LOCUS       sequence-416840-        7187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses TadA8e and SpRY cas9N by the specific TNT4 promoter and 
            incorporation of the miR122 target sequences (miR122TS) into the 3' 
            untranslated region (3' UTR).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7187)
  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 7187)
  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-miR122 TS
FEATURES             Location/Qualifiers
     source          1..7187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     143..162
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    530..551
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(566..571,572..589,590..596)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    604..620
                     /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)."
     primer_bind     609..631
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     628..644
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     628..644
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   682..811
                     /label=AAV2 ITR
     primer_bind     complement(858..878)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     regulatory      1496..1505
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1508..1528
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2123..3838
                     /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(4669..4688)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4698..4779
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4723..4742
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4918..5047
                     /label=AAV2 ITR
     primer_bind     complement(5066..5083)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5066..5082)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5075..5097)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5224..5679
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5311..5330)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5521..5542
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5705..5809
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5810..5878,5879..6670)
                     /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(6028..6047)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6841..7187,1..242)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
       61 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
      121 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      181 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
      241 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
      301 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
      361 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
      421 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
      481 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
      541 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
      601 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccagat
      661 ttaattaagg ccttaattag gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag
      721 cccgggcgtc gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag
      781 ggagtggcca actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac
      841 ttatctactg cggccgcgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
      901 agcaggcaga agtatgcaaa gcatggacat tgattattga ctcagtccat taggagccag
      961 tagcctggaa gatgtcttta cccccagcat cagttcaagt ggagcagcac ataactcttg
     1021 ccctctgcct tccaagattc tggtgctgag acttatggag tgtcttggag gttgccttct
     1081 gccccccaac cctgctccca gctggccctc ccaggcctgg gttgctggcc tctgctttat
     1141 caggattctc aagagggaca gctggtttat gttgcatgac tgttccctgc atatctgctc
     1201 tggttttaaa tagcttatct gagcagctgg aggaccacat gggcttatat ggcgtggggt
     1261 acatgttcct gtagccttgt ccctggcacc tgccaaaata gcagccaaca ccccccaccc
     1321 ccaccgccat ccccctgccc cacccgtccc ctgtcgcaca ttcctccctc cgcagggctg
     1381 gctcaccagg ccccagccca catgcctgct taaagccctc tccatcctct gcctcaccca
     1441 gtccccgctg agactgagca gacgcctcca ggatctgtcg gcagcgctac cggtcgccac
     1501 catggcccca aagaagaagc ggaaagtctc tgaggtggag ttttcccacg agtactggat
     1561 gagacatgcc ctgaccctgg ccaagagggc acgggatgag agggaggtgc ctgtgggagc
     1621 cgtgctggtg ctgaacaata gagtgatcgg cgagggctgg aacagagcca tcggcctgca
     1681 cgacccaaca gcccatgccg aaattatggc cctgagacag ggcggcctgg tcatgcagaa
     1741 ctacagactg attgacgcca ccctgtacgt gacattcgag ccttgcgtga tgtgcgccgg
     1801 cgccatgatc cactctagga tcggccgcgt ggtgtttggc gtgaggaact caaaaagagg
     1861 cgccgcaggc tccctgatga acgtgctgaa ctaccccggc atgaatcacc gcgtcgaaat
     1921 taccgaggga atcctggcag atgaatgtgc cgccctgctg tgcgatttct atcggatgcc
     1981 tagacaggtg ttcaatgctc agaagaaggc ccagagctcc atcaactccg gaggatctag
     2041 cggaggctcc tctggctctg agacacctgg cacaagcgag agcgcaacac ctgaaagcag
     2101 cgggggcagc agcggggggt cagacaagaa gtacagcatc ggcctggcca tcggcaccaa
     2161 ctctgtgggc tgggccgtga tcaccgacga gtacaaggtg cccagcaaga aattcaaggt
     2221 gctgggcaac accgaccggc acagcatcaa gaagaacctg atcggagccc tgctgttcga
     2281 cagcggcgaa acagccgaga gaacccggct gaagagaacc gccagaagaa gatacaccag
     2341 acggaagaac cggatctgct atctgcaaga gatcttcagc aacgagatgg ccaaggtgga
     2401 cgacagcttc ttccacagac tggaagagtc cttcctggtg gaagaggata agaagcacga
     2461 gcggcacccc atcttcggca acatcgtgga cgaggtggcc taccacgaga agtaccccac
     2521 catctaccac ctgagaaaga aactggtgga cagcaccgac aaggccgacc tgcggctgat
     2581 ctatctggcc ctggcccaca tgatcaagtt ccggggccac ttcctgatcg agggcgacct
     2641 gaaccccgac aacagcgacg tggacaagct gttcatccag ctggtgcaga cctacaacca
     2701 gctgttcgag gaaaacccca tcaacgccag cggcgtggac gccaaggcca tcctgtctgc
     2761 cagactgagc aagagcagac ggctggaaaa tctgatcgcc cagctgcccg gcgagaagaa
     2821 gaatggcctg ttcggaaacc tgattgccct gagcctgggc ctgaccccca acttcaagag
     2881 caacttcgac ctggccgagg atgccaaact gcagctgagc aaggacacct acgacgacga
     2941 cctggacaac ctgctggccc agatcggcga ccagtacgcc gacctgtttc tggccgccaa
     3001 gaacctgtcc gacgccatcc tgctgagcga catcctgaga gtgaacaccg agatcaccaa
     3061 ggcccccctg agcgcctcta tgatcaagag atacgacgag caccaccagg acctgaccct
     3121 gctgaaagct ctcgtgcggc agcagctgcc tgagaagtac aaagagattt tcttcgacca
     3181 gagcaagaac ggctacgccg gctacattga cggcggagcc agccaggaag agttctacaa
     3241 gttcatcaag cccatcctgg aaaagatgga cggcaccgag gaactgctcg tgaagctgaa
     3301 cagagaggac ctgctgcgga agcagcggac cttcgacaac ggcagcatcc cccaccagat
     3361 ccacctggga gagctgcacg ccattctgcg gcggcaggaa gatttttacc cattcctgaa
     3421 ggacaaccgg gaaaagatcg agaagatcct gaccttccgc atcccctact acgtgggccc
     3481 tctggccagg ggaaacagca gattcgcctg gatgaccaga aagagcgagg aaaccatcac
     3541 cccctggaac ttcgaggaag tggtggacaa gggcgcttcc gcccagagct tcatcgagcg
     3601 gatgaccaac ttcgataaga acctgcccaa cgagaaggtg ctgcccaagc acagcctgct
     3661 gtacgagtac ttcaccgtgt ataacgagct gaccaaagtg aaatacgtga ccgagggaat
     3721 gagaaagccc gccttcctga gcggcgagca gaaaaaggcc atcgtggacc tgctgttcaa
     3781 gaccaaccgg aaagtgaccg tgaagcagct gaaagaggac tacttcaaga aaatcgagtg
     3841 cttcgactcc gtggaaatct ccggcgtgga agatcggttc aacgcctccc tgggcacata
     3901 ccacgatctg ctgaaaatta tcaaggacaa ggacttcctg gacaatgagg aaaacgagga
     3961 cattctggaa gatatcgtgc tgaccctgac actgtttgag gacagagaga tgatcgagga
     4021 acggctgaaa acctatgccc acctgttcga cgacaaagtg atgaagcagc tgaagcggcg
     4081 gagatacacc ggctggggca ggctgagccg gaagctgatc aacggcatcc gagacaagca
     4141 gagtggaaag acaatcctgg attttcttaa gtccgatgga tttgccaacc ggaacttcat
     4201 gcagttgatc catgatgact ctctcacctt taaggaggac atccagaaag cacaagtttg
     4261 tctggctggc gatactctca ttaccctggc cgatggacga cgagtgccta ttagagaact
     4321 ggtgtcacag cagaattttt ccgtgtgggc tctgaatcct cagacttacc gcctggagag
     4381 ggctagagtg agtagagctt tctgtaccgg catcaaacct gtgtaccgcc tcaccactag
     4441 actggggaga tccattaggg ccactgccaa ccaccgattt ctcacacctc agggctggaa
     4501 acgagtcgat gaactccagc ctggagatta cctggctctg cctaggagaa tccctactgc
     4561 ctcctgatct cgacggtcaa acaccattgt cacactccat actatcaaac accattgtca
     4621 cactccagat caacaaacac cattgtcaca ctccacgccg tcgagctcga tgagtttgga
     4681 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     4741 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     4801 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta aaacctctac
     4861 aaatgtggta gcggccgcag gtagataagt agcatggcgg gttaatcatt aactacaagg
     4921 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     4981 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     5041 cgcgcagcct taattaacct aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     5101 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     5161 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     5221 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     5281 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     5341 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     5401 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     5461 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     5521 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     5581 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     5641 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     5701 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     5761 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     5821 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     5881 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     5941 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     6001 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     6061 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     6121 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     6181 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     6241 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     6301 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
     6361 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     6421 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     6481 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     6541 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     6601 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     6661 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     6721 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     6781 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     6841 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     6901 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     6961 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     7021 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     7081 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     7141 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagct
//
