleotides to the four (G, A, T and C) found in standard DNA. These form four orthogonal additional pairs between AEGIS complements that allow AEGIS DNA to bind to complementary DNA but not to standard GACT DNA [Benner et al. 2010] joined by “non-standard hydrogen bonding patterns” These are illustrated in  FIG. 4 , with the hydrogen bonding pattern defined by the prefix “py” (to indicate a heterocycle with a single six-membered ring) or “pu” (to indicate a heterocycle with a single six membered ring fused to a single five-membered ring) followed by “donor” or “acceptor”, proceeding from the major groove to the minor groove. The pattern is “non standard” if it differs from the patterns of hydrogen bonding groups found in G, A, aminoA, C, T, or U, or other heterocycles that implement the same hydrogen bonding pattern, regardless of the choice of the heterocycle upon which to implement that hydrogen bonding pattern. A teaching of this specification is that the same hydrogen bonding pattern can be implemented by more than one heterocycle. 
     A EGIS  pairs have been designed to not suffer from many of the problems found in natural DNA. First, all nucleobase pairs are joined by three hydrogen bonds ( FIG. 4 ), addressing Problem (A) in natural DNA. This means that AEGIS nucleobase pairs are not sometimes strong and sometimes weak (the standard C:G pair is strong; the T:A pair is weak), but rather are uniformly strong. 
     Second, the AEGIS nucleobases are designed so as to not have Hoogsteen and other major groove non-canonical hydrogen bonding possibilities ( FIG. 2 ). Thus, in regions where AEGIS nucleotides are incorporated, Problem (B) cannot confound desired hybridization. 
     Third, adding nucleotide letters to an expanded genetic alphabet increases the information density of the resulting DNA sequences, addressing Problem (D). With six or eight different nucleotide letters, and certainly with 10 or 12, even a mixture of 10,000 fragments, each 100 nucleotides long, does not have close “off-target” mismatches that can, kinetically, slow the rate of hybridization or, thermodynamically, generate undesired hybrids that compete with the formation of desired hybrids. 
     The added information density can be used strategically to solve Problem (C). For example, if a segment of DNA is built entirely from standard GACT nucleotides, and if AEGIS nucleotides are at the ends where ligation will take place, it is impossible for hairpin structures to form, and therefore impossible for hairpin structures to compete with desired ligation ( FIG. 3 ). 
     As reduction to practice, the oligonucleotides built from A EGIS  components are prepared by standard phosphoramidite-based phosphoramidite synthesis from phosphoramidites [Yang et al 2011, and references cited therein]. These procedures are described in the following publications and patents, and publications that these cite, all incorporated in their entirety by reference. 
     (2) Optional Conversion 
     Of course, any construct that has self-assembled autonomously around AEGIS fragments will not at this point be completely natural DNA. It will contain unnatural AEGIS nucleotides embedded throughout the LS-DNA construct. 
     This unnaturalness need not necessarily limit the application of such constructs should bacteria become available that accept AEGIS DNA. Nor will it be an issue should the constructs be used to support nanostructures that need not enter natural biological systems downstream. 
     However, many synthetic biologists want entirely natural LS-DNA constructs. Therefore, the process of the invention can optionally include a step that converts AEGIS pairs to give standard pairs. This conversion exploits the recipes disclosed in U.S. patent application Ser. No. 12/653,613, which is incorporated in its entirety herein by reference. While not wishing to be bound by theory, the key to this conversion is the ability of a polymerase, if it does not have available a complementary non-standard nucleoside triphosphate, to create the best Watson-Crick mismatch between a non-standard AEGIS component in a template and a standard nucleotide, based on (for example) alternative protonation/deprotonation states and/or alternative tautomeric forms. 
     Conversion in its various embodiments is exemplified by two examples, one involving the dZ:dP pair, and the other involving the dS:dB AEGIS pair. While not wishing to be bound by theory, protonation of the dC:dP mismatch allows the misincorporation of dC by a polymerase opposite dP in a template ( FIG. 5 ) in the absence of a complement to the template dP. Deprotonation of the dG:dZ mismatch allows the misincorporation of dG by a polymerase opposite dZ in a template, again in absence of dPTP. In the next cycle of copying, the newly incorporated C then directs the incorporation of dG, finishing the conversion of dP:dZ pairs to dG:dC pairs. 
     Polymerases can, again according to theory, use this deprotonated pair to direct the misincorporation of dG opposite dZ in a template In the next cycle of copying, the newly incorporated dG then directs the incorporation of dC, finishing the conversion of dZ:dP pairs to dC:dG pairs. This can be done in two or three steps, three steps if small amounts of dZTP and/or dPTP are added. 
     While not wishing to be bound by theory, for dS and dB, a minor tautomeric form of the puDDA hydrogen bonding pattern, when implemented using the isoguanine heterocycle, supports a mismatch with thymine ( FIG. 6 ). Polymerases can, again according to theory, use this minor tautomer to direct the misincorporation of dT opposite dB in a template in the absence of a complement to the major tautomer of isoguanine. In the next cycle of copying, the newly incorporated dT then directs the incorporation of dA, finishing the conversion of dS:dB pairs to dT:dA pairs. 
     Since dS does not have an analogous mechanism to support an dS:dA mismatch, the presently preferred conversion process includes a small amount of disoGTP, to match to disoC in the template. The newly incorporated isoguanine then, in its minor tautomeric form, is mismatched against thymine, and the overall conversion of these dS:dB pairs to dT:dA pairs is completed with three copying steps. 
     Conversion can also be effected with RNA assemblies involving reverse transcriptase, and other implementations of the AEGIS non-standard hydrogen bonding schemes. 
     Thus, after autonomous self-assembly of independently synthesized fragments using the orthogonality of AEGIS nucleotides, the AEGIS nucleotides are removed to give an entirely natural full-length DNA product. Of course, the synthetic fragments must be designed so as to have Z present at sites where C is desired in the final LS-DNA product, P is present at sites where G is desired in the final LS-DNA product, S is present at sites where T is desired in the final LS-DNA product, and B is present at sites where A is desired in the final LS-DNA product. Additional rules can be specified depending on the nature of the conversion process. 
     (3) References 
     
         
         Benner, S. A., Yang, Z., Chen, F. (2010) Synthetic biology, tinkering biology, and artificial biology. What are we learning? Comptes Rendus 14, 372-387 
         Benner, S. A. (2010) Non-standard nucleobases implementing the isocytidine and isoguanosine hydrogen bonding patterns. U.S. Pat. No. 7,741,294 
         Gibson, D. G. (2009) Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides.  Nucl. Acids Res.  37, 6984-6990. 
         Gibson, D. G. (2011) Enzymatic assembly of overlapping DNA fragments.  Methods Enzymol . 498, 349-361 
         Szostak, J. W., Orrweaver, T. L., Rothstein, R. J., Stahl, F. W. (1983) The double-strand-break repair model for recombination.  Cell : 33, 25-35 
         Yang, Z., Chen, F., Chamberlin, S. G., Benner, S. A. (2010) Expanded genetic alphabets in the polymerase chain reaction. Angew. Chem. 49, 177-180 
         Yang, Z., Chen, F., Alvarado, J. B., Benner, S. A. (2011) Amplification, mutation, and sequencing of a six-letter synthetic genetic system.  J. Am. Chem. Soc.  133, 15105-15112 
       
    
     (4) Examples 
     Synthesis of Preselected DNA Oligonucleotide Fragments 
     The invention was demonstrated by the assembly of a gene that encodes a protein that confers resistance on kanamycin. The following fragments were synthesized on solid phase. 
     
       
         
               
             
           
               
                 SEQ ID NO 01 
               
               
                 CACCATGAGCCATATTCAACGGGAAACGTCGAGGCCGCGATTAAATTCCAA 
               
               
                   
               
               
                 CATGGASGCSGASTT 
               
               
                   
               
               
                 SEQ ID NO 02 
               
               
                 SGASTGCCCGACBTTATCGCGAGCCCATTTATACCCATATAABTCBGCBTC 
               
               
                   
               
               
                 C 
               
               
                   
               
               
                 SEQ ID NO 03 
               
               
                 SGTCGGGCABTCBGGTGCGACAATCTATCGCTTGTATGGGAAGCCCGASGC 
               
               
                   
               
               
                 GCCBGAG 
               
               
                   
               
               
                 SEQ ID NO 04 
               
               
                 BACBTCBTTGGCBACGCTACCTTTGCCATGTTTCAGAAACAACTCSGGCGC 
               
               
                   
               
               
                 BT 
               
               
                   
               
               
                 SEQ ID NO 05 
               
               
                 SGCCAASGASGTSACAGATGAGATGGTCAGACTAAACTGGCTGACGGABTT 
               
               
                   
               
               
                 TATGCCSCTSC 
               
               
                   
               
               
                 SEQ ID NO 06 
               
               
                 TCBTCBGGBGTBCGGATAAAATGCTTGATGGTCGGBAGBGGCATAAAST 
               
               
                   
               
               
                 SEQ ID NO 07 
               
               
                 SACSCCSGASGATGCATGGTTACTCACCACTGCGATCCCCGGBAAAACBGC 
               
               
                   
               
               
                 BTT 
               
               
                   
               
               
                 SEQ ID NO 08 
               
               
                 CBACAATBTTSTCBCCTGAATCAGGATATTCTTCTAATACCTGGAASGCSG 
               
               
                   
               
               
                 TTTTSC 
               
               
                   
               
               
                 SEQ ID NO 09 
               
               
                 SGABAASATTGTSGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCASTCGAT 
               
               
                   
               
               
                 TCCTGTST 
               
               
                   
               
               
                 SEQ ID NO 10 
               
               
                 GBGCGAGBCGAAATACGCGATCGCTGTTAAAAGGACAATTACABACAGGAA 
               
               
                   
               
               
                 TCGABT 
               
               
                   
               
               
                 SEQ ID NO 11 
               
               
                 TCGSCTCGCSCAGGCGCAATCACGAATGAATAACGGSTTGGTSGASG 
               
               
                   
               
               
                 SEQ ID NO 12 
               
               
                 CSTGSTCBACBGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCBTCBA 
               
               
                   
               
               
                 CCAABC 
               
               
                   
               
               
                 SEQ ID NO 13 
               
               
                 SGTSGABCABGTCTGGAAAGAAATGCASAABCTSTTGCCBT 
               
               
                   
               
               
                 SEQ ID NO 14 
               
               
                 BTCBAGSGAGAABTCACCATGAGTGACGACTGAATCCGGTGAGAASGGCAA 
               
               
                   
               
               
                 BAGSTTBT 
               
               
                   
               
               
                 SEQ ID NO 15 
               
               
                 ASTTCTCBCTSGASAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTA 
               
               
                   
               
               
                 TTGASGTTGGACGBGT 
               
               
                   
               
               
                 SEQ ID NO 16 
               
               
                 CBAGBTCCTGGTASCGGTCTGCGATTCCGACSCGTCCAACBTCA 
               
               
                   
               
               
                 SEQ ID NO 17 
               
               
                 BTACCAGGASCTSGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCSTC 
               
               
                   
               
               
                 BTTACAGAABC 
               
               
                   
               
               
                 SEQ ID NO 18 
               
               
                 STTBTTCATBTCBGGATTATCAATACCATATTTTTGAAAAAGCCGSTTCTG 
               
               
                   
               
               
                 TAASGABG 
               
               
                   
               
               
                 SEQ ID NO 19 
               
               
                 CSGASATGAASAABTTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAAC 
               
               
                   
               
               
                 AGGATCCGCBCGBCSAG 
               
               
                   
               
               
                 SEQ ID NO 20 
               
               
                 CTAGSGGSCGBTCSGTCCGTCCTGTCAGCTGCTBGSCGSGCG 
               
             
          
         
       
     
     The sequence for the protein that encodes kanamycin resistance was obtained from the literature. Using a software tool designed for this purpose, a gene that encodes the protein was broken down to 20 single stranded oligonucleotide fragments containing S (5-methyl-2′-deoxyisocytidine) and B (2′-deoxyisoguanosine) nucleotides at their 5′ and 3′ ends, ranging in size from 41 to 67 nucleotides (nts) (Table 1). These were the DNA oligonucleotide “fragments” with pre-selected sequences that, with partial overlap, would self-assemble in hybridization including S:B pairs, as the first step in the anneal-extend-ligate process. 
     Additionally, the LS-DNA product was designed to have a CACC tetranucleotide immediately upstream from the start codon; this assisted cloning into a TOPO expression vector. It was also designed to have a Bam HI region downstream of the stop codon. The complete designed sequence that is the intended product from the process of the instant invention was therefore 863 nts (Table 2). 
     Annealing, Extending and Ligating the Preselected DNA Oligonucleotide Fragments 
     The “anneal-extend-ligate” procedure was then executed with these 20 fragments, prepared by solid phase phosphoramidite synthesis. The procedure followed two different methods (AEL #1 and AEL #2). In each case, DNA (125-250 ng or each fragment) was used. Working stocks of 10 μM dNTPs were mixed with 5×ISO buffer and water to a final volume of 40 μL (one μL each oligo, 8 μL 5×ISO buffer, 12 μL water) in duplicate, and heated with the fragments, and then slowly cooled. Incubation programs were: 
     AEL #1: 95° C. for 5 minutes, temperature reduced at 0.1° C./second to 45° C. 
     AEL #2: 75° C. for 20 minutes, temperature reduced at 0.1° C./second to 60° C., held 30 minutes, temperature reduced 0.1° C./second to 4° C. 
     Aliquots (5 μL) of each annealing mixture were transferred to a new tube to which was added enzyme mixture containing polymerases, ligases, and substrates (15 μL). The mixtures were then incubated at 48° C. for 60 minutes to permit primer extension (using a non-strand displacing polymerase active at this temperature, preferably Phusion) in a procedure resembling that of [Gibson 2011]. The samples were then cooled and stored at 4° C. until PCR amplified. 
     PCR Amplification with Conversion of the Ligated LS-DNA Product 
     PCR reactions set up with 1 μL assembly mixture and either no added disoGTP (dBTP) or 0.5 μL added dBTP. The forward and reverse primers were: 
     KanR For: CACCATGAGCCATATTCAACGG SEQ ID NO 21 
     KanR Rev: GTCCGTCCTGTCAGCTGC SEQ ID NO 22 
     As the reverse primer was designed to be upstream of the final AEGIS nucleotides, the final PCR product was 849 bp. The PCR cycling program was: 95° C. 2 minutes, followed by 30 cycles of 95° C. 40 seconds, 55° C. 20 seconds, 72° C. 2 minutes, with a final extension of 72° C. 15 minutes. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 PCR Recipe 
               
             
          
           
               
                   
                 Item 
                 Per rxn 
                 MMix (×6) 
               
               
                   
               
               
                   
                 Taq Buffer, 10X 
                 5 μL 
                 30 μL 
               
               
                   
                 dNTP (10 mM) 
                 1 μL 
                  6 μL 
               
               
                   
                 KanR For (10 μM) 
                 2 μL 
                 12 μL 
               
               
                   
                 KanR Rev (10 μM) 
                 2 μL 
                 12 μL 
               
               
                   
                 Taq polymerase 
                 0.4 μL   
                 2.4 μL  
               
               
                   
                 DNA or water 
                 1 μL 
                 — 
               
               
                   
                 Water 
                 38.6 μL   
                 231.6 μL   
               
               
                   
                 dBTP (10 mM) 
                 0.3 or 0 μL 
                 — 
               
               
                   
               
             
          
         
       
     
     Two methods were used for the PCR amplification (with conversion) of the annealed, extended, and ligated construct. In the second, 2′-deoxyisoguanosine triphosphate (dBTP) is present in small amounts; in the first, it is absent. Gel electrophoretic analysis of the products showed the largest at the expected length of 863 bp. Ladder bands were observed, and presumed to represent incomplete assemblies. The same ladder structure is evident for both incubation methods, and both methods resulted in strong doublet products as the top of the gel. 
     The second method (where dBTP is present) appears to give more of the desired product, and is presently preferred. While not wishing to be bound by theory, we interpret this result as evidence that the presence of dBTP facilitates the copying of templates containing dS. 
     The PCR product was then ligated into a TOPO expression vector. This was used to transform Top10 cells by electroporation. Transformed cells were grown in the presence of kanamycin, harvested, and the plasmid recovered, and a sampling of the recovered plasmids was sequenced. The sequences (Table 2) showed essentially no error in the annealing, extension, ligation, or conversion steps; they did show errors common in sequences at the ends of long reads. 
     To show that the LS-DNA was functional,  E. coli  cells containing it were plated on LB/agar containing kanamycin (100 μg/mL) and IPTG (0.1 mm) from cultures prepared from BL21(DE3) cells transformed with pET TOPO expression vector containing the insert encoding for the kanamycin resistance protein. A negative control plate was spread with a culture of cells containing vector but no insert, a fact confirmed with PCR using vector primers. 
     
       
         
               
             
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                  5 Sequences of gene for kanamycin resistance. AEGIS bases are highlighted 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 KanR_AEGIS 
                 CTAGTGGSCGBTCTGSCCGTCCTGTCAGCTGCTBGSCGSGCGGATCCTGT SEQ ID NO 23 
               
               
                 KanR_normal 
                 -------------------------------------------------T SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 --------------GTCCGACCTGTCAGCTGCTAGTCGTGCGGATCCTGT SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 --------------GTCCGTCCTGTCAGCTGCTAGTCGTGCGGATCCCGT SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 ------------------GTCCTGCCAGCTGCTATTCGGGGGGATCCTGT SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 --------------GTCCGTCCTGTCAGCTGCTAGTCGTGCGGATCCTGT SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 --------------GTCCGTCCTGTCAGCTGCTAGTCGTGCGGATCCTGT SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 TAGAAAAACTCATCGAGCATCAAATGAAACTGCAASTTBTTCATBTCBGG SEQ ID NO 23 
               
               
                 KanR_normal 
                 TAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGG SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 CAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTGTTCATATCCGG SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 TAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGG SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 TAGATCC-TTCATCGAGCATCATATGAAACTGCAATTTATTCATATCAGG SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 TAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGG SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 TAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGG SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 ATTATCAATACCATATTTTTGAAAAAGCCGSTTCTGTAASGABGGAGAAA SEQ ID NO 23 
               
               
                 KanR_normal 
                 ATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAA SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 ATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGACAAA SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 ATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAA SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 ATTATCAATACCATATTTTTGAAAAAGCTTTTTCTGCAATGACCGAAAAA SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 ATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAA SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 ATTATCAATACCATATTTTTGAAAAAGCCGCTTCTGTAATGAAGGAGAAA SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 ACTCACCGAGGCAGTTCCATAGGATGGCBAGBTCCTGGTASCGGTCTGCG SEQ ID NO 23 
               
               
                 KanR_normal 
                 ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCG SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCG SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCG SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCG SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCG SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 ACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCG SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 ATTCCGACSCGSCCBACBTCAATACAACCTATTAATTTCCCCTCGTCAAA SEQ ID NO 23 
               
               
                 KanR_normal 
                 ATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAA SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 ATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAA SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 ATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAA SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 ATTCCGACTCGTCCAACATCAATACAACCTATTA-TTTCCCCTCGTCAAA SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 ATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAA SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 ATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAA SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 AATAAGGTTBTCBAGSGAGAABTCACCATGAGTGACGACTGAATCCGGTG SEQ ID NO 23 
               
               
                 KanR_normal 
                 AATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTG SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 AATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCCGTG SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 AATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTG SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 AATAAGGTTATCAAGAGAGAAATCTCCATGAGTGACGACTGAATTTTGTA SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 AATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTG SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 AATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTG SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 AGAASGGCAABAGSTTBTGCATTTCTTTCCAGACSTGSTCBACBGGCCAG SEQ ID NO 23 
               
               
                 KanR_normal 
                 AGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAG SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 AGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAG SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 AGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAG SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 AGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGATCAACAGGCCAG SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 AGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAG SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 AGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAG SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 CCATTACGCTCGTCATCAAAATCACTCGCBTCBACCAABCCGTTATTCAT SEQ ID NO 23 
               
               
                 KanR_normal 
                 CCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCAT SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 CCATTACGCTCGTCATCAAAATCACTCGCATCAACCAACCCGTTATTCAT SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 CCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCAT SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 CCATTACGOTCGTCATCAAAATCACTCGCATCAACCAACCCGTTATTCAT SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 CCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCAT SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 CCATTACGCTCGTCATCAAAATCACTCGCATCCACCAAACCGTTATTCAT SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 TCGTGATTGCGCCTGBGCGAGBCGAAATACGCGATCGCTGTTAAAAGGAC SEQ ID NO 23 
               
               
                 KanR_normal 
                 TCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGAC SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 TCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGAC SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 TCGTGATTGCGCCCGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGAC SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 TCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGAC SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 TCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGAC SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 TCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGAC SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 AATTACBAACBGGAATCGABTGCAACCGGCGCAGGAACACTGCCAGCGCA SEQ ID NO 23 
               
               
                 KanR_normal 
                 AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCA SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCA SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCA SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCA SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCA SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 AATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCA SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 TCBACAATBTTSTCBCCTGAATCAGGATATTCTTCTAATACCTGGAASGC SEQ ID NO 23 
               
               
                 KanR_normal 
                 TCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGC SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 TCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGC SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 TCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGC SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 TCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGC SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 TCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGC SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 TCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGC SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 SGTSTTSCCGGGGATCGCAGTGGTGAGTAACCATGCATCBTCBGGBGTBC SEQ ID NO 23 
               
               
                 KanR_normal 
                 TGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTAC SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 TGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTAC SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 TGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTAC SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 TGTTTTTCCOGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTAC SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 TGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTAC SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 TGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTAC SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 GGATAAAATGCTTGATGGTCGGBAGBGGCATAAA-STCCGTCAGCCAGTT SEQ ID NO 23 
               
               
                 KanR_normal 
                 GGATAAAATGCTTGATGGTCGGAAGAGGCATAAA-TTCCGTCAGCCAGTT SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 GGATAAAATGCTTGATGGTCGGAAGAGGCATAAA-TTCCGTCAGCCAGTT SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 GGATAAAATGCTTGATGGTCGGAAGAGGCATAAA-TTCCGTCAGCCAGTT SEQ ID NO 26 
               
               
                 Kan14 with dETP 
                 GGATAAAATGCTTGATGGTCGGAAGAGGCATAAA-TTCCGTCAGCCAGTT SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 GGATAAAATGCTTGATGGTCGGAAGAGGCATAAAGTCCCGTCAGCCAGTT SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 GGATAAAATGCTTGATGGTCGGAAGAGGCATAAA-TTCCGTCAGCCAGTT SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 TAGTCTGACCATCTCATCTGTBACBTCBTTGGCABCGCT-ACCTTTGCCA SEQ ID NO 23 
               
               
                 KanR_normal 
                 TAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT-ACCTTTGCCA SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 TAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT-ACCTTTGCCA SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 TAGTCTGACCATCTCATCTGTCACATCATTGGCAACGCT-ACCTTTGCCA SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 TAGTCTGACCATCTCATCTGTAACATCATTGGCCACGCT-ACCTTTGCCA SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 TAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTTACCTTTGCCA SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 TAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCT-ACCTTTGCCA SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 TGTTT-CAGAAACAACTCS-GGCGCBTCGGG-CTTCCCA-TACAAGCGAT SEQ ID NO 23 
               
               
                 KanR_normal 
                 TGTTT-CAGAAACAACTCT-GGCGCATCGGG-CTTCCCA-TACAAGCGAT SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 TGTTT-CAGAAACAACTCG-GGCGCATCGGG-CTTCCCA-TACAAGCGAT SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 TGTTT-CAGAAACAACTCT-GGCGCATCGGG-CTTCCCA-TACAAGCGAT SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 TGTTT-CAGAAACAACTCT-GGCGCATCGGG-CTTCCCA-TACAAGCGAT SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 TGTTTTCAGAAACAACTCT-GGCGCATCGGG-CTTCCCAATACAAGCGAT SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 TGTTT-CAGAAACAACTCTTGGCGCATCGGGGCTTCCCCATACAAGCGAT SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 A-GATT-GTC-GCACCS-GASTGCCC-GACBTT-ATCGCG--AGCCCATT SEQ ID NO 23 
               
               
                 KanR_nomal 
                 A-GATT-GTC-GCACCT-GATTGCCC-GACATT-ATCGCG--AGCCCATT SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 A-GATT-GTC-GCACCT-GATTGCCC-GACATT-ATCGCA--AGCCCATT SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 A-GATT-GTC-GCACCT-GATTGCCC-GACATT-ATCGCG--AGCCCATT SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 A-GATT-GTC-GCACCT-GATTGCCC-GACATT-ATCGCG--AGCCCATT SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 ATGATT-GTCCGCACCC-GAGTGCCCCGACATTTATCGCGGAGGCCCATT SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 TAGATTTGTC-GCACCCTGATTGCCCCGACCTTTATCGCG--AGCCCATT  
               
               
                   
               
               
                 KanR_AEGIS 
                 T-ATACCCAT-ATAAB-TCBGCBTCC-ATGTT--GGAATTTAAT-CG--C SEQ ID NO 23 
               
               
                 KanR_normal 
                 T-ATACCCAT-ATAAA-TCAGCATCC-ATGTT--GGAATTTAAT-CG--C SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 T-ATACCCAT-ATAAA-TCAGCATCC-ATGTT--GGAATTTAAT-CG--C SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 T-ATACCCAT-ATAAA-TCAGCCTCC-ATGTT--GGAATTTAAT-CG--C SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 T-ATACCCAT-ATAAA-TCAGCATCC-ATGTT--GGAATTTAAT-CG--C SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 TTATACCCATTATAAAATCACCATCCCATGTTTGGGAATTTAAT-CGGCG SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 TTATACCCAT-ATAAA-TCAGCATCC-ATGTT-GGAAATTTAATTCG--C SEQ ID NO 29 
               
               
                   
               
               
                 KanR_AEGIS 
                 GGCCTC-GACGTTTCCC--GTTGAATATGGCTCATGGTG-- 863 SEQ ID NO 23 
               
               
                 KanR_normal 
                 GGCCTC-GACGTTTCCC--GTTGAATATGGCTCAT------ 810 SEQ ID NO 24 
               
               
                 Kan09 with dBTP 
                 GGCCTC-GACGTTTCCC--GTTGAATATGGCTCATGGTG-- 849 SEQ ID NO 25 
               
               
                 Kan11 with dBTP 
                 GGCCTC-GACGTTTCCC--GTTGAATATGGCTCATGGTG-- 849 SEQ ID NO 26 
               
               
                 Kan14 with dBTP 
                 GGCCTC-GACGTTTCCC--GTTGAATATGGCTCATGGTG-- 843 SEQ ID NO 27 
               
               
                 Kan12 without dBTP 
                 GGCCTCCAACGTTTCCCCGGTTGAATATGGCTTCTAGGGTG 872 SEQ ID NO 28 
               
               
                 Kan13 without dBTP 
                 GGCCTCCGACGTTTTCCC-GTTGAATATGGCTCATGGGA-- 862 SEQ ID NO 29