LOCUS       sequence-423077-        8089 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for expression of ISDra2-TnpB driven by chicken 
            beta-actin promoter and mCherry driven by CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8089)
  AUTHORS   Li Z, Guo R, Sun X, Li G, Shao Z, Huo X, Yang R, Liu X, Cao X, Zhang
            H, Zhang W, Zhang X, Ma S, Zhang M, Liu Y, Yao Y, Shi J, Yang H, Hu 
            C, Zhou Y, Xu C
  TITLE     Engineering a transposon-associated TnpB-omegaRNA system for 
            efficient gene editing and phenotypic correction of a tyrosinaemia 
            mouse model.
  JOURNAL   Nat Commun. 2024 Jan 27;15(1):831. doi: 10.1038/s41467-024-45197-z.
  PUBMED    38280857
REFERENCE   2  (bases 1 to 8089)
  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 
            Commun."; date: "2024-01-27"; volume: "15(1):831. doi"; pages: " 
            10.1038/s41467-024-45197-z"
COMMENT     Sequence Label: ISDra2-TnpB-mCherry-U6-omega-RNA*
FEATURES             Location/Qualifiers
     source          1..8089
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   50..112
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        421..706
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        708..985
                     /label=chicken beta-actin promoter
     intron          986..1213
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     primer_bind     1224..1243
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1224..1242
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1258..1267
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1273..1293
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2530..2577
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2584..2607
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2623..2676
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             2677..3387
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     DDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2822..2840)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3394..3528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3431..3450)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3485..3504
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3535..3783
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3535..3555
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3706..3725
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     ncRNA           3907..3974
                     /product="hepatitis delta virus self-cleaving ribozyme 
                     (Ferre-D'Amare and Scott, 2010)"
                     /label=HDV ribozyme
                     /ncRNA_class="ribozyme"
     promoter        4031..4242
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             4258..5001
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             5011..5067
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             5068..5664
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     5561..5581
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    5671..5895
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6252..6286
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        6317..6421
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6422..6490,6491..7282)
                     /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(6640..6659)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7453..8041
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7942..7961
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttgctcaca tgttctttcc tgcgttatcc cctgattctc tcgagttaac cctagaaaga
       61 taatcatatt gtgacgtacg ttaaagataa tcatgcgtaa aattgacgca tgtgttttat
      121 cgatctgtat atcgaggttt atttattaat ttgaatagat attaagtttt attatattta
      181 cacttacata ctaataataa attcaacaaa caatttattt atgtttattt atttattaaa
      241 aaaaaacaaa aactcaaaat ttcttttata aagtaacaaa acttttaaac attctctctt
      301 ttacaaaaat aaacttattt tgtactttaa aaacagtcat gttgtattat aaaataagta
      361 attagcttaa cttatacata atagaaacaa attatactta ttaattaatc tagaggtacc
      421 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      481 gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
      541 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
      601 cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt
      661 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc
      721 cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt
      781 tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg
      841 cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa
      901 tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta
      961 taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg
     1021 ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt
     1081 gagcgggcgg gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt
     1141 taagggatgg ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat
     1201 cacttttttt caggttggga aattaatacg actcactata gggaccggtg cggccgcgcc
     1261 accatgggat ctccaaaaaa gaaacggaag gtgggatcta tgatccggaa caaggccttc
     1321 gtggtgaggc tgtaccccaa cgccgcccag acagagctga tcaatagaac cctgggcagc
     1381 gccaggttcg tgtacaatca cttcctggcc aggaggatcg ccgcctacaa ggagtccggc
     1441 aagggcctga catacggcca gacatccagc gagctgaccc tgctgaagca ggccgaggag
     1501 acatcctggc tgagcgaggt ggacaagttt gccctgcaga acagcctgaa gaatctggag
     1561 accgcctaca agaacttctt cagaacagtg aagcagagcg gcaagaaggt gggctttccc
     1621 aggttcagaa agaagagaac aggcgagagc tacaggacac agtttacaaa taacaacatc
     1681 cagatcggcg agggcaggct gaagctgcct aagctgggct gggttaagac aaagggccag
     1741 caggatattc agggcaagat cctgaatgtg acagtgagaa ggattcatga gggccactac
     1801 gaggccagcg tgctgtgtga ggtggagatc ccctacctgc ccgccgctcc taagttcgcc
     1861 gccggagttg atgtgggcat caaggacttt gccatcgtga ccgacggcgt gagatttaag
     1921 cacgagcaga accctaagta ctacaggagc acactgaaga ggctgaggaa ggcccagcag
     1981 acactgtcca gaagaaagaa gggcagcgcc cggtacggca aggccaagac aaagctggcc
     2041 aggattcata agagaatcgt gaataagagg caggactttc tgcacaagct gaccacaagc
     2101 ctggtgaggg agtacgagat catcggcaca gagcacctga agcctgataa catgaggaag
     2161 aacaggagac tggccctgtc catcagcgat gccggctggg gagagtttat cagacagctg
     2221 gagtacaagg ccgcctggta cggcagactg gtgagcaagg tgagccctta cttccccagc
     2281 tcccagctgt gtcacgactg cggcttcaag aatcccgagg tgaagaacct ggccgtgaga
     2341 acctggacct gtcccaactg cggcgagacc cacgacagag atgaaaatgc cgccctgaac
     2401 atcaggaggg aggccctggt ggccgccgga attagcgaca cactgaatgc ccacggcggc
     2461 tacgtgagac ctgcctctgc cggaaatggc ctgaggtccg agaaccacgc caccctggtg
     2521 gtgggttcca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaaggga
     2581 tctgattaca aagacgatga cgataaggaa ttcggtagtg gggagggcag aggaagtctg
     2641 ctaacatgcg gtgacgtcga ggagaatcct ggcccaatgg tgagcaaggg cgaggaggat
     2701 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     2761 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     2821 gccaagctga aggtgaccaa gggtggcccc ctgcccttcg cctgggacat cctgtcccct
     2881 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     2941 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga tgacggcggc
     3001 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     3061 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaaaac catgggctgg
     3121 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     3181 aggctgaagc tgaaggacgg cggccactac gacgctgagg tcaagaccac ctacaaggcc
     3241 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagttgga catcacctcc
     3301 cacaacgagg actacaccat cgtggaacag tacgaacgcg ccgagggccg ccactccacc
     3361 ggcggcatgg acgagctgta caagtgaaag cttaacttgt ttattgcagc ttataatggt
     3421 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     3481 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatcac tagtgagggc
     3541 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     3601 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     3661 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     3721 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     3781 accgtggtgg ctgcgggaat ctcagacacc ttaaacgctc atggaggcta tgaaaatggt
     3841 ctgcgaagtg agaatcacgc gactttagtc gtgtgaggtt caaatgtctt cctgggacga
     3901 agacaaggcc ggcatggtcc cagcctcctc gctggcgccg gctgggcaac atgcttcggc
     3961 atggcgaatg ggactttttt tacgcgttag gtcttgaaag gagtgggaat tggctccggt
     4021 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
     4081 ggcaattgat ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
     4141 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
     4201 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggacgctag cgccaccatg
     4261 agccggctgg acaagagcaa agtgatcaac agcgccctgg aactgctgaa cggcgtgggc
     4321 atcgagggcc tgaccacccg gaagctggcc cagaaactgg gcgtggaaca gcccaccctg
     4381 tactggcacg tgaagaacaa gcgggccctg ctggacgccc tgcccatcga gatgctggac
     4441 cggcaccaca cccacagctg ccctctggaa ggcgagagct ggcaggactt cctgcggaac
     4501 aacgccaaga gctacagatg cgccctgctg agccaccggg acggcgccaa agtgcacctg
     4561 ggcaccagac ccaccgagaa gcagtacgag acactggaaa accagctggc cttcctgtgc
     4621 cagcagggct tcagcctgga aaacgccctg tacgccctga gcgccgtggg ccacttcacc
     4681 ctgggctgcg tgctggaaga acaggaacac caggtcgcca aagaggaaag agagacaccc
     4741 accaccgaca gcatgccccc cctgctgaag caggccatcg agctgttcga cagacagggc
     4801 gccgagcccg ccttcctgtt cggcctggaa ctgatcatct gcggcctcga gaagcagctg
     4861 aagtgcgaga gcggcggacc caccgacgcc ctggacgact tcgacctgga catgctgccc
     4921 gccgatgccc tggatgattt tgatctggat atgctccctg ctgacgctct cgatgacttc
     4981 gatctcgata tgctgccagg cggtagtggg gcaacaaact tctctctgct gaaacaagcc
     5041 ggagatgtcg aagagaatcc tggaccgacc gagtacaagc ccacggtgcg cctcgccacc
     5101 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     5161 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     5221 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     5281 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     5341 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     5401 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg agtctcgccc
     5461 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     5521 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     5581 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     5641 atgacccgca agcccggtgc ctgacttaag ctgtgccttc tagttgccag ccatctgttg
     5701 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     5761 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     5821 gggtggggca ggacagcaag ggggaggatt gggaaaacaa tagcaggcat gctggggatg
     5881 cggtgggctc tatgggtcga cggcgcgccc tagacgaagc agctccagcc tacacaatcg
     5941 ctcaagacgt gtaatgcttt tattatatat tagtcacgat atctataaca agaaaatata
     6001 tatataataa gttatcacgt aagtagaaca tgaaataaca atataattat cgtatgagtt
     6061 aaatcttaaa agtcacgtaa aagataatca tgcgtcattt tgactcacgc ggtcgttata
     6121 gttcaaaatc agtgacactt accgcattga caagcacgcc tcacgggagc tccaagcggc
     6181 gactgagatg tcctaaatgc acagcgacgg attcgcgcta tttagaaaga gagagcaata
     6241 tttcaagaat gcatgcgtca attttacgca gactatcttt ctagggttaa ggatccgcac
     6301 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6361 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     6421 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     6481 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     6541 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     6601 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6661 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6721 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6781 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6841 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6901 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6961 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     7021 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     7081 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     7141 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     7201 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     7261 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     7321 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     7381 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7441 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     7501 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     7561 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     7621 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7681 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7741 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7801 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7861 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7921 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7981 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     8041 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcct
//
