LOCUS       sequence-411113-        5572 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one AAV vector expressing ISTfu1 TnpB and its reRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5572)
  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 5572)
  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-ISTfu1-NLS-3xHA-bGHpA;U6-BbsI-reRNA
FEATURES             Location/Qualifiers
     source          1..5572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   11..140
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        164..543
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        544..747
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     698..718
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      766..775
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             778..798
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2014..2061
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2068..2148
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2176..2193)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2182..2389
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(2588..2836)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2646..2665)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2816..2836)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2845..2985
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2845..2974
                     /label=AAV2 ITR
     rep_origin      3060..3515
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3147..3166)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3357..3378
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3532..3551)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3651..3673
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3711..3729)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3797..3901
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3902..3970,3971..4762)
                     /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(4120..4139)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4933..5521
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5422..5441
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gctcacatgt cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg
       61 ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa
      121 ctccatcact aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact
      181 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc
      241 gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg
      301 acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa
      361 tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca
      421 agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac
      481 atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc
      541 atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga
      601 tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg
      661 gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta
      721 cggtgggagg tctatataag cagagctctc tggctaacta ccggtgccac catggcccca
      781 aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccatgctgac cgggagaagg
      841 tacctgctgg ccttcacccc cgagcaggag gagttcgccg agaagatcgg cgacgcctgc
      901 agagtggtgt ggaacacagc cctggagcag agacgtcagt acagaagaag aggagcattc
      961 atcggctacg tggagcaggc aagacagatg gcagaggcca agaaggactt cccatggctg
     1021 gccgaggccc caagccacac cctgcagcag accctgagag acctggagag agcctgcaag
     1081 acacacggca cattcaaggt gagatggaga agcaagagaa agaacgcccc aaccttcaga
     1141 tttcccgacc ccaaacagat caccgtggaa agaataagca gaagatgggg aagagtgaaa
     1201 ctcccaaaac tgggctggac cagattcaga tggacaagac cactgggcgg ccagctgaga
     1261 aacgcaaccg tgctcaagga cggcggaaga tggtacatca gcttctgcgt ggaggacggc
     1321 ctgatggaaa gtgctcccaa cggcaagccc cccgtgggcg ttgatagagg cgttaccgtg
     1381 gccgtggcta ccagcagcgg atggatgagg gacagggagt ttgtgacccc cggcgaggct
     1441 gtgagactga agagactgca gcagcagctg gctagacaga gaaaaggaag taacaggaga
     1501 gccgcaacaa gagcaaagct gacaaagctg aacgcaagaa taagagcaag aagaacagac
     1561 ttcctggcat ggaccgcaaa cagactgacc tgcgaccacg gactggtggt ggtggaggac
     1621 ctgaacatca gaaacatgac agcaagcgcc aagggcaccc tggcagagcc cggacgaaac
     1681 gtggccgcca aagctggcct gaacagagcc atactggcta aagggtgggg agggctgctc
     1741 acagccctgg aacacaaggc aagataccac ggaagcagaa tactgagagt gcccccagcc
     1801 ttcaccagcc aaacctgcca cgcctgtggc cactgtgccc ctgacaaccg ggaatcccaa
     1861 gccgaattta gatgcagagc ctgcggccat caagcaaacg ccgacgtgaa cgccgccaag
     1921 aacatcctcg ccgccggcct ggccgtgacc ggaaggggag atctggctgt gggcagaagc
     1981 gtgaagagac agccacccaa ggtggaagcc gccaaaaggc cggcggccac gaaaaaggcc
     2041 ggccaggcaa aaaagaaaaa gggatcctac ccatacgatg ttccagatta cgcttaccca
     2101 tacgatgttc cagattacgc ttacccatac gatgttccag attacgctta agaattccta
     2161 gagctcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2221 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2281 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2341 aggacagcaa gggggaggat tgggaagaga atagcaggca tgctggggag gtaccaaaaa
     2401 attgtcttcg tcccaggaag acatttgact tcctccccac gcctgaaggc gggggattcc
     2461 cgtggctgag gcgcgttcac gctgcctcta ccttgggtgg ttggcgcttc acagaccgcc
     2521 caacggcgag gtctccacgc cctgtcaccg ccagcccggc ggcgaggatg ttcttcgcgg
     2581 cgttcacggt gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc
     2641 aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc
     2701 caaaaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca
     2761 aattaattcc aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca
     2821 tgggaaatag gccctcgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc
     2881 tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg
     2941 cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt
     3001 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     3061 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3121 tacacttgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3181 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3241 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     3301 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     3361 actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata
     3421 agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     3481 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     3541 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     3601 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3661 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     3721 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     3781 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3841 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3901 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3961 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     4021 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     4081 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     4141 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4201 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4261 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4321 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4381 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4441 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4501 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4561 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag
     4621 ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4681 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4741 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4801 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4861 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4921 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4981 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     5041 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     5101 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     5161 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5221 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5281 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5341 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5401 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5461 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5521 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt tt
//
