US2019002914A1PendingUtilityA1

Methods and compositions for transposition using minimal segments of the eukaryotic transformation vector piggybac

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Assignee: UNIV NOTRE DAME DU LACPriority: Oct 31, 2000Filed: Aug 23, 2018Published: Jan 3, 2019
Est. expiryOct 31, 2020(expired)· nominal 20-yr term from priority
Inventors:Malcolm J. FraserXu Li
C12N 15/90C12N 15/85C12N 2800/90C12N 2800/204
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Claims

Abstract

Isolated nucleic acid molecules that include a minimally-sized, functional (minimally-functional) piggyBac transposon can incorporate (i) a 5′ internal domain (ID) comprising a nucleotide fragment that is substantially homologous to a native piggyBac transposon sequence; (ii) a 5′ terminal repeat domain (TRD) comprising a 5′ terminal repeat (TR) sequence, a 5′ spacer sequence, and a 5′ internal repeat (IR) sequence; (ii) a sequence of interest; (iv) a 3′ TRD comprising a 3′ IR sequence, a 3′ spacer sequence, and a 3′ TR sequence; and (iv) a 3′ ID comprising a nucleotide fragment that is substantially homologous to the native piggyBac transposon sequence. The 5′ TRD and the 3′ TRD can be optionally linked by a sequence comprising a multiple cloning site.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . An isolated nucleic acid molecule comprising a minimally-sized, functional (minimally-functional) piggyBac transposon, the minimally-functional piggyBac transposon comprising the following genetic elements in a 5′ to 3′ direction:
 a 5′ internal domain (ID) comprising a nucleotide fragment that is substantially homologous to a native piggyBac transposon sequence; 
 a 5′ terminal repeat domain (TRD) comprising a 5′ terminal repeat (TR) sequence, a 5′ spacer sequence, and a 5′ internal repeat (IR) sequence; 
 a sequence of interest; 
 a 3′ TRD comprising a 3′ IR sequence, a 3′ spacer sequence, and a 3′ TR sequence; and 
 a 3′ ID comprising a nucleotide fragment that is substantially homologous to the native piggyBac transposon sequence. 
 
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , wherein the 5′ TRD and the 3′ TRD are linked by a sequence comprising a multiple cloning site. 
     
     
         3 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleotide fragments of the 5′ ID and the 3′ ID are each at least 66-bp in length. 
     
     
         4 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleotide fragment of the 5′ ID corresponds to a sequence selected from the group consisting of: SEQ ID NO: 192, SEQ ID NO: 198, SEQ ID NO: 199, or SEQ ID NO: 200. 
     
     
         5 . The isolated nucleic acid molecule of  claim 1 , wherein the 5′ TRD comprises a 35-bp sequence corresponding to SEQ ID NO: 193. 
     
     
         6 . The isolated nucleic acid molecule of  claim 1 , wherein the 5′ TR sequence comprises a 13-bp sequence corresponding to SEQ ID NO: 194. 
     
     
         7 . The isolated nucleic acid molecule of  claim 1 , wherein the 5′ IR sequence comprises a 19-bp sequence corresponding to SEQ ID NO: 195. 
     
     
         8 . The isolated nucleic acid molecule of  claim 1 , wherein the 5′ spacer sequence comprises the nucleotide sequence 5′-GTC-3′. 
     
     
         9 . The isolated nucleic acid molecule of  claim 1 , wherein the 3′ TRD comprises a 63-bp sequence corresponding to SEQ ID NO: 71 
     
     
         10 . The isolated nucleic acid molecule of  claim 1 , wherein the 3′ TR sequence comprises a 13-bp sequence corresponding to SEQ ID NO: 194. 
     
     
         11 . The isolated nucleic acid molecule of  claim 1 , wherein the 3′ IR sequence comprises a 19-bp sequence corresponding to SEQ ID NO: 195. 
     
     
         12 . The isolated nucleic acid molecule of  claim 1 , wherein the 5′ TR and the 3′ TR comprise a same 13-bp sequence corresponding to SEQ ID NO: 194. 
     
     
         13 . The isolated nucleic acid molecule of  claim 1 , wherein the 5′ IR and the 3′ IR comprise a same 19-bp sequence corresponding to SEQ ID NO: 195. 
     
     
         14 . The isolated nucleic acid molecule of  claim 1 , wherein the nucleotide fragment of the 3′ ID comprises SEQ ID NO: 196. 
     
     
         15 . The isolated nucleic acid molecule of  claim 1 , wherein the 3′ spacer sequence comprises SEQ ID NO: 197. 
     
     
         16 . A plasmid selected from the group consisting of: pBSII-ECFP-R1/L5, pBSII-ECFP-R2/L5, pBSII-ECFP-R3/L5, and pBSII-ECFP-R4/L5.\ 
     
     
         17 . The plasmid of  claim 16 , wherein the plasmid is pBSII-ECFP-R1/L5. 
     
     
         18 . The plasmid of  claim 16 , wherein the plasmid is pBSII-ECFP-R2/L5. 
     
     
         19 . The plasmid of  claim 16 , wherein the plasmid is pBSII-ECFP-R3/L5. 
     
     
         20 . The plasmid of  claim 16 , wherein the plasmid is pBSII-ECFP-R4/L5.

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