Iterative nucleic acid assembly using activation of vector-encoded traits

Abstract

Certain aspects of the present invention provide methods for assembling nucleic acid molecules using iterative activation of one or more vector-encoded traits to progressively assemble a longer nucleic acid insert. Aspects of the invention also provide kits, compositions, devices, and systems for assembling synthetic nucleic acids using iterative activation of one or more vector-encoded traits.

1 .- 33 . (canceled) 
     
     
         34 . A composition for generating a nucleic acid having a predetermined sequence, the composition comprising a plurality of double-stranded nucleic acid fragments, wherein each fragment has a first restriction enzyme recognition site at its 3′ end and a second restriction enzyme recognition site at its 5′ end, wherein the first restriction enzyme recognition site and the second restriction enzyme recognition site are different from each other, and wherein, upon digestion with the restriction enzymes and assembly, the plurality of digested double-stranded nucleic acid fragments together form the nucleic acid having a predetermined sequence. 
     
     
         35 . The composition of  claim 34 , wherein the plurality of double-stranded nucleic acid fragments comprises a first and a second fragment, wherein each fragment comprises a first end and a second end, and wherein the first and second fragments comprise the same restriction enzyme recognition site at their first ends and different restriction enzyme recognition sites at their second ends. 
     
     
         36 . The composition of  claim 35 , wherein the first and second fragments are any two fragments that are consecutive to each other in the nucleic acid having the predetermined sequence. 
     
     
         37 . A composition for generating a nucleic acid having a predetermined sequence, the composition comprising:
 (i) a vector comprising one or more restriction enzyme recognition sites;   (ii) a plurality of double-stranded nucleic acid fragments, wherein each fragment has a first restriction enzyme recognition site at its 3′ end and a second restriction enzyme recognition site at its 5′ end, wherein the first restriction enzyme recognition site and the second restriction enzyme recognition site are different from each other, wherein each of the plurality of double-stranded nucleic acid fragments comprises a different fragment of the nucleic acid having a predetermined sequence, and wherein, upon digestion with the restriction enzymes and assembly, the plurality of digested double-stranded nucleic acid fragments together form the nucleic acid having a predetermined sequence.   
     
     
         38 . The composition of  claim 37 , wherein the plurality of double-stranded nucleic acid fragments comprises a first and a second fragment, wherein each fragment comprises a first end and a second end, and wherein the first and second fragments comprise the same restriction enzyme recognition site at their first ends and different restriction enzyme recognition site at their second ends. 
     
     
         39 . The composition of  claim 38 , wherein the first and second fragments are any two fragments that are consecutive to each other in the nucleic acid having the predetermined sequence. 
     
     
         40 . The composition of  claim 37 , wherein the vector is linearized and has restriction site overhangs produced by digestion with the one or more restriction enzymes. 
     
     
         41 . The composition of  claim 40 , wherein the linearized vector comprises
 (i) at its 3′ end a coding sequence of a first marker gene 5′ of a first restriction site, and (ii) at its 5′ end a coding sequence of a second marker gene 3′ of a second restriction site.   
     
     
         42 . The composition of  claim 41 , wherein the first and second marker genes are antibiotic resistance genes. 
     
     
         43 . The composition of  claim 37 , wherein the one or more restriction enzyme recognition sites are type II restriction enzyme recognition sites. 
     
     
         44 . The composition of  claim 37 , wherein the one or more restriction enzyme recognition sites are type IIs restriction enzyme recognition sites. 
     
     
         45 . The composition of  claim 37 , wherein the plurality of double-stranded nucleic acid fragments are cloned nucleic acids. 
     
     
         46 . The composition of  claim 37 , wherein the plurality of double-stranded nucleic acid fragment are PCR-derived nucleic acids. 
     
     
         47 . The composition of  claim 37 , wherein the plurality of double-stranded nucleic acid fragments are at least about 1 kb in length. 
     
     
         48 . The composition of  claim 37 , wherein the nucleic acid having the predetermined sequence is at least about 50 kb in length. 
     
     
         49 . The composition of  claim 41 , wherein expression of the first marker gene is regulated by a first activation sequence and expression of the second marker gene is regulated by a second activation sequence. 
     
     
         50 . The composition of  claim 49 , wherein the first activation sequence is a promoter, terminator, or fragment thereof. 
     
     
         51 . The composition of  claim 49 , wherein the second activation sequence is a promoter, terminator, or fragment thereof. 
     
     
         52 . The composition of  claim 41 , wherein the first marker gene is a selectable marker. 
     
     
         53 . The composition of  claim 41 , wherein the second marker gene is a selectable marker. 
     
     
         54 . The composition of  claim 37 , wherein the plurality of double-stranded nucleic acid fragments comprises at least one primer binding sequence at the 5′ end and/or the 3′ end.