LOCUS       sequence-423080-        6172 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV vector for encoding an ISDra2-TnpB driven by EFs promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6172)
  AUTHORS   Li Z, Guo R, Sun X, Li G, Shao Z, Huo X, Yang R, Liu X, Cao X, Zhang
            H, Zhang W, Zhang X, Ma S, Zhang M, Liu Y, Yao Y, Shi J, Yang H, Hu 
            C, Zhou Y, Xu C
  TITLE     Engineering a transposon-associated TnpB-omegaRNA system for 
            efficient gene editing and phenotypic correction of a tyrosinaemia 
            mouse model.
  JOURNAL   Nat Commun. 2024 Jan 27;15(1):831. doi: 10.1038/s41467-024-45197-z.
  PUBMED    38280857
REFERENCE   2  (bases 1 to 6172)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2024-01-27"; volume: "15(1):831. doi"; pages: " 
            10.1038/s41467-024-45197-z"
COMMENT     Sequence Label: AAV_U6-omega-RNA*_ISDra2-TnpB-mNG
FEATURES             Location/Qualifiers
     source          1..6172
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        74..322
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     74..94
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     245..264
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     ncRNA           446..513
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     promoter        570..781
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     primer_bind     792..811
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        792..810
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      826..835
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             841..861
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2098..2145
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2152..2175
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2191..2244
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2245..2952
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="VSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEE
                     LNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDG
                     ASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIST
                     FKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK
                     AFTDVMGMDELYK"
     regulatory      2497..2506
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3015..3035)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3210..3344
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3247..3266)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3301..3320
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3362..3491
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(3553..4141)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3633..3652)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4312..5103,5104..5172))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4935..4954
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5173..5277)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5345..5363
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5401..5423)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5523..5542
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5559..6014)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5696..5717)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5908..5927
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   join(6089..6172,1..57)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   join(6100..6172,1..57)
                     /label=AAV2 ITR
ORIGIN
        1 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg
       61 gccgcttaat taagagggcc tatttcccat gattccttca tatttgcata tacgatacaa
      121 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
      181 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
      241 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
      301 cttgtggaaa ggacgaaaca ccgtggtggc tgcgggaatc tcagacacct taaacgctca
      361 tggaggctat gaaaatggtc tgcgaagtga gaatcacgcg actttagtcg tgtgaggttc
      421 aaatgtcttc ctgggacgaa gacaaggccg gcatggtccc agcctcctcg ctggcgccgg
      481 ctgggcaaca tgcttcggca tggcgaatgg gacttttttt acgcgttagg tcttgaaagg
      541 agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga
      601 gaagttgggg ggaggggtcg gcaattgatc cggtgcctag agaaggtggc gcggggtaaa
      661 ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta
      721 tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca
      781 ggacgggaaa ttaatacgac tcactatagg gaccggtgcg gccgcgccac catgggatct
      841 ccaaaaaaga aacggaaggt gggatctatg atccggaaca aggccttcgt ggtgaggctg
      901 taccccaacg ccgcccagac agagctgatc aatagaaccc tgggcagcgc caggttcgtg
      961 tacaatcact tcctggccag gaggatcgcc gcctacaagg agtccggcaa gggcctgaca
     1021 tacggccaga catccagcga gctgaccctg ctgaagcagg ccgaggagac atcctggctg
     1081 agcgaggtgg acaagtttgc cctgcagaac agcctgaaga atctggagac cgcctacaag
     1141 aacttcttca gaacagtgaa gcagagcggc aagaaggtgg gctttcccag gttcagaaag
     1201 aagagaacag gcgagagcta caggacacag tttacaaata acaacatcca gatcggcgag
     1261 ggcaggctga agctgcctaa gctgggctgg gttaagacaa agggccagca ggatattcag
     1321 ggcaagatcc tgaatgtgac agtgagaagg attcatgagg gccactacga ggccagcgtg
     1381 ctgtgtgagg tggagatccc ctacctgccc gccgctccta agttcgccgc cggagttgat
     1441 gtgggcatca aggactttgc catcgtgacc gacggcgtga gatttaagca cgagcagaac
     1501 cctaagtact acaggagcac actgaagagg ctgaggaagg cccagcagac actgtccaga
     1561 agaaagaagg gcagcgcccg gtacggcaag gccaagacaa agctggccag gattcataag
     1621 agaatcgtga ataagaggca ggactttctg cacaagctga ccacaagcct ggtgagggag
     1681 tacgagatca tcggcacaga gcacctgaag cctgataaca tgaggaagaa caggagactg
     1741 gccctgtcca tcagcgatgc cggctgggga gagtttatca gacagctgga gtacaaggcc
     1801 gcctggtacg gcagactggt gagcaaggtg agcccttact tccccagctc ccagctgtgt
     1861 cacgactgcg gcttcaagaa tcccgaggtg aagaacctgg ccgtgagaac ctggacctgt
     1921 cccaactgcg gcgagaccca cgacagagat gaaaatgccg ccctgaacat caggagggag
     1981 gccctggtgg ccgccggaat tagcgacaca ctgaatgccc acggcggcta cgtgagacct
     2041 gcctctgccg gaaatggcct gaggtccgag aaccacgcca ccctggtggt gggttccaaa
     2101 aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc tgattacaaa
     2161 gacgatgacg ataaggaatt cggtagtggg gagggcagag gaagtctgct aacatgcggt
     2221 gacgtcgagg agaatcctgg cccagtgagc aagggcgagg aggataacat ggcctctctc
     2281 ccagcgacac atgagttaca catctttggc tccatcaacg gtgtggactt tgacatggtg
     2341 ggtcagggca ccggcaatcc aaatgatggt tatgaggagt taaacctgaa gtccaccaag
     2401 ggtgacctcc agttctcccc ctggattctg gtccctcata tcgggtatgg cttccatcag
     2461 tacctgccct accctgacgg gatgtcgcct ttccaggccg ccatggtaga tggctccgga
     2521 taccaagtcc atcgcacaat gcagtttgaa gatggtgcct cccttactgt taactaccgc
     2581 tacacctacg agggaagcca catcaaagga gaggcccagg tgaaggggac tggtttccct
     2641 gctgacggtc ctgtgatgac caactcgctg accgctgcgg actggtgcag gtcgaagaaa
     2701 acttacccca acgacaaaac catcatcagt acctttaagt ggagttacac cactggaaat
     2761 ggcaagcgct accggagcac tgcgcggacc acctacacct ttgccaagcc aatggcggct
     2821 aactatctga agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa gcactccaag
     2881 accgagctca acttcaagga gtggcaaaag gcctttaccg atgtgatggg catggacgag
     2941 ctgtacaagt gaaagcttga taatcaacct ctggattaca aaatttgtga aagattgact
     3001 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     3061 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggtta
     3121 gttcttgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     3181 ctgttgggca ctgacaattc cgtggtgtta acttgtttat tgcagcttat aatggttaca
     3241 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3301 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatcggcgcg ccagcggccg
     3361 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     3421 gccgggcgac caaaggtcgc ccgacgcccg ggcggcctca gtgagcgagc gagcgcgcag
     3481 ctgcctgcag gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3541 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3601 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3661 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3721 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3781 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3841 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3901 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3961 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4021 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4081 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4141 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4201 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4261 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4321 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4381 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4441 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4501 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4561 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4621 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4681 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4741 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4801 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4861 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4921 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4981 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5041 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5101 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5161 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5221 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5281 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     5341 taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa
     5401 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     5461 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta
     5521 tgcggcatca gagcagattg tactgagagt gcaccataaa attgtaaacg ttaatatttt
     5581 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat agaccgaaat
     5641 cggcaaaatc ccttataaat caaaagaata gcccgagata gagttgagtg ttgttccagt
     5701 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     5761 ctatcagggc gatggcccac tacgtgaacc atcacccaaa tcaagttttt tggggtcgag
     5821 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     5881 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctaaggc
     5941 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     6001 gctacagggc gcgtactatg gttgctttga cgtatgcggt gtgaaatacc gcacagatgc
     6061 gtaaggagaa aataccgcat caggcgcccc tgcaggcagc tgcgcgctcg ctcgctcact
     6121 gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg gtcgcccggc ct
//
