LOCUS       sequence-411123-        5507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one AAV vector expressing ISAam1 TnpB and its reRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5507)
  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 5507)
  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-ISAam1-NLS-3xHA-bGHpA;U6-BbsI-reRNA
FEATURES             Location/Qualifiers
     source          1..5507
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   6..135
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        159..538
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        539..742
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     693..713
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      761..770
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             773..793
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1925..1972
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             1979..2059
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2087..2104)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2093..2300
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(2518..2766)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2576..2595)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2746..2766)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2775..2915
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2775..2904
                     /label=AAV2 ITR
     rep_origin      2990..3445
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3077..3096)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3287..3308
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3462..3481)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3581..3603
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3641..3659)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3727..3831
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3832..3900,3901..4692)
                     /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(4050..4069)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4863..5451
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5352..5371
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga
       61 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
      121 tcactagggg ttcctgcggc ctctagactc gaggcgttga cattgattat tgactagtta
      181 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      241 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      301 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      361 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      421 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      481 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      541 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      601 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      661 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      721 ggaggtctat ataagcagag ctctctggct aactaccggt gccaccatgg ccccaaagaa
      781 gaagcggaag gtcggtatcc acggagtccc agcagccatg gtgaacaaga gctacaagtt
      841 caggctgtac cccaccaagg agcaggagca gctgctggct aagaccttcg gctgcgtgag
      901 gttcgtgtac aacaagatgc tggaggagag gattcaaatc tacgagaaat tcaaggacga
      961 caaggaggcc ctgaagaagc agaccttccc cacccctgcc aaatacaaga aggagttccc
     1021 ctggctgaag gaggtggaca gcctggctct ggctaacgct cagctgaacc tgcagaaggc
     1081 ctttcagaac ttcttctccg gccgcgccgg ctttcctaag ttcaagaaca gaaaggccaa
     1141 gcagagctac accacaaacg tggtgaacgg gaacatccag ctgagcgacg gatacatcaa
     1201 gctgcccaag ctgaaatggg tgaagttcaa gcagcacaga gagatcccag cccaccacat
     1261 catcaaggcc tgcaccatca agaaaaccaa gaccggcaaa tactacgtga gcatcctgac
     1321 cgaatacgaa caccagcccg tgcccaagga aatccagacc gtggtgggac tggacttcag
     1381 catgaacggg ctgttcgtgg acagcgaagg caagagggcc aactatccca gattctacag
     1441 acaggccctg gagaaactgg ccaaggagca gagaatcctg agtagacgga aaaagggaag
     1501 caacagatgg cataagcaga gactgaaagt ggccaagctg cacgaaaaga tcgccaacca
     1561 gagaaaggac ttcctgcaca agaagagcta cgagctggcc aaacagtacg actgcgtggt
     1621 gatcgagaac ctgaacatga aaggaatgag tcaggtgctg aacttcggca agagcgtgca
     1681 cgacaacggc tggggaatgt tcacaacctt tctgcagtac aaactggagg agcagggaaa
     1741 aaagctgatc aagatcgaca agtggttccc cagcagcaag acctgcagct gctgcgacca
     1801 ggtgaaggag agcctgagcc tgagcgagag aaccttcaga tgcgagtgcg gcttcgagag
     1861 cgacagagac gtgaacgccg ccatcaacat caagcacgag ggcatgaaga gactggccat
     1921 cgccaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa agggatccta
     1981 cccatacgat gttccagatt acgcttaccc atacgatgtt ccagattacg cttacccata
     2041 cgatgttcca gattacgctt aagaattcct agagctcgct gatcagcctc gactgtgcct
     2101 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2161 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2221 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagag
     2281 aatagcaggc atgctgggga ggtaccaaaa aattgtcttc gtcccaggaa gacatgtgac
     2341 atgctcccac cacctacgct tcgcttagag gtgggggctt ctcaggtaat cccatctcat
     2401 gacgggaagt tgactgagcg atccccgtgt gtcccacggt tcgaggacaa gttaggctat
     2461 tgctaatcgt ttcatgccct catgtttgat attgattgcc gcattgacgt ccctgtcggt
     2521 gtttcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt
     2581 aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta
     2641 ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattcc
     2701 aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag
     2761 gccctcgcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg
     2821 ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc
     2881 ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct
     2941 tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt
     3001 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     3061 agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     3121 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     3181 cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga
     3241 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     3301 caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg
     3361 ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     3421 aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc
     3481 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
     3541 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
     3601 aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt
     3661 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     3721 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3781 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3841 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3901 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3961 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     4021 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     4081 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     4141 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     4201 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     4261 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     4321 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     4381 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4441 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4501 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc
     4561 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4621 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4681 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4741 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4801 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4861 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4921 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4981 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     5041 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     5101 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     5161 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     5221 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     5281 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     5341 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     5401 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5461 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctca
//
