LOCUS       sequence-411112-        5616 bp    DNA     circular SYN 14-APR-2025
DEFINITION  All-in-one AAV vector expressing ISAba30 TnpB and its reRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5616)
  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 5616)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-ISAba30-NLS-3xHA-bGHpA;U6-BbsI-reRNA
FEATURES             Location/Qualifiers
     source          1..5616
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   16..145
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        169..548
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        549..752
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     703..723
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      771..780
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             783..803
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2046..2093
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2100..2180
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2208..2225)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2214..2421
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(2637..2885)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2695..2714)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2865..2885)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   2894..3034
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2894..3023
                     /label=AAV2 ITR
     rep_origin      3109..3564
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3196..3215)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3406..3427
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3581..3600)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3700..3722
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3760..3778)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3846..3950
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3951..4019,4020..4811)
                     /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(4169..4188)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4982..5570
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5471..5490
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cttttgctca catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg
       61 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg
      121 gccaactcca tcactagggg ttcctgcggc ctctagactc gaggcgttga cattgattat
      181 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      241 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      301 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      361 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      421 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      481 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      541 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      601 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      661 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      721 gtgtacggtg ggaggtctat ataagcagag ctctctggct aactaccggt gccaccatgg
      781 ccccaaagaa gaagcggaag gtcggtatcc acggagtccc agcagccatg aagcggctgc
      841 aggctttcaa gtacgagatg gtgcccaacg gacagcagga gaggcagatg agaagattcg
      901 ccgggagctg ccggttcgtg tacaacaagg ccctggccct gcagaaggag agatacgaga
      961 gaggagaaaa gaagctgggg tacgccggcc tgtgcaagct gctgacagag tggaggaaca
     1021 gcagcgagat ggcttggctg gccgacgctc ctgtgcatcc tctgcagcag accctgaagg
     1081 atctggagag agcctacagc aatttcttcg ccaagcgcgc cgactttccc agattcaaaa
     1141 agaagggctg tcactccagc ttccgctacc ccgatcccaa acagatcaaa ctggagcagc
     1201 acaacagcag aatcttcctg ccaaagctgg gctggattag ataccgcaag agtagagacg
     1261 tgctgggagc cgtgaaaaac gtgaccgtga gtctgagcgg cgggaaatgg ttcgtgagca
     1321 tccagacaga gcgggaagtg gagcagccca tccctcaggg aggagctgtg ggaattgaca
     1381 tggggatcgc cagattcgct accattagcg acggaaagtt ctacgctccc ctgaacagct
     1441 tcaaaagaca cgagaagaga ctgagatgcg cccagcaggc cctgtctaga aaggtgaagt
     1501 tcagcaacaa ctggcagaaa gccaaagcca gaatccagag aatccatagc agaatcggca
     1561 acgccagaag agacttcctg cacaagacca gcaccgccat cagccagaac catgccatgg
     1621 tgtgcatcga ggacctgcag gtgagaaaca tgagcaagag cgccgccgga acccctgaac
     1681 agcctggaag aaacgtgaga gccaaaagcg gcctgaacaa gagcatcctg gaccagggat
     1741 ggttcgaatt tagaagacag ctggactaca agctggcctg gaacggaggc tggctggtgg
     1801 ctgtgcctcc taaaaacacc agcagaacct gcccctgctg cgctcatgtg agcgctgata
     1861 acagacagac ccaggagaga tttgagtgca tcgagtgtgg cttcgagggc aacgctgacg
     1921 tggtgggagc tatcaacgtg ctgagagccg gccacgctag actggcttgt gaggtgagcg
     1981 acgccgtggg aagtccagct gctggaacac accggagcga ctctgatcag gcccactgta
     2041 gggtgaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagggatcct
     2101 acccatacga tgttccagat tacgcttacc catacgatgt tccagattac gcttacccat
     2161 acgatgttcc agattacgct taagaattcc tagagctcgc tgatcagcct cgactgtgcc
     2221 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2281 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2341 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2401 gaatagcagg catgctgggg aggtaccaaa aaatgagacc tgccgtggtc tccttgacat
     2461 cctccccggc ctgaaggccg gagattccta cggcgctcag acgcggcaat gagcctggtc
     2521 tgagtcgctt cggtgggttc ctgctgctgg cgaccctact gcatcgctca cttcacaggc
     2581 taaccgggcg tgccccgccc ttagtacatt gatcgcgccg accacatcgg cattacggtg
     2641 tttcgtcctt tccacaagat atataaagcc aagaaatcga aatactttca agttacggta
     2701 agcatatgat agtccatttt aaaacataat tttaaaactg caaactaccc aagaaattat
     2761 tactttctac gtcacgtatt ttgtactaat atctttgtgt ttacagtcaa attaattcca
     2821 attatctctc taacagcctt gtatcgtata tgcaaatatg aaggaatcat gggaaatagg
     2881 ccctcgcggc cgcaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc
     2941 tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc
     3001 tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc tgatgcggta ttttctcctt
     3061 acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac gcgccctgta
     3121 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     3181 gcgccttagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     3241 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     3301 acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca tcgccctgat
     3361 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     3421 aaactggaac aacactcaac tctatctcgg gctattcttt tgatttataa gggattttgc
     3481 cgatttcggt ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     3541 acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc tctgatgccg
     3601 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     3661 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     3721 ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
     3781 tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa
     3841 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3901 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc
     3961 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     4021 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     4081 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     4141 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg
     4201 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     4261 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     4321 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     4381 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     4441 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa
     4501 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     4561 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     4621 cggctggctg gtttattgct gataaatctg gagccggtga gcgtggaagc cgcggtatca
     4681 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     4741 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     4801 agcattggta actgtcagac caagtttact catatatact ttagattgat ttaaaacttc
     4861 atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc
     4921 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4981 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     5041 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     5101 tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact
     5161 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     5221 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     5281 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     5341 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     5401 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5461 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5521 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5581 acgcggcctt tttacggttc ctggcctttt gctggc
//
