Systems and methods for engineering nucleic acid constructs using scoring techniques

Abstract

Systems and methods are provided for defining a nucleic acid construct for integration at locus L of an organism. Nucleic acid requests are received, each such request specifying a genetic change to L. The request are expanded into component polynucleotides which are then arranged into {AR 1 , . . . , AR m } different arrangements, each AR i in {AR 1 , . . . , AR m } defining a different arrangement of the component polynucleotides. A score S i for each AR i in {AR 1 , . . . , AR m } is determined based on whether source constructs encoding a portion of AR i are physically present. An AR f in {AR 1 , . . . , AR m } is selected based on the score for AR f . Primer pairs are calculated to amplify the portions of AR f not represented in the source constructs. The portions of AR f amplified by the primer pairs and the portions of AR f in the source constructs, ordered by AR f , define the nucleic acid construct.

1 . A method of defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell, the method comprising:
 (A) receiving a plurality of nucleic acid requests {NR 1 , . . . , NR n }, wherein n is a positive integer greater than 1, each nucleic acid request NR i  in {NR 1 , . . . , NR n } specifying a genetic change to L;   (B) expanding each NR i  in {NR 1 , . . . , NR n } into a corresponding component polynucleotide, thereby forming a plurality of component polynucleotides;   (C) arranging the plurality of component polynucleotides into a contiguous arrangement AR i , wherein the arranging uses linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into the contiguous arrangement AR i ;   (D) repeating the arranging (C) until a set of {AR 1 , . . . , AR m } contiguous arrangements are formed, wherein m is a positive integer greater than 1, the set of {AR 1 , . . . , AR m } contiguous arrangements representing a plurality of different contiguous arrangements of the component polynucleotides in the plurality of component polynucleotides;   (E) determining a score S i  for each respective contiguous arrangement AR i  in {AR 1 , . . . , AR m }, wherein, for each respective contiguous arrangement AR i , a contribution to the score S i  is made when one or more source constructs are identified as being physically present in a freezer store, wherein each of the one or more physically present source constructs encodes one or more of the component polynucleotides, and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs is bound to a corresponding linker nucleic acid sequence that was used for the corresponding component polynucleotide in the arranging (C) to form the AR i ;   (F) selecting a final contiguous arrangement AR f  in {AR 1 , . . . , AR m } having a score S i  that meets a selection criterion as an optimal contiguous arrangement; and   (G) calculating, in response to selection of the AR f , one or more primer pairs based upon the AR f , wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of the AR f  not represented in the one or more physically present source constructs identified for the AR f , wherein the portions of the contiguous arrangement AR f  amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for the AR f , in the order specified in the AR f , collectively define the engineered nucleic acid construct,   wherein at least one of the receiving (A), expanding (B), arranging (C), repeating (D), determining (E), selecting (F), and calculating (G) is performed using one or more suitably programmed computers.   
     
     
         2 . The method of  claim 1 , wherein
 the arranging (C) comprises inserting a selectable marker having a nucleic acid sequence into the contiguous arrangement AR i , and   the set of contiguous arrangements {AR 1 , . . . , AR m } represents a plurality of different contiguous arrangements of the component polynucleotides in the plurality of component polynucleotides and the selectable marker with respect to each other.   
     
     
         3 . The method of  claim 1 , wherein the score S i  for each respective AR i  is a function of a number of component polynucleotides already arranged according to the contiguous arrangement specified by AR i  in the one or more physically present constructs selected by AR i  by the determining (E). 
     
     
         4 . The method of  claim 1 , wherein a nucleic acid request in the plurality of nucleic acid requests specifies insertion of an insertion sequence at L. 
     
     
         5 . The method of  claim 4 , wherein the insertion sequence comprises a promoter and a gene to be expressed by the promoter. 
     
     
         6 . The method of  claim 4 , wherein the insertion sequence comprises a divergent promoter and a first gene and a second gene driven by the divergent promoter. 
     
     
         7 . The method of  claim 6 , wherein the divergent promoter is a back-to-back divergent promoter, an overlapping divergent promoter, or a face-to-face divergent promoter. 
     
     
         8 . The method of  claim 4 , wherein the insertion sequence comprises a promoter, a gene, a terminator, an open reading frame, a codon substitution, a nucleic acid substitution, a point mutation, an insertion mutation, or a deletion mutation. 
     
     
         9 . The method of  claim 4 , wherein the insertion sequence comprises a fusable open reading frame without a stop codon. 
     
     
         10 . The method of  claim 4 , wherein the nucleic acid request specifies that the entire genomic locus L is to be replaced by the insertion sequence. 
     
     
         11 . The method of  claim 4 , wherein the nucleic acid request specifies that a promoter and a gene at L is to be replaced by the insertion sequence. 
     
     
         12 . The method of  claim 4 , wherein the nucleic acid request specifies that a divergent promoter and a first gene and a second gene driven by the divergent promoter at L is to be replaced by the insertion sequence. 
     
     
         13 . The method of  claim 12 , wherein the divergent promoter is a back-to-back divergent promoter, an overlapping divergent promoter, or a face-to-face divergent promoter. 
     
     
         14 . The method of  claim 4 , wherein the nucleic acid request specifies that a promoter, a gene, a terminator, an open reading frame, a codon substitution, a nucleic acid substitution, a point mutation, an insertion mutation, or a deletion mutation at L is to be replaced by the insertion sequence. 
     
     
         15 . The method of  claim 4 , wherein the nucleic acid request specifies that a fusible open reading frame without a stop codon is to be replaced by the insertion sequence. 
     
     
         16 . The method of  claim 4 , wherein the insertion sequence includes a first copy of a gene in a 3′ to 5′ orientation and a second copy of the gene in a 5′ to 3′ orientation, and a bi-directional promoter between the first copy and the second copy. 
     
     
         17 . The method of  claim 1 , wherein the arranging (C) comprises barring an AR i  that would cause a repeat sequence of greater than a predetermined number of bases to arise in the engineered nucleic acid construct. 
     
     
         18 . The method of  claim 1 , wherein the calculating (G) comprises applying at least one design rule to identify primers in the one or more primer pairs. 
     
     
         19 . The method of  claim 18 , wherein the at least one design rule is (i) avoidance of hair pin termini, (ii) avoidance of self-dimerization, (iii) primer length between 17 and 28 bases, (iv) percent G+C content between fifty and sixty percent, (v) melting temperature between 55° C. and 80° C., or (vi) avoidance of runs of three or more Cs or Gs at the 3′ terminus. 
     
     
         20 . The method of  claim 1 , wherein a first component polynucleotide is identical to a second component polynucleotide in the plurality of component polynucleotides and wherein the arranging (C) comprises barring a contiguous arrangement that would cause the first component polynucleotide and the second component polynucleotide to run in the same direction in the engineered nucleic acid construct. 
     
     
         21 . The method of  claim 2 , wherein an identity of the selectable marker is received with the plurality of nucleic acid requests in the receiving (A). 
     
     
         22 . The method of  claim 2 , wherein an identity of the selectable marker is determined without human intervention from a predetermined list of selectable markers by the arranging (C). 
     
     
         23 . The method of  claim 1 , wherein the calculating (G) comprises encoding a linker nucleic acid sequence specified for contiguous arrangement AR f  by the arranging (C) into a primer in the one or more primer pairs calculated for AR f . 
     
     
         24 . The method of  claim 1 , wherein the expanding (B) comprises expanding a first nucleic acid request in {NR 1 , . . . , NR n } into a first component polynucleotide and a second component polynucleotide, wherein the first component polynucleotide is a promoter and the second component polynucleotide is a gene. 
     
     
         25 . The method of  claim 1 , wherein
 the determining (E) comprises determining whether a source construct having all or a subset of the plurality of component polynucleotides, in the contiguous order specified by the contiguous arrangement AR i , is present in the freezer store, and   a contribution the source construct makes to the score S i  for the contiguous arrangement AR i  is dependent upon a number of component polynucleotides in the source construct that are in the contiguous order specified by the contiguous arrangement AR i .   
     
     
         26 . The method of  claim 1 , wherein each source construct in the freezer store comprises a linker nucleic acid sequence, selected from a predetermined library of linker nucleic acid sequences, bound to a 3′ end or a 5′ end of a component polynucleotide. 
     
     
         27 . The method of  claim 1 , wherein
 the determining (E) comprises identifying a first source construct in the freezer store, the first source construct having a first subset of the plurality of component polynucleotides, in the contiguous order specified by the contiguous arrangement AR i ,   the determining (E) comprises identifying a second source construct in the freezer store, the second source construct having a second subset of the plurality of component polynucleotides, in the contiguous order specified by the contiguous arrangement AR i ,   there is no overlap between the first subset and the second subset,   a first contribution to the score S i  for the contiguous arrangement AR i  is based upon a number of component polynucleotides in the first source construct that are in the contiguous order specified by the contiguous arrangement AR i , and   a second contribution to the score S i  for the contiguous arrangement AR i  is based upon a number of component polynucleotides in the second source construct that are in the contiguous order specified by the contiguous arrangement AR i .   
     
     
         28 . The method of  claim 1 , wherein:
 the determining (E) comprises identifying a set of {C 1 , . . . , C q } source constructs in the freezer store, wherein q is a positive integer greater than 1, for a contiguous arrangement Ar i  in the set of contiguous arrangements {AR 1 , . . . , AR m }, each respective source construct C i  in {C 1 , . . . , C q } having a corresponding subset S i  of component polynucleotides in the plurality of component polynucleotides, in the contiguous order specified by the contiguous arrangement Ar i , wherein the corresponding subset S i  of component polynucleotides is not found in any other source construct in {C 1 , . . . , C q }, and   a contribution to the score S i  for AR i  from each respective C i  in {C 1 , . . . , C q } is based upon a number of component polynucleotides in C i  that are in a contiguous order specified by AR i .   
     
     
         29 . The method of  claim 1 , wherein the freezer store comprises 1000 source constructs. 
     
     
         30 . The method of  claim 1 , wherein the freezer store comprises 10,000 source constructs. 
     
     
         31 . The method of  claim 1 , wherein the plurality of nucleic acid requests {NR 1 , . . . , NR n } comprises between 2 and 12 nucleic acid requests. 
     
     
         32 . The method of  claim 1 , wherein a nucleic acid request in {NR 1 , . . . , NR n } specifies a point mutation in a gene at genomic locus L and wherein the expanding (B) obtains a nucleic acid sequence of the gene and modifies the nucleic acid sequence of the gene to form a component polynucleotide in the plurality of component polynucleotides. 
     
     
         33 . The method of  claim 1 , wherein a nucleic acid request in {NR 1 , . . . , NR n } is a request that an exogenous gene be inserted at L, and wherein the expanding (B) comprises obtaining a sequence of the exogenous gene from a database of nucleic acid sequences to form a component polynucleotide in the plurality of component polynucleotides. 
     
     
         34 . The method of  claim 2 , wherein the engineered nucleic acid construct comprises a first PCR product having a first part of the selectable marker and a second PCR product, having a second part of the selectable marker, wherein the first PCR product, running in the 5′ to 3′ direction, combines with the second PCR product, running in the 3′ to 5′ direction, to form the engineered nucleic acid construct with the selectable marker. 
     
     
         35 . The method of  claim 1 , wherein, for each respective nucleic acid component in the plurality of component polynucleotides, the arranging (C) comprises incorporating a first linker nucleic acid sequence from a predetermined library of linker nucleic acid sequences at or near a 5′ end of the respective component polynucleotide and a second linker nucleic acid sequence from a predetermined library of linker nucleic acid sequences at or near the 3′ end of the respective component polynucleotide, thereby forming a bounded component polynucleotide; and
 the determining (E) comprises computing a score S i  for each respective contiguous arrangement AR i  in {AR 1 , . . . , AR m } based on a number of bounded component polynucleotides in the respective contiguous arrangement AR i  that are in the freezer store in the order specified by the contiguous arrangement AR i . 
 
     
     
         36 . The method of  claim 1 , wherein the engineered nucleic acid construct comprises a first integration sequence and a second integration sequence for L, the method further comprising:
 synthesizing the engineered nucleic acid construct; and   contacting the engineered nucleic acid construct with the genome of the target organism or host cell under conditions suitable for homologous recombination, thereby achieving the plurality of {NR 1 , . . . , NR n } nucleic acid requests at L.   
     
     
         37 . The method of  claim 1 , wherein the linker nucleic acid sequences are selected from a predetermined library of linker nucleic acid sequences consisting of 100 linker nucleic acid sequences or less. 
     
     
         38 . The method of  claim 1 , wherein the linker nucleic acid sequences are selected from a predetermined library of linker nucleic acid sequences consisting of 50 linker nucleic acid sequences or less. 
     
     
         39 . The method of  claim 1 , wherein a contiguous arrangement AR i  in the set of {AR 1 , . . . , AR m } contiguous arrangements comprises, in a 5′ to 3′ orientation,
   A=an ordered set {X 1 , . . . ,X t }, 
 wherein,
 t is a positive integer greater than 1, 
 each i is an integer in the set of integers {1, . . . , t}, 
 each X i  comprises 5′-LA i -NA i -LB i -3′, 
 each LB i  is a linker nucleic acid sequence in the selected from a predetermined library of linker nucleic acid sequences, 
 each NA i  is a component polynucleotide in the plurality of component polynucleotides, 
 each LB i  for i less than t, upon denaturation to single stranded form, is capable of hybridizing to the complement of LA i+1 , thereby forming a nucleic acid construct comprising the nucleic acid sequence: 
 5′-LA 1 -NA 1 , . . . , LB n−1 -NA n -LB n -3′. 
 
 
     
     
         40 . The method of  claim 40 , wherein the contiguous arrangement AR i  further comprises:
   B=NA 0 -LB 0 , and     C=LA t+1 -NA t+1 ,   wherein
 LB 0  is a linker nucleic acid sequence selected from a predetermined library of linker nucleic acid sequences, 
 NA 0  and NA t+1  are each component polynucleotides in the plurality of component polynucleotides, the contiguous arrangement AR i  comprising, in a 5′ to 3′ orientation,
   A,B,C, and 
 
   wherein
 LB 0 , upon denaturation to single stranded form, is capable of hybridizing to the complement of LA 1 , and 
 LB t , upon denaturation to single stranded form, is capable of hybridizing to the complement of LA t+1 , so that the nucleic acid construct comprises the nucleic acid sequence: 
 5′-NA 0 -LB O , . . . , LB t−1 -NA t -LB t -NA t+1 -3′. 
   
     
     
         41 . The method of  claim 39 , wherein the determining (E) comprises determining whether a source construct in the freezer store comprises an X i  in the ordered set {X 1 , . . . , X t }. 
     
     
         42 . The method of  claim 39 , wherein the determining (E) comprises determining whether a source construct in the freezer store comprises a plurality of X i  in a contiguous order specified by the ordered set {X 1 , . . . , X t }. 
     
     
         43 . The method of  claim 39 , wherein, upon denaturation of X i  in A, each LB i , for i less than t, is capable of selectively hybridizing to the complement of LA i+1  compared to each other linker nucleic acid sequence LA y  or LB y , or their complements, in A, wherein each y is an integer, other than i, in the set of integers {1, . . . , t}. 
     
     
         44 . The method of  claim 39 , wherein an LA i  or an LB i  of an X i  in A is at least 24 nucleotides in length and has a melting temperature of at least 60° C. 
     
     
         45 . The method of  claim 1 , wherein a contiguous arrangement AR i  in the set of {AR 1 , . . . , AR m } contiguous arrangements comprises, in a 5′ to 3′ orientation,
   D=an ordered set {Q 1 , . . . ,Q a }. 
 wherein,
 a is a positive integer greater than 1, 
 each i is an integer in the set of integers {1, . . . , a}, 
 each Q i  comprises 5′-RA i -LA i -NA i -LB i -RB i -3′, 
 each LB i  is a linker nucleic acid sequence selected from a predetermined library of linker nucleic acid sequences, 
 each NA i  is a component polynucleotide in the plurality of component polynucleotides, and 
 each LB i , for i less than a, upon denaturation to single stranded form and upon cleavage of each restriction site RA i  and RB i , is capable of hybridizing to the complement of LA i+1 , thereby forming the engineered nucleic acid construct comprising the nucleic acid sequence:
 5′-LA 1 -NA 1 , . . . , LB a−1 -NA a -LB a -3′. 
 
 
 
     
     
         46 . The method of  claim 45 , wherein each restriction site RA i  and each restriction site RB i  is independently cleavable by one or more type IIS restriction endonucleases, wherein each of restriction site RA i  and each restriction site RB i  is oriented so that cleavage of a respective restriction site RA i  and a respective restriction site RB i  separates their recognition and cleavage sites from the resulting nucleic acid molecule LA i -NA i -LB i . 
     
     
         47 . The method of  claim 45 , wherein the contiguous arrangement AR i  further comprises:
   E=RA 0 -NA 0 -LB 0 -RB 0 , and     F=RA a+1 -LA a+1 -NA a+1 -RB a+1 ,   wherein   LB 0  is a linker nucleic acid sequence selected from a predetermined library of linker nucleic acid sequences,   restriction sites RA 0 , RB 0 , RA a+1  and RB a+1  are each independently cleavable by one or more type IIS restriction endonucleases,   restriction sites RA 0  and RB 0  are oriented so that cleavage of RA 0  and RB 0  separates their respective recognition and cleavage sites from the resulting nucleic acid molecule that comprises NA 0 -LB 0 ,   restriction sites RA a+1  and RB a+1  are oriented so that cleavage of RA a+1  and RB a+1  separates their respective recognition and cleavage sites from the resulting nucleic acid molecule that comprises NA a+1  LB a+1 , and   NA 0  and NA a+1  are each component polynucleotides in the plurality of component polynucleotides, the contiguous arrangement AR i  comprising, in a 5′ to 3′ orientation,
   D,E,F, and 
   wherein   LB 0 , upon denaturation to single stranded form and upon cleavage of RB 0 , is capable of hybridizing to the complement of LA 1 , and   LB n , upon denaturation to single stranded form and upon cleavage of RB a , is capable of hybridizing to the complement of LA a+1 , so that the nucleic acid construct comprises the nucleic acid sequence:   5′-NA 0 -LB 0 , . . . , LB a−i -NA a -LB a -NA a+1 -3′.   
     
     
         48 . The method of  claim 45 , wherein the determining (E) comprises determining whether a source construct in the freezer store comprises a Q i  in the ordered set {Q i , . . . , Q a }. 
     
     
         49 . The method of  claim 45 , wherein the determining (E) comprises determining whether a source construct in the freezer store comprises a plurality of Q i  in a contiguous order specified by the ordered set {Q 1 , . . . , Q a }. 
     
     
         50 . The method of  claim 45  wherein, upon denaturation of Q i  in D and upon cleavage of each restriction site RA i  and RB i  in D, each LB i , for i less than a, is capable of selectively hybridizing to the complement of LA i+1  compared to each of the other linker nucleic acid sequences LA y  or LB y , or their complements, in D, wherein each y is an integer, other than i, in the set of integers {1, . . . , a}. 
     
     
         51 . The method of  claim 45 , wherein an LA i  or an LB i  in a Q i  in D is at least 24 nucleotides in length and has a melting temperature of at least 60° C. 
     
     
         52 . The method of  claim 45 , wherein each RA i  and each RB i  in Q is cleavable by SapI or LguI restriction endonuclease. 
     
     
         53 . The method of  claim 1 , wherein a source construct in the one or more physically present source constructs is circular. 
     
     
         54 . The method of  claim 1 , wherein the set of contiguous arrangements {AR 1 , . . . , AR m } consists of each possible unique complete contiguous arrangement of the component polynucleotides in the plurality of component polynucleotides. 
     
     
         55 . The method of  claim 1 , wherein the set of contiguous arrangements {AR 1 , . . . , AR m } consists of a randomized subset of all possible unique complete contiguous arrangements of the component polynucleotides in the plurality of component polynucleotides. 
     
     
         56 . The method of  claim 1 , wherein a nucleic acid request references a gene that is to be mutated, deleted from, or integrated in L and wherein the expanding (B) comprises validating that the gene exists in an electronic gene database. 
     
     
         57 . The method of  claim 1 , wherein the expanding (B) comprises obtaining a nucleic acid segment specified by a nucleic acid request NR i  in {NR 1 , . . . , NR n } from an electronic database of nucleic acid sequences and incorporating the nucleic acid segment into the component polynucleotide associated with the nucleic acid request NR i . 
     
     
         58 . The method of  claim 57 , wherein the nucleic acid segment is a portion of a gene, a promoter, a terminator, or a gene. 
     
     
         59 . The method of  claim 1 , wherein the nucleic acid request NR i  specifies a nucleic acid segment having an approximate start point or an approximate end point and wherein the expanding (B) defines an exact start point or an exact endpoint for the nucleic acid segment for incorporation into a component polynucleotide corresponding to NR i  based on one or more endpoint selection criteria. 
     
     
         60 . The method of  claim 59 , wherein an endpoint selection criterion in the one or more endpoint selection criteria is avoiding low complexity DNA sequence or avoiding a restriction site. 
     
     
         61 . The method of  claim 1 , wherein the expanding (B) comprises:
 obtaining a nucleic acid segment specified by a nucleic acid request NR i  in {NR 1 , . . . , NR n }, and   inverting the nucleic acid segment relative to a naturally occurring orientation of the nucleic acid segment prior to incorporation of the nucleic acid segment into a component polynucleotide associated with the nucleic acid request NR i .   
     
     
         62 . The method of  claim 1 , wherein a nucleic acid request NR i  in {NR 1 , . . . , NR n } specifies an inline sequence to be incorporated into a component polynucleotide corresponding to NR i  during said expanding (B). 
     
     
         63 . The method of  claim 1 , wherein the genetic change in a nucleic acid request NR i  in {NR 1 , . . . , NR n } specifies a nucleic acid segment within a gene that is to be rewritten with one or more synonymous codons before incorporation into the component polynucleotide corresponding to NR i  during said expanding (B). 
     
     
         64 . The method of  claim 63 , wherein the genetic change in the nucleic acid request specifies that the nucleic acid segment is to be rewritten, before incorporation into the component polynucleotide, with synonymous codons so that the nucleic acid segment is maximally dissimilar relative to a naturally occurring instance of the nucleic acid segment. 
     
     
         65 . The method of  claim 1 , wherein the plurality of nucleic acid requests {NR 1 , . . . , NR n } is in a data input construct, and wherein the data input construct further comprises one or more pragmas to be used in performing the expanding (B), arranging (C) or calculating (G). 
     
     
         66 . The method of  claim 65 , wherein a pragma in the one or more pragmas specifies a predetermined library of linker nucleic acid sequences. 
     
     
         67 . The method of  claim 65 , wherein a pragma in the one or more pragmas specifies whether the engineered nucleic acid construct is:
 (i) a single construct or,   (ii) a two part construct comprising a first PCR product having a first part of a selectable marker and a second PCR product, having a second part of the selectable marker, wherein the first PCR product, running in the 5′ to 3′ direction, combines with the second PCR product, running in the 3′ to 5′ direction, to form the engineered nucleic acid construct with the selectable marker.   
     
     
         68 . The method of  claim 65 , wherein a pragma in the one or more pragmas specifies a selectable marker having a nucleic acid sequence to be incorporated into each contiguous arrangement AR i  in {AR 1 , . . . , AR m }. 
     
     
         69 . The method of  claim 65 , wherein a pragma is between a first nucleic acid request and a second nucleic acid request in the data input construct and wherein the pragma directs the arranging (C) to not place a linker nucleic acid sequence between a first component polynucleotide specified by the first nucleic acid request and a second component polynucleotide specified by the second nucleic acid request in each contiguous arrangement AR i  in {AR 1 , . . . , AR m }. 
     
     
         70 . The method of  claim 69 , wherein the first component polynucleotide comprises a promoter and the second component polynucleotide comprises a gene. 
     
     
         71 . The method of  claim 65 , wherein a pragma in the one or more pragmas specifies a reference genome to be used by the expanding (B) to generate the corresponding component polynucleotide for a nucleic acid request in {NR 1 , . . . , NR n }. 
     
     
         72 . The method of  claim 1 , wherein the selection criterion is a score that is higher than the score S i  of any other contiguous arrangement AR i  in {AR i , . . . , AR m }. 
     
     
         73 . The method of  claim 1 , wherein the selection criterion is a score that is lower than the score S i  of any other contiguous arrangement AR i  in {AR i , . . . , AR m }. 
     
     
         74 . The method of  claim 1 , wherein the selection criterion is identification of the first AR i  in {AR i , . . . , AR m } to have a score that exceeds a predetermined threshold. 
     
     
         75 . The method of  claim 1 , wherein the selection criterion is identification of the first AR i  in {AR i , . . . , AR m } to have a score that is less than a predetermined threshold. 
     
     
         76 . The method of  claim 1 , the method comprising outputting the nucleic acid construct to a tangible memory or a computer monitor. 
     
     
         77 . The method of  claim 1 , wherein the expanding (B) iterates between (i) expansion of {NR 1 , . . . , NR n } to a parse tree and (ii) using the parse tree to rewrite {NR 1 , . . . , NR n } in simpler form, until no NR i  in {NR 1 , . . . , NR n } can be rewritten in simpler form. 
     
     
         78 . The method of  claim 2 , wherein the engineered nucleic acid construct comprises a first PCR product having a first part of the selectable marker, and a second PCR product having a second part of the selectable marker, wherein the first PCR product, oriented in a 5′ to 3′ direction, combines with the second PCR product, oriented in a 3′ to 5′ direction, to form the engineered nucleic acid construct with the selectable marker, and wherein the one or more primer pairs comprises a first primer pair and a second primer pair, wherein the first primer pair defines the termini of the first PCR product and the second primer pair defines the termini of the second PCR product. 
     
     
         79 . The method of  claim 1 , wherein one or more instances of the instructions for arranging (C) are performed concurrently. 
     
     
         80 . The method of  claim 1 , wherein the one or more physically present source constructs selected for an AR i  in {AR 1 , . . . , AR m } collectively encode a portion of AR i . 
     
     
         81 . The method of  claim 80 , wherein the one or more component polynucleotides in the one or more physically present source constructs identified for AR i  is less than 90 percent of a nucleic acid sequence defined by AR i . 
     
     
         82 . The method of  claim 80 , wherein the one or more component polynucleotides in the one or more physically present source constructs identified for AR i  is less than 80 percent of a nucleic acid sequence defined by AR i . 
     
     
         83 . The method of  claim 80 , wherein the one or more component polynucleotides in the one or more physically present source constructs identified for AR i  is less than 60 percent of a nucleic acid sequence defined by AR i . 
     
     
         84 . The method of  claim 80 , wherein the one or more component polynucleotides in the one or more physically present source constructs identified for AR i  is less than 20 percent of a nucleic acid sequence defined by AR i . 
     
     
         85 . The method of  claim 1 , wherein
 the selecting (F) further comprises selecting a plurality of contiguous arrangements in {AR 1 , . . . , AR m }, including AR f , each contiguous arrangement AR i  in the plurality of contiguous arrangements having a score S i  that meets a selection criterion, and   the calculating (G) further comprises calculating, for each respective contiguous arrangement AR i  in the plurality of contiguous arrangements, one or more primer pairs based upon the respective contiguous arrangement, the one or more primer pairs collectively capable of amplifying the portions of the respective contiguous arrangement AR i  not represented in the one or more component polynucleotides in the physically present source constructs identified for AR i , wherein the portions of the contiguous arrangement amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for AR i , in the order specified in the contiguous arrangement AR i , collectively define an instance of the engineered nucleic acid construct.   
     
     
         86 . The method of  claim 85 , wherein the plurality of contiguous arrangements comprises five contiguous arrangements. 
     
     
         87 . The method of  claim 85 , wherein the plurality of contiguous arrangements comprises ten contiguous arrangements. 
     
     
         88 . The method of  claim 1 , wherein the target organism is yeast,  E. coli , or baculovirus. 
     
     
         89 . An apparatus for performing the method of  claim 1 . 
     
     
         90 . A method of generating a host cell, the method comprising the steps of:
 (1) assembling an engineered nucleic acid according to the method of  claim 1 ;   (2) transforming a host cell with the engineered nucleic acid construct; and   (3) selecting a host cell comprising the engineered nucleic acid construct.   
     
     
         91 . The method of  claim 90 , wherein the engineered nucleic acid construct comprises a selectable marker having a nucleic acid sequence and the selecting (3) comprises propagating the transformed host cell on selectable media. 
     
     
         92 . The method of  claim 90 , wherein the receiving a plurality of {NR 1 , . . . , NR n } nucleic acid requests (A) comprises receiving each nucleic acid request NR i  in {NR 1 , . . . , NR n } in electronic alphanumeric format. 
     
     
         93 . An apparatus comprising one or more memories and one or more processors, wherein the one or more memories and the one or more processors are in electronic communication with each other, the one or more memories tangibly encoding a set of instructions for defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell using the one or more processors, the set of instructions comprising:
 (A) instructions for receiving a plurality of nucleic acid requests {NR 1 , . . . , NR n }, wherein n is a positive integer greater than 1, each nucleic acid request NR i  in {NR 1 , . . . , NR n } specifying a genetic change to L;   (B) instructions for expanding each NR i  in {NR 1 , . . . , NR n } into a corresponding component polynucleotide, thereby forming a plurality of component polynucleotides;   (C) instructions for arranging the plurality of component polynucleotides into a contiguous arrangement AR i , wherein the arranging (C) uses linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into a contiguous arrangement AR i ;   (D) instructions for repeating the instructions for arranging (C) until a set of {AR 1 , . . . , AR m } contiguous arrangements are formed, wherein m is a positive integer greater than 1, the set of {AR 1 , . . . , AR m } contiguous arrangements representing a plurality of different contiguous arrangements of the component polynucleotides in the plurality of component polynucleotides;   (E) instructions for determining a score S i  for each respective contiguous arrangement AR i  in {AR 1 , . . . , AR m }, wherein, for each respective contiguous arrangement AR i , a contribution to the score S i  is made when one or more source constructs are identified as being physically present in a freezer store, wherein each of the one or more physically present source constructs encodes one or more of the component polynucleotides, and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs is bound to a corresponding linker that was used for the corresponding component polynucleotide in the arranging (C) to form AR i ;   (F) instructions for selecting a final contiguous arrangement AR f  in {AR 1 , . . . , AR m } having a score S i  that meets a selection criterion as an optimal contiguous arrangement; and   (G) instructions for calculating, in response to completion of the instructions for selecting, one or more primer pairs based upon the final AR f , wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of the AR f  not represented in the one or more physically present source constructs identified for the AR f , wherein the portions of the contiguous arrangement amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for AR f , in the order specified in the contiguous arrangement AR f , collectively define the engineered nucleic acid construct.   
     
     
         94 . A method of defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell, the method comprising:
 (A) receiving a plurality of nucleic acid requests {NR 1 , . . . , NR n }, wherein n is a positive integer greater than 1, each nucleic acid request NR i  in {NR 1 , . . . , NR n } specifying a genetic change to L;   (B) expanding each NR i  in {NR 1 , . . . , NR n } into a corresponding component polynucleotide, thereby forming a plurality of component polynucleotides;   (C) arranging the plurality of component polynucleotides into a contiguous arrangement AR i , wherein the arranging (C) uses linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into the AR i ;   (D) selecting, in response to the arranging, one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein each of the one or more physically present source constructs encode one or more of the component polynucleotides, and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs is bound to a corresponding linker that was used for the corresponding component polynucleotide in the arranging (C) to form the AR i ; and   (E) calculating one or more primer pairs based upon AR i , wherein each primer pair is capable of amplifying a portion of AR i  not represented in the one or more physically present source constructs identified for AR i , wherein the portions of the AR i  amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for the AR i , in the order specified by the AR i , collectively define the engineered nucleic acid construct, wherein   at least one of the expanding (B), arranging (C), selecting (D), and calculating (E) is performed using one or more suitably programmed computers.   
     
     
         95 . The method of  claim 94 , wherein the arranging (C) comprises inserting a selectable marker having a nucleic acid sequence into the contiguous arrangement Ar i . 
     
     
         96 . The method of  claim 94 , wherein a nucleic acid request in {NR 1 , . . . , NR n } specifies insertion of an insertion sequence at L. 
     
     
         97 . The method of  claim 96 , wherein the insertion sequence comprises a promoter and a gene to be expressed by the promoter. 
     
     
         98 . The method of  claim 96 , wherein the insertion sequence comprises a divergent promoter and a first gene and a second gene driven by the divergent promoter. 
     
     
         99 . The method of  claim 98 , wherein the divergent promoter is a back-to-back divergent promoter, an overlapping divergent promoter, or a face-to-face divergent promoter. 
     
     
         100 . The method of  claim 96 , wherein the insertion sequence comprises a promoter, a gene, a terminator, an open reading frame, a codon substitution, a nucleic acid substitution, a point mutation, an insertion mutation, or a deletion mutation. 
     
     
         101 . The method of  claim 96 , wherein the insertion sequence comprises a fusable open reading frame without a stop codon. 
     
     
         102 . The method of  claim 96 , wherein the nucleic acid request specifies that the entire genomic locus L is to be replaced by the insertion sequence. 
     
     
         103 . The method of  claim 96 , wherein the nucleic acid request specifies that a promoter and a gene at L are to be replaced by the insertion sequence. 
     
     
         104 . The method of  claim 96 , wherein the nucleic acid request specifies that a divergent promoter and a first gene and a second gene driven by the divergent promoter at L are to be replaced by the insertion sequence. 
     
     
         105 . The method of  claim 104 , wherein the divergent promoter is a back-to-back divergent promoter, an overlapping divergent promoter, or a face-to-face divergent promoter. 
     
     
         106 . The method of  claim 96 , wherein the nucleic acid request specifies that a promoter, a gene, a terminator, an open reading frame, a codon substitution, a nucleic acid substitution, a point mutation, an insertion mutation, or a deletion mutation at L is to be replaced by the insertion sequence. 
     
     
         107 . The method of  claim 96 , wherein the nucleic acid request specifies that a fusible open reading frame without a stop codon is to be replaced by the insertion sequence. 
     
     
         108 . The method of  claim 96 , wherein the insertion sequence includes a first copy of a gene in a 3′ to 5′ orientation and a second copy of the gene in a 5′ to 3′ orientation, and a bi-directional promoter between the first copy and the second copy. 
     
     
         109 . The method of  claim 94 , wherein the arranging (C) comprises barring an AR i  that would cause a repeat sequence of greater than a predetermined number of bases to arise in the engineered nucleic acid construct. 
     
     
         110 . The method of  claim 94 , wherein the calculating (E) comprises applying at least one design rule to identify primers in the one or more primer pairs. 
     
     
         111 . The method of  claim 110 , wherein the at least one design rule is (i) avoidance of hairpin termini, (ii) avoidance of self-dimerization, (iii) a primer length between 17 and 28 bases, (iv) a percent G+C content between fifty and sixty percent, (v) a melting temperature between 55° C. and 80° C., or (vi) avoidance of runs of three or more cytosine or guanines at the 3′ terminus of a primer. 
     
     
         112 . The method of  claim 94 , wherein a first component polynucleotide is identical to a second component polynucleotide in the plurality of component polynucleotides and wherein the arranging (C) comprises barring a contiguous arrangement that would cause the first component polynucleotide and the second component polynucleotide to run in the same direction in the engineered nucleic acid construct. 
     
     
         113 . The method of  claim 95 , wherein an identity of the selectable marker is received with the plurality of nucleic acid requests in the receiving (A). 
     
     
         114 . The method of  claim 95 , wherein an identity of the selectable marker is determined without human intervention from a predetermined list of selectable markers by the arranging (C). 
     
     
         115 . The method of  claim 94 , wherein the calculating (E) comprises encoding a linker nucleic acid sequence from a predetermined library of linker nucleic acid sequences specified for the AR i  into one or more primers in the one or more primer pairs calculated for the AR i . 
     
     
         116 . The method of  claim 94 , wherein the expanding (B) comprises expanding a first nucleic acid request in {NR 1 , . . . , NR n } into a first component polynucleotide and a second component polynucleotide, wherein the first component polynucleotide is a promoter and the second component polynucleotide is a gene. 
     
     
         117 . An apparatus comprising one or more memories and one or more processors, wherein the one or more memories and the one or more processors are in electronic communication with each other, the one or more memories tangibly encoding a set of instructions for defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell using the one or more processors, the set of instructions comprising:
 (A) instructions for receiving a plurality of nucleic acid requests {NR 1 , . . . , NR n }, wherein n is a positive integer greater than 1, each nucleic acid request NR i  in {NR 1 , . . . , NR n } specifying a genetic change to L;   (B) instructions for expanding each NR i  in {NR 1 , . . . , NR n } into a corresponding component polynucleotide having a nucleic acid sequence, thereby forming a plurality of component polynucleotides;   (C) instructions for arranging the plurality of component polynucleotides into a contiguous arrangement AR i , wherein the arranging (C) uses linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into a contiguous arrangement AR i ;   (D) instructions for selecting one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein each of the one or more physically present source constructs encode one or more of the component polynucleotides in the plurality of component polynucleotides, and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs is bound to a corresponding linker nucleic acid that was used for the respective component polynucleotide in the arranging (C) to form the AR i ; and   (E) instructions for calculating one or more primer pairs based upon the AR i , wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of the AR i  not represented in the one or more physically present source constructs identified for the AR i , wherein the portions of the AR i  amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for the AR i , in the order specified by the AR i , collectively define the engineered nucleic acid construct.   
     
     
         118 . A method of defining a plurality of engineered nucleic acid constructs {EN 1 , . . . , EN k }, wherein k is a positive integer greater than 1, each engineered nucleic acid construct EN i  in {EN 1 , . . . , EN k } for integration into a genomic locus L of a target organism or a host cell, the method comprising:
 (A) receiving, for each respective EN i  in {EN 1 , . . . , EN k }, a corresponding plurality of nucleic acid requests {NR i,1 , . . . , NR i,n }, each nucleic acid request NR i,j  in {NR i,1 , . . . , NR i,n } specifying a genetic change to L, wherein, for each respective EN i  in {EN 1 , . . . , EN k }, n is a positive integer that is the same or different as n for each other EN m  in {EN 1 , . . . , EN k };   (B) expanding, for each respective EN i  in {EN 1 , . . . , EN k }, each NR i,j  in {NR i,1 , . . . , NR i,n } into a corresponding component polynucleotide having a nucleic acid sequence, thereby forming a corresponding plurality of component polynucleotides;   (C) arranging, for each respective EN i  in {EN 1 , . . . , EN k }, the corresponding plurality of component polynucleotides from the expanding (B) into a contiguous arrangement AR i , wherein the arranging (C) uses linker nucleic acid sequences to combine component polynucleotides in the corresponding plurality of component polynucleotides into AR i , thereby forming a plurality of contiguous arrangements {AR 1 , . . . , AR m }, each AR i  in {AR 1 , . . . , AR m } representing an EN i  in {EN 1 , . . . , EN k };   (D) selecting, for each respective EN i  in {EN 1 , . . . , EN k }, one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein each of the one or more physically present source constructs for a respective EN i  in {EN 1 , . . . , EN k } encode one or more of the component polynucleotides in the plurality of component polynucleotides for the respective EN i , and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs for a respective EN i  is bound to a corresponding linker nucleic acid that was used for the respective component polynucleotide in the arranging (C) to form AR i ; and   (E) calculating, for each respective EN i  in {EN 1 , . . . , EN k }, one or more primer pairs based upon the AR i  in {AR 1 , . . . , AR m } that represents EN i , wherein each primer pair in the one or more primer pairs for an AR i  is capable of amplifying a portion of AR i  not represented in the one or more physically present source constructs identified for AR i , wherein the portions of the contiguous arrangement AR i  amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for AR i , in the order specified by AR i , collectively define the engineered nucleic acid construct EN i , wherein
 at least one of the expanding (B), arranging (C), selecting (D), and calculating (E) is performed using one or more suitably programmed computers. 
   
     
     
         119 . The method of  claim 118 , the method further comprising:
 (F) synthesizing, for each respective EN i  in {EN 1 , . . . , EN k }, EN i , as defined by the AR i  in {AR 1 , . . . , AR m } that represents EN i , using the one or more primer pairs calculated for AR i  in the calculating (E) and the one or more physically present source constructs selected for AR i  in the selecting (D);   (G) transforming each respective EN i  in {EN 1 , . . . , EN k } synthesized in the synthesizing (F) into a different host cell; and   (H) selecting a plurality of host cells, wherein each host cell in the plurality of host cells comprises an EN i  in {EN 1 , . . . , EN k } such that the plurality of host cells represents at least sixty percent of {EN 1 , . . . , EN k }.   
     
     
         120 . The method of  claim 119 , wherein the engineered nucleic acid construct EN i  comprises a selectable marker having a nucleic acid sequence and the selecting (H) comprises propagating the transformed host cell on selectable media. 
     
     
         121 . The method of  claim 119 , wherein the plurality of {EN 1 , . . . , EN k } engineered nucleic acid constructs comprises one hundred engineered nucleic acid constructs and wherein the transforming (G) is performed within two weeks of the expanding (B). 
     
     
         122 . The method of  claim 119 , wherein the plurality of {EN 1 , . . . , EN k } engineered nucleic acid constructs comprises two hundred engineered nucleic acid constructs and wherein the transforming (G) is performed within three weeks of the expanding (B). 
     
     
         123 . The method of  claim 119 , wherein the plurality of {EN 1 , . . . , EN k } engineered nucleic acid constructs comprises three hundred engineered nucleic acid constructs and wherein the transforming (G) is performed within three weeks of the expanding (B). 
     
     
         124 . The method of  claim 119 , wherein the plurality of {EN 1 , . . . , EN k } engineered nucleic acid constructs comprises four hundred engineered nucleic acid constructs and wherein the transforming (G) is performed within three weeks of the expanding (B). 
     
     
         125 . The method of  claim 119 , wherein the plurality of {EN 1 , . . . , EN k } engineered nucleic acid constructs comprises five hundred engineered nucleic acid constructs and wherein the transforming (G) is performed within three weeks of the expanding (B). 
     
     
         126 . The method of  claim 118  wherein, for at least one NR i  in {NR 1 , . . . , NR n },
 (i) the arranging (C) comprises arranging the plurality of corresponding component polynucleotides corresponding to NR i  from the expanding (B) into a set of temporary contiguous arrangements {TAR 1 , . . . , TAR z } wherein, for each TAR i  in {TAR 1 , . . . , TAR z }, the arranging (C) uses linker nucleic acid sequences from a predetermined library of linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into TAR i , 
 (ii) a score S k  is determined for each respective TAR k  in {TAR 1 , . . . , TAR z }, wherein, for each respective TAR k  in {TAR 1 , . . . , TAR z }, the corresponding score S k  is determined by a method comprising (a) selecting one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein the one or more constructs collectively encode all a portion of TAR k ; and (b) calculating S k  based on an amount of TAR k  represented by the one or more source constructs, and 
 (iii) selecting the contiguous arrangement TAR f  in {TAR 1 , . . . , TAR m } having a score S f  that meets a selection criterion as the optimal contiguous arrangement, wherein the selected TAR f  is deemed to be the contiguous arrangement AR i  for EN i , 
 thereby forming {AR 1 , . . . . AR k }, wherein each AR i  in {AR 1 , . . . , AR k } is for a different NR m  in {NR 1 , . . . , NR n }. 
 
     
     
         127 . An apparatus comprising one or more memories and one or more processors, wherein the one or more memories and the one or more processors are in electronic communication with each other, the one or more memories encoding a set of instructions for defining a plurality of {EN 1 , . . . , EN k } engineered nucleic acid constructs, wherein k is a positive integer greater than 1, each engineered nucleic acid construct EN i  in {EN 1 , . . . , EN k } for integration into a genomic locus L of a target organism or a host cell, using the one or more processors, the set of instructions comprising:
 (A) instructions for receiving, for each respective EN i  in {EN 1 , . . . , EN k }, a corresponding plurality of {NR i,1 , . . . , NR i,n } nucleic acid requests, each nucleic acid request NR i,j  in {NR i,1 , . . . , NR i,n } specifying a genetic change to L, wherein, for each respective EN i  in {EN 1 , . . . , EN k }, n is a positive integer that is the same or different as n for each other EN m  in {EN 1 , . . . , EN k };   (B) instructions for expanding, for each respective EN i  in {EN 1 , . . . , EN k }, each NR i,j  in {NR i,1 , . . . , NR i,n } into a corresponding component polynucleotide having a nucleic acid sequence, thereby forming a corresponding plurality of component polynucleotides for each respective EN i  in {EN 1 , . . . , EN k };   (C) instructions for arranging, for each respective EN i  in {EN 1 , . . . , EN k }, the corresponding plurality of component polynucleotides from the expanding (B) into a contiguous arrangement AR i , wherein the arranging (C) uses linker nucleic acid sequences to combine component polynucleotides in the corresponding plurality of component polynucleotides into AR i , thereby forming a plurality of contiguous arrangements {AR 1 , . . . , AR m }, each AR i  in {AR 1 , . . . , AR m } representing a EN i  in {EN 1 , . . . , EN k };   (D) instructions for selecting, for each respective EN i  in {EN 1 , . . . , EN k }, one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein each of the one or more physically present source constructs for a respective EN i  in {EN 1 , . . . , EN k } encode one or more of the component polynucleotides in the plurality of component polynucleotides for the respective EN i , and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs for a respective EN i  is bound to a corresponding linker nucleic acid that was used for the respective component polynucleotide in the arranging (C) to form AR i ; and   (E) instructions for calculating, for each respective EN i  in {EN 1 , . . . , EN k }, one or more primer pairs based upon the AR i  in {AR 1 , . . . , AR m } that represents EN i , wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of AR i  not represented in the one or more physically present source constructs identified for AR i , wherein the portions of AR i  amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for AR i , in the order specified by AR i , collectively define the engineered nucleic acid construct EN i .   
     
     
         128 . An apparatus comprising one or more memories and one or more processors, wherein the one or more memories and the one or more processors are in electronic communication with each other, the one or more memories encoding a set of instructions for defining a plurality of engineered nucleic acid constructs {EN 1 , . . . , EN k }, wherein k is an integer greater than 1, each engineered nucleic acid construct EN i  in {EN 1 , . . . , EN k } for integration into a genomic locus L of a target organism or a host cell, the set of instructions comprising:
 (A) instructions for receiving, for each respective EN i  in {EN 1 , . . . , EN k }, a corresponding plurality of nucleic acid requests {NR i,1 , . . . , NR i,n } in digital alphanumeric format, each nucleic acid request NR i,j  in {NR i,1 , . . . , NR i,n } specifying a genetic change to L, wherein, for each respective EN i  in {EN 1 , . . . , EN k }, n is a positive integer that is the same or different as n for each other EN m  in {EN 1 , . . . , EN k };   (B) instructions for expanding, for each respective EN i  in {EN 1 , . . . , EN k }, each NR i,j  in {NR i,1 , . . . , NR i,n } into a corresponding component polynucleotide having a nucleic acid sequence, thereby forming a corresponding plurality of component polynucleotides for each respective EN i  in {EN 1 , . . . , EN k };   (C) instructions for arranging, for each respective EN i  in {EN 1 , . . . , EN k }, the corresponding plurality of component polynucleotides from the instructions for expanding (B) into a contiguous arrangement AR i , wherein the instructions for arranging (C) use linker nucleic acid sequences to combine component polynucleotides in the plurality of corresponding component polynucleotides into AR i , thereby forming a plurality of contiguous arrangements {AR 1 , . . . , AR m }, each AR i  in {AR 1 , . . . , AR m } representing an EN i  in {EN 1 , . . . , EN k };   (D) instructions for selecting, for each respective EN i  in {EN 1 , . . . , EN k }, one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein each of the one or more physically present source constructs for a respective EN i  in {EN 1 , . . . , EN k } encode one or more of the component polynucleotides in the plurality of component polynucleotides for the respective EN i , and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs for a respective EN i  is bound to a corresponding linker nucleic acid that was used for the respective component polynucleotide in the arranging (C) to form AR i ; and   (E) instructions for calculating, for each respective EN i  in {EN 1 , . . . , EN k }, one or more primer pairs based upon the AR i  in {AR 1 , . . . , AR m } that represents EN i , wherein each primer pair in the one or more primer pairs for an AR i  is capable of amplifying a portion of AR i  not represented in the one or more source constructs identified for AR i , wherein the portions of AR i  amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for AR i , in the order specified by AR i , collectively define the engineered nucleic acid construct EN i .   
     
     
         129 . The apparatus of  claim 128 , wherein the set of instructions further comprises:
 (F) instructions for outputting to a non-volatile computer memory, a non-transitory computer memory, a persistent data storage, a monitor, or a printer, for each respective EN i  in {EN 1 , . . . , EN k }, one or more primer pairs based upon the AR i  in {AR 1 , . . . . AR k } that represents EN i , and the one or more source constructs identified by the instructions for calculating (E) for E i .   
     
     
         130 . The apparatus of  claim 128  wherein, for each NR i  in {NR 1 , . . . , NR n },
 (i) the instructions for arranging (C) comprise instructions for arranging the plurality of corresponding component polynucleotides corresponding to NR i  from the instructions for expanding (B) into a set of temporary contiguous arrangements {TAR 1 , . . . , TAR z } wherein z is a positive integer greater than 1 and wherein, for each TAR i  in {TAR 1 , . . . , TAR z }, the instructions for arranging (C) uses linker nucleic acid sequences from a predetermined library of linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into TAR i , 
 (ii) a score S k  is determined for each respective TAR k  in {TAR 1 , . . . , TAR z }, wherein, for each respective TAR k  in {TAR 1 , . . . , TAR z }, the corresponding score S k  is determined by a method comprising (a) selecting one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein the one or more constructs collectively encode all a portion of TAR k ; and (b) calculating S k  based on an amount of TAR k  represented by the one or more source constructs, and 
 (iii) selecting the contiguous arrangement TAR f  in {TAR 1 , . . . , TAR z } having a score S f  that meets a selection criterion as the optimal contiguous arrangement, wherein the selected TAR f  is deemed to be the contiguous arrangement AR i  for EN i , 
 thereby forming {AR 1 , . . . . AR k }, wherein each AR i  in {AR 1 , . . . . AR k } is for a different NR m  in {NR 1 , . . . , NR n }. 
 
     
     
         131 . A method for defining a plurality of {EN 1 , . . . , EN k } engineered nucleic acid constructs, wherein k is an integer greater than 1, each engineered nucleic acid construct EN i  in {EN 1 , . . . , EN k } for integration into a genomic locus L of a target organism or a host cell, the method comprising:
 (A) receiving, for each respective EN i  in {EN 1 , . . . , EN k }, a corresponding plurality of {NR i,1 , . . . , NR i,n } nucleic acid requests in digital alphanumeric format, each nucleic acid request NR i,j  in {NR i,1 , . . . , NR i,n } specifying a genetic change to L, wherein, for each respective EN i  in {EN 1 , . . . , EN k }, n is a positive integer that is the same or different as n for each other EN m  in {EN 1 , . . . , EN k };   (B) expanding, for each respective EN i  in {EN 1 , . . . , EN k }, each NR i,j  in {NR i,1 , . . . , NR i,n } into a corresponding component polynucleotide having a nucleic acid sequence, thereby forming a corresponding plurality of component polynucleotides for each respective EN i  in {EN 1 , . . . , EN k };   (C) arranging, for each respective EN i  in {EN 1 , . . . , EN k }, the corresponding plurality of component polynucleotides from the expanding (B) into a contiguous arrangement AR i , wherein the arranging (C) uses linker nucleic acid sequences to combine component polynucleotides in the plurality of corresponding component polynucleotides into AR i , thereby forming a plurality of contiguous arrangements {AR 1 , . . . . AR k }, each AR i  in {AR 1 , . . . . AR k } representing an EN i  in {EN 1 , . . . , EN k };   (D) selecting, for each respective EN i  in {EN 1 , . . . , EN k }, one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein the one or more physically present source constructs collectively encode a portion of the AR i  corresponding to EN i ;   (E) calculating, for each respective EN i  in {EN 1 , . . . , EN k }, one or more primer pairs based upon the AR i  in {AR 1 , . . . . AR k } that represents the respective EN i , wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of AR i  not represented in the one or more physically present source constructs identified for AR i , wherein the portions of AR i  amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for AR i , in the order specified by AR i , collectively define the engineered nucleic acid construct EN i ; and   (F) outputting to a non-transitory computer memory, a persistent data storage, a monitor, or a printer, for each respective EN i  in {EN 1 , . . . , EN k }, one or more primer pairs based upon the AR i  in {AR 1 , . . . . AR k } that represents the respective EN i , and the one or more source constructs identified by the calculating (E) for the respective EN i , wherein   at least one of the expanding (B), arranging (C), selecting (D) or calculating (E) is performed using a suitably programmed computer.   
     
     
         132 . The method of  claim 131  wherein, for each NR i  in {NR 1 , . . . , NR n },
 (i) the arranging (C) comprises arranging the plurality of corresponding component polynucleotides corresponding to NR i  from the expanding (B) into a set of temporary contiguous arrangements {TAR 1 , . . . , TAR z } wherein, for each TAR i  in {TAR 1 , . . . , TAR z }, the arranging (C) uses linker nucleic acid sequences from a predetermined library of linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into TAR i , 
 (ii) a score S k  is determined for each respective TAR k  in {TAR 1 , . . . , TAR z }, wherein z is a positive integer greater than 1, wherein, for each respective TAR k  in {TAR 1 , . . . , TAR z }, the corresponding score S k  is determined by a method comprising (a) selecting one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein the one or more constructs collectively encode all a portion of TAR k ; and (b) calculating S k  based on an amount of TAR k  represented by the one or more source constructs, and 
 (iii) selecting the contiguous arrangement TAR f  in {TAR 1 , . . . , TAR z } having a score S f  that meets a selection criterion as the optimal contiguous arrangement, wherein the selected TAR f  is deemed to be the contiguous arrangement AR i  for EN i , 
 thereby forming {AR 1 , . . . , AR k }, wherein each AR i  in {AR 1 , . . . , AR k } is for a different NR q  in {NR 1 , . . . , NR n }. 
 
     
     
         133 . The method of  claim 131 , wherein k is ten or greater. 
     
     
         134 . The method of  claim 131 , wherein k is one hundred or greater. 
     
     
         135 . The method of  claim 131 , wherein k is one thousand or greater. 
     
     
         136 . An apparatus comprising one or more memories and one or more processors, wherein the one or more memories and the one or more processors are in electronic communication with each other, the one or more memories encoding a set of instructions for defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell, using the one or more processors, the set of instructions comprising:
 (A) instructions for listing as a table on a display in electronic communication with the one or more processors, a first plurality of component polynucleotides physically present in a freezer store;   (B) instructions for receiving a first selection of a first component polynucleotide from the table by a user;   (C) instructions for displaying, responsive to the first selection, an icon on the display for the first component polynucleotide, wherein the icon for the first component polynucleotide specifies an identity of a 5′ linker nucleic acid sequence and an identity of a 3′ linker nucleic acid sequence bound to the first component polynucleotide, wherein the 5′ linker nucleic acid sequence and the 3′ linker nucleic acid sequence are present in an electronic library of linker nucleic acid sequences that is stored in non-transitory form in the one or more memories; and   (D) instructions for updating the table on the display, responsive to the first selection, to list a second plurality of component polynucleotides physically present in a freezer store, wherein each component polynucleotide in the second plurality of component polynucleotides comprises a 5′ linker nucleic acid sequence or a 3′ linker nucleic acid sequence that upon denaturation to single stranded form, is capable of hybridizing to the complement of the 5′ linker nucleic acid sequence or the complement of the 3′ linker nucleic acid sequence of the first component polynucleotide;   (E) instructions for receiving a second selection of a second component polynucleotide from the table by a user;   (F) instructions for displaying, responsive to the second selection, an icon on the display for the second component polynucleotide, wherein the icon for the second component polynucleotide specifies an identity of the 5′ linker nucleic acid sequence and an identity of a the ′ linker nucleic acid sequence bound to the second component polynucleotide; wherein   when the 5′ linker nucleic acid sequence of the second component polynucleotide upon denaturation to single stranded form, is capable of hybridizing to the complement of the 3′ linker of the first component polynucleotide, the second icon is shown to the right of the first icon on the display, and   when the 3′ linker nucleic acid sequence of the second component polynucleotide upon denaturation to single stranded form, is capable of hybridizing to the complement of the 5′ linker of the first component polynucleotide, the second icon is shown to the left of the first icon on the display, and   wherein the defined engineered nucleic acid construct comprises the first component polynucleotide and the second component polynucleotide.   
     
     
         137 . The apparatus of  claim 136 , wherein the electronic library of linker nucleic acid sequences consists of 100 linker nucleic acid sequences or less. 
     
     
         138 . The apparatus of  claim 136 , wherein the electronic library of linker nucleic acid sequences consists of 50 linker nucleic acid sequences or less. 
     
     
         139 . The apparatus of  claim 136 , wherein the engineered nucleic acid construct comprises, in a 5′ to 3′ orientation,
   A=an ordered set of component polynucleotides {X 1 , . . . ,X n } having the sequential order displayed in the display, 
 wherein,
 n is a positive integer greater than 1, 
 each i is an integer in the set of integers {1, . . . , n}, 
 each X i  comprises 5′-LA i -NA i -LB i -3′, 
 each LB i  is a linker nucleic acid sequence in the electronic library of linker nucleic acid sequences, 
 each NA i  is a component polynucleotide, 
 each LB i , for i less than n, upon denaturation to single stranded form, is capable of hybridizing to the complement of LA i+1 , thereby forming the engineered nucleic acid construct comprising the nucleic acid sequence: 
 5′-LA 1 -NA 1 , . . . , LB n−1 -NA n -LB n -3′. 
 
 
     
     
         140 . The apparatus of  claim 139 , wherein:
   B=NA 0 -LB 0 , and     C=LA n+1 -NA n+1 ,   wherein   LB 0  is a linker nucleic acid sequence in the electronic library of linker nucleic acid sequences,   NA 0  and NA n+1  are each component polynucleotides, the contiguous arrangement AR i  comprising, in a 5′ to 3′ orientation,
   A,B,C, and 
   wherein   LB 0 , upon denaturation to single stranded form, is capable of hybridizing to the complement of LA 1 , and   LB n , upon denaturation to single stranded form, is capable of hybridizing to the complement of LA n+1 , so that the engineered nucleic acid construct comprises the nucleic acid sequence:   5′-NA 0 -LB O , . . . , LB n−1 -NA n -LB n -NA n+1 -3′.   
     
     
         141 . An apparatus comprising one or more memories and one or more processors, wherein the one or more memories and the one or more processors are in electronic communication with each other, the one or more memories encoding a set of instructions for determining whether a nucleic acid sequence is present in any source construct in a plurality of source constructs physically present in a freezer store, using the one or more processors, the set of instructions comprising:
 (A) instructions for receiving a first nucleic acid sequence in electronic alphanumeric format using a display in electronic communication with the one or more memories;   (B) instructions for receiving a matching threshold criterion using the display; and   (C) instructions for comparing the first nucleic acid sequence with a sequence of each respective source construct in the plurality of source constructs, wherein, when a second nucleic acid sequence that satisfies the matching threshold criterion is found within the sequence of a respective source construct, the instructions for comparing further include instructions for displaying an identity of the respective source construct.   
     
     
         142 . The apparatus of  claim 141 , wherein the matching threshold criterion is 100 percent sequence identity. 
     
     
         143 . The apparatus of  claim 141 , wherein the matching threshold criterion is at least 90 percent identity. 
     
     
         144 . The apparatus of  claim 141 , wherein the plurality of source constructs comprises 1000 source constructs. 
     
     
         145 . The apparatus of  claim 141 , wherein the plurality of source constructs comprises 10,000 source constructs. 
     
     
         146 . The apparatus of  claim 141 , wherein the plurality of source constructs comprises 100,000 source constructs. 
     
     
         147 . The apparatus of  claim 141  wherein, when a second nucleic acid sequence that satisfies the matching threshold criterion is found within the sequence of a respective source construct, the instructions for comparing further include instructions for displaying an alignment of the second nucleic acid sequence against the first nucleic acid sequence. 
     
     
         148 . A non-transitory computer readable storage medium storing one or more programs configured for execution by one or more processors of a system, the one or more programs for defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell, the one or more programs comprising:
 (A) instructions for receiving a plurality of nucleic acid requests {NR 1 , . . . , NR n }, wherein n is a positive integer greater than 1, each nucleic acid request NR i  in {NR 1 , . . . , NR n } specifying a genetic change to L;   (B) instructions for expanding each NR i  in {NR 1 , . . . , NR n } into a corresponding component polynucleotide, thereby forming a plurality of component polynucleotides;   (C) instructions for arranging the plurality of component polynucleotides into a contiguous arrangement AR i , wherein the arranging (C) uses linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into a contiguous arrangement AR i ;   (D) instructions for repeating the instructions for arranging (C) until a set of {AR 1 , . . . , AR m } contiguous arrangements are formed, wherein m is a positive integer greater than 1, the set of {AR 1 , . . . , AR m } contiguous arrangements representing a plurality of different contiguous arrangements of the component polynucleotides in the plurality of component polynucleotides;   (E) instructions for determining a score S i  for each respective contiguous arrangement AR i  in {AR 1 , . . . , AR m }, wherein, for each respective contiguous arrangement AR i , a contribution to the score S i  is made when one or more source constructs are identified as being physically present in a freezer store, wherein each of the one or more physically present source constructs encodes one or more of the component polynucleotides, and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs is bound to a corresponding linker that was used for the corresponding component polynucleotide in the arranging (C) to form AR i ;   (F) instructions for selecting a final contiguous arrangement AR f  in {AR 1 , . . . , AR m } having a score S i  that meets a selection criterion as an optimal contiguous arrangement; and   (G) instructions for calculating, in response to completion of the instructions for selecting, one or more primer pairs based upon the final AR f , wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of the AR f  not represented in the one or more physically present source constructs identified for the AR f , wherein the portions of the contiguous arrangement amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for AR f , in the order specified in the contiguous arrangement AR f , collectively define the engineered nucleic acid construct.   
     
     
         149 . A non-transitory computer readable storage medium storing one or more programs configured for execution by one or more processors of a system, the one or more programs for defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell, the one or more programs comprising:
 (A) instructions for receiving a plurality of nucleic acid requests {NR 1 , . . . , NR n }, wherein n is a positive integer greater than 1, each nucleic acid request NR i  in {NR 1 , . . . , NR n } specifying a genetic change to L;   (B) instructions for expanding each NR i  in {NR 1 , . . . , NR n } into a corresponding component polynucleotide having a nucleic acid sequence, thereby forming a plurality of component polynucleotides;   (C) instructions for arranging the plurality of component polynucleotides into a contiguous arrangement AR i , wherein the arranging (C) uses linker nucleic acid sequences to combine component polynucleotides in the plurality of component polynucleotides into a contiguous arrangement AR i ;   (D) instructions for selecting one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein each of the one or more physically present source constructs encode one or more of the component polynucleotides in the plurality of component polynucleotides, and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs is bound to a corresponding linker nucleic acid that was used for the respective component polynucleotide in the arranging (C) to form the AR i ; and   (E) instructions for calculating one or more primer pairs based upon the AR i , wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of the AR i  not represented in the one or more physically present source constructs identified for the AR i , wherein the portions of the AR i  amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for the AR i , in the order specified by the AR i , collectively define the engineered nucleic acid construct.   
     
     
         150 . A non-transitory computer readable storage medium storing one or more programs configured for execution by one or more processors of a system, the one or more programs for defining a plurality of {EN 1 , . . . , EN k } engineered nucleic acid constructs, wherein k is a positive integer greater than 1, each engineered nucleic acid construct EN i  in {EN 1 , . . . , EN k } for integration into a genomic locus L of a target organism or a host cell, the one or more programs comprising:
 (A) instructions for receiving, for each respective EN i  in {EN 1 , . . . , EN k }, a corresponding plurality of {NR i,1 , . . . , NR i,n } nucleic acid requests, each nucleic acid request NR i,j  in {NR i,1 , . . . , NR i,n } specifying a genetic change to L, wherein, for each respective EN i  in {EN 1 , . . . , EN k }, n is a positive integer that is the same or different as n for each other EN m  in {EN 1 , . . . , EN k };   (B) instructions for expanding, for each respective EN i  in {EN 1 , . . . , EN k }, each NR i,j  in {NR i,1 , . . . , NR i,n } into a corresponding component polynucleotide having a nucleic acid sequence, thereby forming a corresponding plurality of component polynucleotides for each respective EN i  in {EN 1 , . . . , EN k };   (C) instructions for arranging, for each respective EN i  in {EN 1 , . . . , EN k }, the corresponding plurality of component polynucleotides from the expanding (B) into a contiguous arrangement AR i , wherein the arranging (C) uses linker nucleic acid sequences to combine component polynucleotides in the corresponding plurality of component polynucleotides into AR i , thereby forming a plurality of contiguous arrangements {AR 1 , . . . , AR m }, each AR i  in {AR 1 , . . . , AR m } representing a EN i  in {EN 1 , . . . , EN k };   (D) instructions for selecting, for each respective EN i  in {EN 1 , . . . , EN k }, one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein each of the one or more physically present source constructs for a respective EN i  in {EN 1 , . . . , EN k } encode one or more of the component polynucleotides in the plurality of component polynucleotides for the respective EN i , and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs for a respective EN i  is bound to a corresponding linker nucleic acid that was used for the respective component polynucleotide in the arranging (C) to form AR i ; and   (E) instructions for calculating, for each respective EN i  in {EN 1 , . . . , EN k }, one or more primer pairs based upon the AR i  in {AR 1 , . . . , AR m } that represents EN i , wherein each primer pair in the one or more primer pairs is capable of amplifying a portion of AR i  not represented in the one or more physically present source constructs identified for AR i , wherein the portions of AR i  amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for AR i , in the order specified by AR i , collectively define the engineered nucleic acid construct EN i .   
     
     
         151 . A non-transitory computer readable storage medium storing one or more programs configured for execution by one or more processors of a system, the one or more programs for defining a plurality of engineered nucleic acid constructs {EN 1 , . . . , EN k }, wherein k is an integer greater than 1, each engineered nucleic acid construct EN i  in {EN 1 , . . . , EN k } for integration into a genomic locus L of a target organism or a host cell, the one or more programs comprising:
 (A) instructions for receiving, for each respective EN i  in {EN 1 , . . . , EN k }, a corresponding plurality of nucleic acid requests {NR i,1 , . . . , NR i,n } in digital alphanumeric format, each nucleic acid request NR i,j  in {NR i,1 , . . . , NR i,n } specifying a genetic change to L, wherein, for each respective EN i  in {EN 1 , . . . , EN k }, n is a positive integer that is the same or different as n for each other EN m  in {EN 1 , . . . , EN k };   (B) instructions for expanding, for each respective EN i  in {EN 1 , . . . , EN k }, each NR i,j  in {NR i,1 , . . . , NR i,n } into a corresponding component polynucleotide having a nucleic acid sequence, thereby forming a corresponding plurality of component polynucleotides for each respective EN i  in {EN 1 , . . . , EN k };   (C) instructions for arranging, for each respective EN i  in {EN 1 , . . . , EN k }, the corresponding plurality of component polynucleotides from the instructions for expanding (B) into a contiguous arrangement AR i , wherein the instructions for arranging (C) use linker nucleic acid sequences to combine component polynucleotides in the plurality of corresponding component polynucleotides into AR i , thereby forming a plurality of contiguous arrangements {AR 1 , . . . , AR m }, each AR i  in {AR 1 , . . . , AR m } representing an EN i  in {EN 1 , . . . , EN k };   (D) instructions for selecting, for each respective EN i  in {EN 1 , . . . , EN k }, one or more source constructs from a plurality of source constructs physically present in a freezer store, wherein each of the one or more physically present source constructs for a respective EN i  in {EN 1 , . . . , EN k } encode one or more of the component polynucleotides in the plurality of component polynucleotides for the respective EN i , and wherein a 3′ or 5′ terminus, or both the 3′ and 5′ termini, of each respective component polynucleotide in the one or more component polynucleotides encoded by the one or more physically present source constructs for a respective EN i  is bound to a corresponding linker nucleic acid that was used for the respective component polynucleotide in the arranging (C) to form AR i ; and   (E) instructions for calculating, for each respective EN i  in {EN 1 , . . . , EN k }, one or more primer pairs based upon the AR i  in {AR 1 , . . . , AR m } that represents EN i , wherein each primer pair in the one or more primer pairs for an AR i  is capable of amplifying a portion of AR i  not represented in the one or more source constructs identified for AR i , wherein the portions of AR i  amplified by the one or more primer pairs and the one or more component polynucleotides in the one or more physically present source constructs identified for AR i , in the order specified by AR i , collectively define the engineered nucleic acid construct EN i .   
     
     
         152 . A non-transitory computer readable storage medium storing one or more programs configured for execution by one or more processors of a system, the one or more programs defining an engineered nucleic acid construct for integration into a genomic locus L of a target organism or a host cell, the one or more programs comprising:
 (A) instructions for listing as a table on a display a first plurality of component polynucleotides physically present in a freezer store;   (B) instructions for receiving a first selection of a first component polynucleotide from the table by a user;   (C) instructions for displaying, responsive to the first selection, an icon on the display for the first component polynucleotide, wherein the icon for the first component polynucleotide specifies an identity of a 5′ linker nucleic acid sequence and an identity of a 3′ linker nucleic acid sequence bound to the first component polynucleotide, wherein the 5′ linker nucleic acid sequence and the 3′ linker nucleic acid sequence are present in an electronic library of linker nucleic acid sequences that is stored in non-transitory form in the one or more memories; and   (D) instructions for updating the table on the display, responsive to the first selection, to list a second plurality of component polynucleotides physically present in a freezer store, wherein each component polynucleotide in the second plurality of component polynucleotides comprises a 5′ linker nucleic acid sequence or a 3′ linker nucleic acid sequence that upon denaturation to single stranded form, is capable of hybridizing to the complement of the 5′ linker nucleic acid sequence or the complement of the 3′ linker nucleic acid sequence of the first component polynucleotide;   (E) instructions for receiving a second selection of a second component polynucleotide from the table by a user;   (F) instructions for displaying, responsive to the second selection, an icon on the display for the second component polynucleotide, wherein the icon for the second component polynucleotide specifies an identity of the 5′ linker nucleic acid sequence and an identity of a the ′ linker nucleic acid sequence bound to the second component polynucleotide; wherein   when the 5′ linker nucleic acid sequence of the second component polynucleotide upon denaturation to single stranded form, is capable of hybridizing to the complement of the 3′ linker of the first component polynucleotide, the second icon is shown to the right of the first icon on the display, and   when the 3′ linker nucleic acid sequence of the second component polynucleotide upon denaturation to single stranded form, is capable of hybridizing to the complement of the 5′ linker of the first component polynucleotide, the second icon is shown to the left of the first icon on the display, and   wherein the defined engineered nucleic acid construct comprises the first component polynucleotide and the second component polynucleotide.   
     
     
         153 . A non-transitory computer readable storage medium storing one or more programs configured for execution by one or more processors of a system, the one or more programs determining whether a nucleic acid sequence is present in any source construct in a plurality of source constructs physically present in a freezer store, the one or more programs comprising:
 (A) instructions for receiving a first nucleic acid sequence in electronic alphanumeric format using a display in electronic communication with the one or more memories;   (B) instructions for receiving a matching threshold criterion using the display; and   (C) instructions for comparing the first nucleic acid sequence with a sequence of each respective source construct in the plurality of source constructs, wherein, when a second nucleic acid sequence that satisfies the matching threshold criterion is found within the sequence of a respective source construct, the instructions for comparing further include instructions for displaying an identity of the respective source construct.