LOCUS       sequence-411122-        5484 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one AAV vector expressing ISYmu1 TnpB and its reRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5484)
  AUTHORS   Xiang G, Li Y, Sun J, Huo Y, Cao S, Cao Y, Guo Y, Yang L, Cai Y, 
            Zhang YE, Wang H
  TITLE     Evolutionary mining and functional characterization of TnpB 
            nucleases identify efficient miniature genome editors.
  JOURNAL   Nat Biotechnol. 2023 Jun 29. doi: 10.1038/s41587-023-01857-x.
  PUBMED    37386294
REFERENCE   2  (bases 1 to 5484)
  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 
            Biotechnol. 2023 Jun 29. doi: 10.1038/s41587-023-01857-x."
COMMENT     Sequence Label: 
            pX601-AAV-CMV-NLS-ISYmu1-NLS-3xHA-bGHpA;U6-BsaI-reRNA
FEATURES             Location/Qualifiers
     source          1..5484
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        157..536
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        537..740
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     691..711
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      759..768
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             771..791
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1962..2009
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2016..2096
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2124..2141)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2130..2337
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(2493..2741)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2551..2570)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2721..2741)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2750..2890
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2750..2879
                     /label=AAV2 ITR
     rep_origin      2965..3420
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3052..3071)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3262..3283
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3437..3456)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3556..3578
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3616..3634)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3702..3806
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3807..3875,3876..4667)
                     /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     complement(4025..4044)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4838..5426
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5327..5346
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc
       61 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      121 actaggggtt cctgcggcct ctagactcga ggcgttgaca ttgattattg actagttatt
      181 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      241 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      301 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      361 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      421 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      481 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      541 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      601 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      661 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      721 aggtctatat aagcagagct ctctggctaa ctaccggtgc caccatggcc ccaaagaaga
      781 agcggaaggt cggtatccac ggagtcccag cagccatgct gcagcacaag gcctacgagt
      841 acagaatcta ccccgacaag aagcaggaga ccctgatcgc caagaccatc ggcagctcca
      901 ggtacgtgta caaccacttc ctggagctgt ggaacaaaga gtacgaggag accggaaagg
      961 ggctgaccta ctacgcctgc agcaaactgc tgaccaagct gaaaagggac cccgagacag
     1021 tgtggctgtg cgaagtggac aagtttagcc tgcagaactc cctgagaaac ctgagcgacg
     1081 ccttcagcag gttcttcaag ggccagaacg agcaccccca gttcaagagc aaaaagagcc
     1141 cccggcagag ctacaccacc cagtatacca acaacaacat cgccgtgagc ggaaactgtc
     1201 tgaagctgcc caagctggga ctggtgaaat tcgccgacag cagagagatg aagggaagaa
     1261 tcctgaacgc cacagtgaga agaaagagca gcggaaagtt cttcgtgagc atcctgtgcg
     1321 aggaggagat ctgcgagctg cctaagaccg atagcagcgt gggaatcgac ctgggaatca
     1381 ttgacttcgc cgtgatgagc gacggatcaa gacacgacaa caaccacttc acccggcaga
     1441 tggaggagag gctgagaaga gaacagagaa aactggccag aagagccctg gccgccgaaa
     1501 aacggggaat cagtctgagc gaggccagaa actaccagaa acagagaaga aaggtggcca
     1561 gactgtacga gaaggtggcc aaccagagaa aagaatacct gaataaactg agcacagaga
     1621 tcgtgaaaaa ccacgacatc atctgcatcg aggacctgaa cgtgaaggga atgatgagaa
     1681 accacaaact ggctaagagc atcagcgacg tgagctggac cagcctggtg tctaagctgc
     1741 agtacaaagc cagctggtac gggaaggagg tgatcagaat cagcagatgg ttccccagca
     1801 gccagatctg tagcgagtgc ggacacaagg acagaaaaaa gcccctgcac gtgagagagt
     1861 ggacctgccc cgtgtgccac gcccaccacg acagagacgt gaacgccgcc agaaacatcc
     1921 tggccgaggg cctgagaatc agagccctga cccccggcag caaaaggccg gcggccacga
     1981 aaaaggccgg ccaggcaaaa aagaaaaagg gatcctaccc atacgatgtt ccagattacg
     2041 cttacccata cgatgttcca gattacgctt acccatacga tgttccagat tacgcttaag
     2101 aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2161 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2221 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2281 ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg ctggggaggt
     2341 accaaaaaat tgtcttcgtc ccaggaagac atttgaaact ctcatgacta aagtcacgag
     2401 attcttggga acagagcata cttaccggct tatttctttg ccggcagagg tttctcttgt
     2461 ttaccaagct acccccgcaa ttcctgcggt tcggtgtttc gtcctttcca caagatatat
     2521 aaagccaaga aatcgaaata ctttcaagtt acggtaagca tatgatagtc cattttaaaa
     2581 cataatttta aaactgcaaa ctacccaaga aattattact ttctacgtca cgtattttgt
     2641 actaatatct ttgtgtttac agtcaaatta attccaatta tctctctaac agccttgtat
     2701 cgtatatgca aatatgaagg aatcatggga aataggccct cgcggccgca ggaaccccta
     2761 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca
     2821 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc
     2881 tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     2941 cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3001 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg
     3061 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3121 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3181 tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3241 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta
     3301 tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa
     3361 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt
     3421 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc
     3481 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     3541 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     3601 gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     3661 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3721 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     3781 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3841 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3901 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3961 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     4021 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     4081 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     4141 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     4201 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     4261 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     4321 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     4381 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     4441 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4501 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     4561 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4621 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4681 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4741 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4801 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4861 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4921 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4981 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     5041 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     5101 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     5161 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     5221 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     5281 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     5341 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     5401 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     5461 ccttttgctg gccttttgct caca
//
