LOCUS       sequence-415448-        7096 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TadA8e and SpRY cas9N by the specific TNT4 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7096)
  AUTHORS   Yang L, Liu Z, Chen G, Chen Z, Guo C, Ji X, Cui Q, Sun Y, Hu X, 
            Zheng Y, Li Y, Gao F, Chen L, Zhou P, Pu WT, Guo Y
  TITLE     MicroRNA-122-Mediated Liver Detargeting Enhances the Tissue 
            Specificity of Cardiac Genome Editing.
  JOURNAL   Circulation. 2024 May 28;149(22):1778-1781. doi: 
            10.1161/CIRCULATIONAHA.123.065438. Epub 2024 May 28.
  PUBMED    38805581
REFERENCE   2  (bases 1 to 7096)
  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"; doi: 
            "10.1161/CIRCULATIONAHA.123.065438"; journalName: "Circulation"; 
            date: "2024-05-28- 28"; volume: "149"; issue: "22"; pages: 
            "1778-1781"
COMMENT     Sequence Label: pAAV-TNT4-TadA8e-SpRY N aa2-713-InteinN
FEATURES             Location/Qualifiers
     source          1..7096
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(32..487)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(169..190)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     381..400
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     614..636
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     628..645
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     629..645
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   664..793
                     /label=AAV2 ITR
     primer_bind     complement(840..860)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     regulatory      1478..1487
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1490..1510
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2105..3820
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     primer_bind     complement(4560..4579)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4589..4670
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4614..4633
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4809..4938
                     /label=AAV2 ITR
     primer_bind     complement(4976..4992)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4976..4992)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4989..5011)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5000..5016
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5024..5030,5031..5048,5049..5054))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5069..5090
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(5378..5966)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5458..5477)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6137..6928,6929..6997))
                     /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     6760..6779
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6998..7096,1..6))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
       61 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
      121 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
      181 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
      241 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
      301 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
      361 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
      421 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
      481 ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
      541 tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
      601 acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt aggttaatta
      661 aggctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
      721 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      781 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac tgcggccgcg
      841 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
      901 aagcatggac attgattatt gactcagtcc attaggagcc agtagcctgg aagatgtctt
      961 tacccccagc atcagttcaa gtggagcagc acataactct tgccctctgc cttccaagat
     1021 tctggtgctg agacttatgg agtgtcttgg aggttgcctt ctgcccccca accctgctcc
     1081 cagctggccc tcccaggcct gggttgctgg cctctgcttt atcaggattc tcaagaggga
     1141 cagctggttt atgttgcatg actgttccct gcatatctgc tctggtttta aatagcttat
     1201 ctgagcagct ggaggaccac atgggcttat atggcgtggg gtacatgttc ctgtagcctt
     1261 gtccctggca cctgccaaaa tagcagccaa caccccccac ccccaccgcc atccccctgc
     1321 cccacccgtc ccctgtcgca cattcctccc tccgcagggc tggctcacca ggccccagcc
     1381 cacatgcctg cttaaagccc tctccatcct ctgcctcacc cagtccccgc tgagactgag
     1441 cagacgcctc caggatctgt cggcagcgct accggtcgcc accatggccc caaagaagaa
     1501 gcggaaagtc tctgaggtgg agttttccca cgagtactgg atgagacatg ccctgaccct
     1561 ggccaagagg gcacgggatg agagggaggt gcctgtggga gccgtgctgg tgctgaacaa
     1621 tagagtgatc ggcgagggct ggaacagagc catcggcctg cacgacccaa cagcccatgc
     1681 cgaaattatg gccctgagac agggcggcct ggtcatgcag aactacagac tgattgacgc
     1741 caccctgtac gtgacattcg agccttgcgt gatgtgcgcc ggcgccatga tccactctag
     1801 gatcggccgc gtggtgtttg gcgtgaggaa ctcaaaaaga ggcgccgcag gctccctgat
     1861 gaacgtgctg aactaccccg gcatgaatca ccgcgtcgaa attaccgagg gaatcctggc
     1921 agatgaatgt gccgccctgc tgtgcgattt ctatcggatg cctagacagg tgttcaatgc
     1981 tcagaagaag gcccagagct ccatcaactc cggaggatct agcggaggct cctctggctc
     2041 tgagacacct ggcacaagcg agagcgcaac acctgaaagc agcgggggca gcagcggggg
     2101 gtcagacaag aagtacagca tcggcctggc catcggcacc aactctgtgg gctgggccgt
     2161 gatcaccgac gagtacaagg tgcccagcaa gaaattcaag gtgctgggca acaccgaccg
     2221 gcacagcatc aagaagaacc tgatcggagc cctgctgttc gacagcggcg aaacagccga
     2281 gagaacccgg ctgaagagaa ccgccagaag aagatacacc agacggaaga accggatctg
     2341 ctatctgcaa gagatcttca gcaacgagat ggccaaggtg gacgacagct tcttccacag
     2401 actggaagag tccttcctgg tggaagagga taagaagcac gagcggcacc ccatcttcgg
     2461 caacatcgtg gacgaggtgg cctaccacga gaagtacccc accatctacc acctgagaaa
     2521 gaaactggtg gacagcaccg acaaggccga cctgcggctg atctatctgg ccctggccca
     2581 catgatcaag ttccggggcc acttcctgat cgagggcgac ctgaaccccg acaacagcga
     2641 cgtggacaag ctgttcatcc agctggtgca gacctacaac cagctgttcg aggaaaaccc
     2701 catcaacgcc agcggcgtgg acgccaaggc catcctgtct gccagactga gcaagagcag
     2761 acggctggaa aatctgatcg cccagctgcc cggcgagaag aagaatggcc tgttcggaaa
     2821 cctgattgcc ctgagcctgg gcctgacccc caacttcaag agcaacttcg acctggccga
     2881 ggatgccaaa ctgcagctga gcaaggacac ctacgacgac gacctggaca acctgctggc
     2941 ccagatcggc gaccagtacg ccgacctgtt tctggccgcc aagaacctgt ccgacgccat
     3001 cctgctgagc gacatcctga gagtgaacac cgagatcacc aaggcccccc tgagcgcctc
     3061 tatgatcaag agatacgacg agcaccacca ggacctgacc ctgctgaaag ctctcgtgcg
     3121 gcagcagctg cctgagaagt acaaagagat tttcttcgac cagagcaaga acggctacgc
     3181 cggctacatt gacggcggag ccagccagga agagttctac aagttcatca agcccatcct
     3241 ggaaaagatg gacggcaccg aggaactgct cgtgaagctg aacagagagg acctgctgcg
     3301 gaagcagcgg accttcgaca acggcagcat cccccaccag atccacctgg gagagctgca
     3361 cgccattctg cggcggcagg aagattttta cccattcctg aaggacaacc gggaaaagat
     3421 cgagaagatc ctgaccttcc gcatccccta ctacgtgggc cctctggcca ggggaaacag
     3481 cagattcgcc tggatgacca gaaagagcga ggaaaccatc accccctgga acttcgagga
     3541 agtggtggac aagggcgctt ccgcccagag cttcatcgag cggatgacca acttcgataa
     3601 gaacctgccc aacgagaagg tgctgcccaa gcacagcctg ctgtacgagt acttcaccgt
     3661 gtataacgag ctgaccaaag tgaaatacgt gaccgaggga atgagaaagc ccgccttcct
     3721 gagcggcgag cagaaaaagg ccatcgtgga cctgctgttc aagaccaacc ggaaagtgac
     3781 cgtgaagcag ctgaaagagg actacttcaa gaaaatcgag tgcttcgact ccgtggaaat
     3841 ctccggcgtg gaagatcggt tcaacgcctc cctgggcaca taccacgatc tgctgaaaat
     3901 tatcaaggac aaggacttcc tggacaatga ggaaaacgag gacattctgg aagatatcgt
     3961 gctgaccctg acactgtttg aggacagaga gatgatcgag gaacggctga aaacctatgc
     4021 ccacctgttc gacgacaaag tgatgaagca gctgaagcgg cggagataca ccggctgggg
     4081 caggctgagc cggaagctga tcaacggcat ccgagacaag cagagtggaa agacaatcct
     4141 ggattttctt aagtccgatg gatttgccaa ccggaacttc atgcagttga tccatgatga
     4201 ctctctcacc tttaaggagg acatccagaa agcacaagtt tgtctggctg gcgatactct
     4261 cattaccctg gccgatggac gacgagtgcc tattagagaa ctggtgtcac agcagaattt
     4321 ttccgtgtgg gctctgaatc ctcagactta ccgcctggag agggctagag tgagtagagc
     4381 tttctgtacc ggcatcaaac ctgtgtaccg cctcaccact agactgggga gatccattag
     4441 ggccactgcc aaccaccgat ttctcacacc tcagggctgg aaacgagtcg atgaactcca
     4501 gcctggagat tacctggctc tgcctaggag aatccctact gcctcctgat ctcgagctcg
     4561 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     4621 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     4681 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     4741 aaaacctcta caaatgtggt agcggccgca ggtagataag tagcatggcg ggttaatcat
     4801 taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     4861 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4921 gagcgagcga gcgcgcagcc taattaaggc cttaattaaa tctggcgtaa tcatggtcat
     4981 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     5041 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     5101 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5161 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     5221 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5281 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5341 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5401 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5461 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5521 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5581 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5641 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5701 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5761 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     5821 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5881 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5941 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6001 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     6061 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     6121 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     6181 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     6241 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     6301 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     6361 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     6421 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     6481 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     6541 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     6601 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     6661 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     6721 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     6781 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     6841 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     6901 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     6961 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     7021 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     7081 ataaacaaat aggggt
//
