LOCUS       sequence-411115-        5571 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one AAV vector expressing ISDge10 TnpB and its reRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5571)
  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 5571)
  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-ISDge10-NLS-3xHA-bGHpA;U6-BsaI-reRNA
FEATURES             Location/Qualifiers
     source          1..5571
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   9..138
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        162..541
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        542..745
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     696..716
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      764..773
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             776..796
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1994..2041
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2048..2128
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2156..2173)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2162..2369
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(2585..2833)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2643..2662)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2813..2833)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2842..2982
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2842..2971
                     /label=AAV2 ITR
     rep_origin      3057..3512
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3144..3163)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3354..3375
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3529..3548)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3648..3670
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3708..3726)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3794..3898
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3899..3967,3968..4759)
                     /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(4117..4136)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4930..5518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5419..5438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcacatgtcc tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg
       61 cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact
      121 ccatcactag gggttcctgc ggcctctaga ctcgaggcgt tgacattgat tattgactag
      181 ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt
      241 tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac
      301 gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
      361 ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag
      421 tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat
      481 gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat
      541 ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt
      601 tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga
      661 ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg
      721 gtgggaggtc tatataagca gagctctctg gctaactacc ggtgccacca tggccccaaa
      781 gaagaagcgg aaggtcggta tccacggagt cccagcagcc atgaccaccc accgcaaggt
      841 gtacagatac cgcatcgagc ccacccccgt gcaggagagc aagctgtaca tgctggccgg
      901 cagccggaga ttcgtgttca actgggccct ggcccggaga agagagcact acgccgaaac
      961 cggcaagacc ctgggataca acgcccaggc aggagagctg accgccctga aaaaccaaga
     1021 agaaaccagc tggctgaaag agtccgactc acaactgctg cagcaggcac tgaaagacgt
     1081 ggaaagagcc ttcgtgaact tcttcgaaaa acgggccaga ttccccagat tcaaaagcaa
     1141 gaaaaccgac acccccagat tcagaatccc ccaaagagtg agaatagagg gcagcagagt
     1201 gtacgtgccc aaggtggggt gggtgaagct gagaaagagt caggagatag aaggaaaaac
     1261 caaaagcgcc accttcaaga gggaggcaga tggccattgg tacgtgctgc tggtgagtga
     1321 gttcgagatg cctgatgtgc ctctgccccc agtgcccgag agcgaggtgg tgggaatcga
     1381 cctgggactg aaggacttct acgtcctgtc tgacgggggc aggaaggagg cccccagatt
     1441 cgctagaaag ggccagcgaa aactgagaag agctgcaaga agacatagca agtgcacccg
     1501 gggaagcaac agaaaggcca aggcaaagag gaagctggca agagtgcaca gacagatcgc
     1561 aaaccagaga aaagacttcg tgcacaaggc caccagcggc ctcgtgcagc agtaccaagg
     1621 cttctgcatc gaaaacctga gcatcaaggg catggccaaa accaaactgt caaaaagcgt
     1681 gctggacgca gccctggggg aatttagaag acagctggcc tataaagccc agtggcatag
     1741 aaagtggctg gcagtgatag acagatggtt ccccagcagc aagctgtgcg gagagtgcgg
     1801 cagcattaac gccgatctga ccctgagcga cagagaatgg acctgcgagt gtggcgcagt
     1861 gcacgacaga gatctgaacg ccgctcgcaa catcaagaga gagggcctga gtcagattgt
     1921 ggtggccgga catgccgaaa ccctgaacgc cagaggggag ggcgtgcgtc ccgccattgc
     1981 cggaagccct agaaaaaggc cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa
     2041 gggatcctac ccatacgatg ttccagatta cgcttaccca tacgatgttc cagattacgc
     2101 ttacccatac gatgttccag attacgctta agaattccta gagctcgctg atcagcctcg
     2161 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2221 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2281 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2341 tgggaagaga atagcaggca tgctggggag gtaccaaaaa atgagacctg ccgtggtctc
     2401 cttgacactc caacggctaa agccgttgga ttctcgcttc atcgagggct gcccgctatc
     2461 gcaggtctga caccctctcc ccgagcgttt aacgtctccg cgtgccccgc gacgacgatt
     2521 tgcgaaagcc cttcccgctt gatgttccgg gcggcgttga ggtcgcggtc gtgaaccgct
     2581 ccacggtgtt tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa tactttcaag
     2641 ttacggtaag catatgatag tccattttaa aacataattt taaaactgca aactacccaa
     2701 gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt acagtcaaat
     2761 taattccaat tatctctcta acagccttgt atcgtatatg caaatatgaa ggaatcatgg
     2821 gaaataggcc ctcgcggccg caggaacccc tagtgatgga gttggccact ccctctctgc
     2881 gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc
     2941 gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt
     3001 ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc
     3061 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     3121 acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     3181 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     3241 tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc
     3301 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     3361 cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg
     3421 gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     3481 gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc
     3541 tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg
     3601 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3661 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg
     3721 cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt
     3781 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     3841 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     3901 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     3961 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     4021 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     4081 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     4141 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     4201 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     4261 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     4321 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     4381 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     4441 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     4501 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     4561 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg
     4621 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     4681 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     4741 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     4801 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4861 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4921 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4981 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5041 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5101 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5161 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5221 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     5281 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5341 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5401 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5461 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5521 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg c
//
