LOCUS       sequence-416839-        7500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SaCas9 by the specific cTNT promoter and sgRNA scaffold by
            U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7500)
  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 7500)
  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: AAV-U6-sgRNA-Scaffold-cTNT-SaCas9-HA-OLLAS
FEATURES             Location/Qualifiers
     source          1..7500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   39..168
                     /label=AAV2 ITR
     promoter        290..538
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     290..310
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     461..480
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        561..636
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     intron          1207..1339
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1322..1341
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1384..1402
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1459..1479
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1504..4659
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             4660..4707
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4714..4740
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4741..4782
                     /codon_start=1
                     /product="Escherichia coli OmpF Linker and mouse Langerin 
                     fusion sequence tag (Park et al., 2008)"
                     /label=OLLAS tag
                     /translation="SGFANELGPRLMGK"
     polyA_signal    4800..4848
                     /note="synthetic polyadenylation signal"
     repeat_region   4896..5025
                     /label=AAV2 ITR
     rep_origin      complement(5262..5850)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5342..5361)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6021..6812,6813..6881))
                     /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     6644..6663
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6882..6986)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(7012..7467)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7149..7170)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7361..7380
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct gcgcgctcgc tcgctcactg
       61 aggccgcccg ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg
      121 agcgagcgcg cagagaggga gtggccaact ccatcactag gggttccttg tagttaatga
      181 ttaacccgcc atgctactta tctacgtagc catgctctgg tgtacaaaaa agcaggcttt
      241 aaaggaacca attcagtcga ctggatccgg taccaaggtc gggcaggaag agggcctatt
      301 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
      361 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      421 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      481 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg
      541 gtcttcacgc gtgaagacca gttttagtac tctggaaaca gaatctacta aaacaaggca
      601 aaatgccgtg tttatctcgt caacttgttg gcgagatttt tttctagacg ggccccccct
      661 cgaggtcggg ataaaagcag tctgggcttt cacatgacag catctggggc tgcggcagag
      721 ggtcgggtcc gaagcgctgc cttatcagcg tccccagccc tgggaggtga cagctggctg
      781 gcttgtgtca gcccctcggg cactcacgta tctccgtccg acgggtttaa aatagcaaaa
      841 ctctgaggcc acacaatagc ttgggcttat atgggctcct gtgggggaag ggggagcacg
      901 gagggggccg gggccgctgc tgccaaaata gcagctcaca agtgttgcat tcctctctgg
      961 gcgccgggca cattcctgct ggctctgccc gccccggggt gggcgccggg gggaccttaa
     1021 agcctctgcc ccccaaggag cccttcccag acagccgccg gcacccaccg ctccgtggga
     1081 cgatccccga agctctagag ctttattgcg gtagtttatc acagttaaat tgctaacgca
     1141 gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc gtgaggcact
     1201 gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa actgggcttg
     1261 tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac tgacatccac
     1321 tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta aggctagagt
     1381 acttaatacg actcactata ggctagccct cgagaagcgg ccgcactact ccgcggacta
     1441 ctagagctca ccatggcccc aaagaagaag cggaaggtcg gtatccacgg agtcccagca
     1501 gccaagcgga actacatcct gggcctggac atcggcatca ccagcgtggg ctacggcatc
     1561 atcgactacg agacacggga cgtgatcgat gccggcgtgc ggctgttcaa agaggccaac
     1621 gtggaaaaca acgagggcag gcggagcaag agaggcgcca gaaggctgaa gcggcggagg
     1681 cggcatagaa tccagagagt gaagaagctg ctgttcgact acaacctgct gaccgaccac
     1741 agcgagctga gcggcatcaa cccctacgag gccagagtga agggcctgag ccagaagctg
     1801 agcgaggaag agttctctgc cgccctgctg cacctggcca agagaagagg cgtgcacaac
     1861 gtgaacgagg tggaagagga caccggcaac gagctgtcca ccaaagagca gatcagccgg
     1921 aacagcaagg ccctggaaga gaaatacgtg gccgaactgc agctggaacg gctgaagaaa
     1981 gacggcgaag tgcggggcag catcaacaga ttcaagacca gcgactacgt gaaagaagcc
     2041 aaacagctgc tgaaggtgca gaaggcctac caccagctgg accagagctt catcgacacc
     2101 tacatcgacc tgctggaaac ccggcggacc tactatgagg gacctggcga gggcagcccc
     2161 ttcggctgga aggacatcaa agaatggtac gagatgctga tgggccactg cacctacttc
     2221 cccgaggaac tgcggagcgt gaagtacgcc tacaacgccg acctgtacaa cgccctgaac
     2281 gacctgaaca atctcgtgat caccagggac gagaacgaga agctggaata ttacgagaag
     2341 ttccagatca tcgagaacgt gttcaagcag aagaagaagc ccaccctgaa gcagatcgcc
     2401 aaagaaatcc tcgtgaacga agaggatatt aagggctaca gagtgaccag caccggcaag
     2461 cccgagttca ccaacctgaa ggtgtaccac gacatcaagg acattaccgc ccggaaagag
     2521 attattgaga acgccgagct gctggatcag attgccaaga tcctgaccat ctaccagagc
     2581 agcgaggaca tccaggaaga actgaccaat ctgaactccg agctgaccca ggaagagatc
     2641 gagcagatct ctaatctgaa gggctatacc ggcacccaca acctgagcct gaaggccatc
     2701 aacctgatcc tggacgagct gtggcacacc aacgacaacc agatcgctat cttcaaccgg
     2761 ctgaagctgg tgcccaagaa ggtggacctg tcccagcaga aagagatccc caccaccctg
     2821 gtggacgact tcatcctgag ccccgtcgtg aagagaagct tcatccagag catcaaagtg
     2881 atcaacgcca tcatcaagaa gtacggcctg cccaacgaca tcattatcga gctggcccgc
     2941 gagaagaact ccaaggacgc ccagaaaatg atcaacgaga tgcagaagcg gaaccggcag
     3001 accaacgagc ggatcgagga aatcatccgg accaccggca aagagaacgc caagtacctg
     3061 atcgagaaga tcaagctgca cgacatgcag gaaggcaagt gcctgtacag cctggaagcc
     3121 atccctctgg aagatctgct gaacaacccc ttcaactatg aggtggacca catcatcccc
     3181 agaagcgtgt ccttcgacaa cagcttcaac aacaaggtgc tcgtgaagca ggaagaaaac
     3241 agcaagaagg gcaaccggac cccattccag tacctgagca gcagcgacag caagatcagc
     3301 tacgaaacct tcaagaagca catcctgaat ctggccaagg gcaagggcag aatcagcaag
     3361 accaagaaag agtatctgct ggaagaacgg gacatcaaca ggttctccgt gcagaaagac
     3421 ttcatcaacc ggaacctggt ggataccaga tacgccacca gaggcctgat gaacctgctg
     3481 cggagctact tcagagtgaa caacctggac gtgaaagtga agtccatcaa tggcggcttc
     3541 accagctttc tgcggcggaa gtggaagttt aagaaagagc ggaacaaggg gtacaagcac
     3601 cacgccgagg acgccctgat cattgccaac gccgatttca tcttcaaaga gtggaagaaa
     3661 ctggacaagg ccaaaaaagt gatggaaaac cagatgttcg aggaaaagca ggccgagagc
     3721 atgcccgaga tcgaaaccga gcaggagtac aaagagatct tcatcacccc ccaccagatc
     3781 aagcacatta aggacttcaa ggactacaag tacagccacc gggtggacaa gaagcctaat
     3841 agagagctga ttaacgacac cctgtactcc acccggaagg acgacaaggg caacaccctg
     3901 atcgtgaaca atctgaacgg cctgtacgac aaggacaatg acaagctgaa aaagctgatc
     3961 aacaagagcc ccgaaaagct gctgatgtac caccacgacc cccagaccta ccagaaactg
     4021 aagctgatta tggaacagta cggcgacgag aagaatcccc tgtacaagta ctacgaggaa
     4081 accgggaact acctgaccaa gtactccaaa aaggacaacg gccccgtgat caagaagatt
     4141 aagtattacg gcaacaaact gaacgcccat ctggacatca ccgacgacta ccccaacagc
     4201 agaaacaagg tcgtgaagct gtccctgaag ccctacagat tcgacgtgta cctggacaat
     4261 ggcgtgtaca agttcgtgac cgtgaagaat ctggatgtga tcaaaaaaga aaactactac
     4321 gaagtgaata gcaagtgcta tgaggaagct aagaagctga agaagatcag caaccaggcc
     4381 gagtttatcg cctccttcta caacaacgat ctgatcaaga tcaacggcga gctgtataga
     4441 gtgatcggcg tgaacaacga cctgctgaac cggatcgaag tgaacatgat cgacatcacc
     4501 taccgcgagt acctggaaaa catgaacgac aagaggcccc ccaggatcat taagacaatc
     4561 gcctccaaga cccagagcat taagaagtac agcacagaca ttctgggcaa cctgtatgaa
     4621 gtgaaatcta agaagcaccc tcagatcatc aaaaagggca aaaggccggc ggccacgaaa
     4681 aaggccggcc aggcaaaaaa gaaaaaggga tcctacccat acgatgttcc agattacgct
     4741 agcggcttcg ccaacgagct tggacccagg ttgatgggaa agtaagaatt cctagagcga
     4801 ataaaagatc tttattttca ttagatctgt gtgttggttt tttgtgtgat gcagctacgt
     4861 agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc
     4921 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg
     4981 cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgca ttaatgaatc
     5041 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     5101 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     5161 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     5221 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     5281 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     5341 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     5401 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     5461 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     5521 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     5581 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     5641 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     5701 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     5761 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     5821 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5881 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5941 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     6001 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     6061 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     6121 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     6181 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     6241 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     6301 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     6361 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     6421 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     6481 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     6541 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     6601 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     6661 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     6721 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     6781 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6841 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6901 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6961 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     7021 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     7081 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     7141 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     7201 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     7261 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     7321 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     7381 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     7441 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
//
