LOCUS       sequence-415445-        7539 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses SaCas9 by the specific cTNT promoter and sgRNA targeted 
            the mouse Camk2d gene by U6 promoter, 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 7539)
  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 7539)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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-msCAMK2d-sgRNA-cTNT-SaCas9-HA-miR122 TS
FEATURES             Location/Qualifiers
     source          1..7539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             2113..2160
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2167..2193
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2293..2341
                     /note="synthetic polyadenylation signal"
     repeat_region   2389..2518
                     /label=AAV2 ITR
     rep_origin      complement(2755..3343)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2835..2854)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3514..4305,4306..4374))
                     /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     4137..4156
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4375..4479)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4505..4960)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4642..4663)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4854..4873
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   5032..5161
                     /label=AAV2 ITR
     promoter        5283..5531
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5283..5303
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5454..5473
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5553..5628
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     intron          6199..6331
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     6314..6333
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        6376..6394
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6451..6471
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(6496..7539,1..2112)
                     /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"
ORIGIN
        1 cagattgcca agatcctgac catctaccag agcagcgagg acatccagga agaactgacc
       61 aatctgaact ccgagctgac ccaggaagag atcgagcaga tctctaatct gaagggctat
      121 accggcaccc acaacctgag cctgaaggcc atcaacctga tcctggacga gctgtggcac
      181 accaacgaca accagatcgc tatcttcaac cggctgaagc tggtgcccaa gaaggtggac
      241 ctgtcccagc agaaagagat ccccaccacc ctggtggacg acttcatcct gagccccgtc
      301 gtgaagagaa gcttcatcca gagcatcaaa gtgatcaacg ccatcatcaa gaagtacggc
      361 ctgcccaacg acatcattat cgagctggcc cgcgagaaga actccaagga cgcccagaaa
      421 atgatcaacg agatgcagaa gcggaaccgg cagaccaacg agcggatcga ggaaatcatc
      481 cggaccaccg gcaaagagaa cgccaagtac ctgatcgaga agatcaagct gcacgacatg
      541 caggaaggca agtgcctgta cagcctggaa gccatccctc tggaagatct gctgaacaac
      601 cccttcaact atgaggtgga ccacatcatc cccagaagcg tgtccttcga caacagcttc
      661 aacaacaagg tgctcgtgaa gcaggaagaa aacagcaaga agggcaaccg gaccccattc
      721 cagtacctga gcagcagcga cagcaagatc agctacgaaa ccttcaagaa gcacatcctg
      781 aatctggcca agggcaaggg cagaatcagc aagaccaaga aagagtatct gctggaagaa
      841 cgggacatca acaggttctc cgtgcagaaa gacttcatca accggaacct ggtggatacc
      901 agatacgcca ccagaggcct gatgaacctg ctgcggagct acttcagagt gaacaacctg
      961 gacgtgaaag tgaagtccat caatggcggc ttcaccagct ttctgcggcg gaagtggaag
     1021 tttaagaaag agcggaacaa ggggtacaag caccacgccg aggacgccct gatcattgcc
     1081 aacgccgatt tcatcttcaa agagtggaag aaactggaca aggccaaaaa agtgatggaa
     1141 aaccagatgt tcgaggaaaa gcaggccgag agcatgcccg agatcgaaac cgagcaggag
     1201 tacaaagaga tcttcatcac cccccaccag atcaagcaca ttaaggactt caaggactac
     1261 aagtacagcc accgggtgga caagaagcct aatagagagc tgattaacga caccctgtac
     1321 tccacccgga aggacgacaa gggcaacacc ctgatcgtga acaatctgaa cggcctgtac
     1381 gacaaggaca atgacaagct gaaaaagctg atcaacaaga gccccgaaaa gctgctgatg
     1441 taccaccacg acccccagac ctaccagaaa ctgaagctga ttatggaaca gtacggcgac
     1501 gagaagaatc ccctgtacaa gtactacgag gaaaccggga actacctgac caagtactcc
     1561 aaaaaggaca acggccccgt gatcaagaag attaagtatt acggcaacaa actgaacgcc
     1621 catctggaca tcaccgacga ctaccccaac agcagaaaca aggtcgtgaa gctgtccctg
     1681 aagccctaca gattcgacgt gtacctggac aatggcgtgt acaagttcgt gaccgtgaag
     1741 aatctggatg tgatcaaaaa agaaaactac tacgaagtga atagcaagtg ctatgaggaa
     1801 gctaagaagc tgaagaagat cagcaaccag gccgagttta tcgcctcctt ctacaacaac
     1861 gatctgatca agatcaacgg cgagctgtat agagtgatcg gcgtgaacaa cgacctgctg
     1921 aaccggatcg aagtgaacat gatcgacatc acctaccgcg agtacctgga aaacatgaac
     1981 gacaagaggc cccccaggat cattaagaca atcgcctcca agacccagag cattaagaag
     2041 tacagcacag acattctggg caacctgtat gaagtgaaat ctaagaagca ccctcagatc
     2101 atcaaaaagg gcaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag
     2161 ggatcctacc catacgatgt tccagattac gcttaacggt caaacaccat tgtcacactc
     2221 catactatca aacaccattg tcacactcca gatcaacaaa caccattgtc acactccacg
     2281 ccgcctagag cgaataaaag atctttattt tcattagatc tgtgtgttgg ttttttgtgt
     2341 gatgcagcta cgtagataag tagcatggcg ggttaatcat taactacaag gaacccctag
     2401 tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa
     2461 aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagct
     2521 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     2581 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     2641 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     2701 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     2761 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     2821 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     2881 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     2941 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     3001 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     3061 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     3121 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     3181 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     3241 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     3301 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     3361 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     3421 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     3481 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     3541 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     3601 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     3661 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     3721 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     3781 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     3841 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     3901 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     3961 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     4021 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     4081 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     4141 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     4201 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     4261 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     4321 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     4381 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     4441 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     4501 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     4561 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     4621 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     4681 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     4741 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     4801 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     4861 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     4921 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     4981 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gctgcgcgct
     5041 cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc gggcgacctt tggtcgcccg
     5101 gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc
     5161 ttgtagttaa tgattaaccc gccatgctac ttatctacgt agccatgctc tggtgtacaa
     5221 aaaagcaggc tttaaaggaa ccaattcagt cgactggatc cggtaccaag gtcgggcagg
     5281 aagagggcct atttcccatg attccttcat atttgcatat acgatacaag gctgttagag
     5341 agataattag aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta
     5401 gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc
     5461 atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag
     5521 gacgaaacac cgtatgaaaa tccctactgg acgttttagt actctggaaa cagaatctac
     5581 taaaacaagg caaaatgccg tgtttatctc gtcaacttgt tggcgagatt tttttctaga
     5641 cgggcccccc ctcgaggtcg ggataaaagc agtctgggct ttcacatgac agcatctggg
     5701 gctgcggcag agggtcgggt ccgaagcgct gccttatcag cgtccccagc cctgggaggt
     5761 gacagctggc tggcttgtgt cagcccctcg ggcactcacg tatctccgtc cgacgggttt
     5821 aaaatagcaa aactctgagg ccacacaata gcttgggctt atatgggctc ctgtggggga
     5881 agggggagca cggagggggc cggggccgct gctgccaaaa tagcagctca caagtgttgc
     5941 attcctctct gggcgccggg cacattcctg ctggctctgc ccgccccggg gtgggcgccg
     6001 gggggacctt aaagcctctg ccccccaagg agcccttccc agacagccgc cggcacccac
     6061 cgctccgtgg gacgatcccc gaagctctag agctttattg cggtagttta tcacagttaa
     6121 attgctaacg cagtcagtgc ttctgacaca acagtctcga acttaagctg cagaagttgg
     6181 tcgtgaggca ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag
     6241 aaactgggct tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt
     6301 actgacatcc actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct
     6361 taaggctaga gtacttaata cgactcacta taggctagcc ctcgagaagc ggccgcacta
     6421 ctccgcggac tactagagct caccatggcc ccaaagaaga agcggaaggt cggtatccac
     6481 ggagtcccag cagccaagcg gaactacatc ctgggcctgg acatcggcat caccagcgtg
     6541 ggctacggca tcatcgacta cgagacacgg gacgtgatcg atgccggcgt gcggctgttc
     6601 aaagaggcca acgtggaaaa caacgagggc aggcggagca agagaggcgc cagaaggctg
     6661 aagcggcgga ggcggcatag aatccagaga gtgaagaagc tgctgttcga ctacaacctg
     6721 ctgaccgacc acagcgagct gagcggcatc aacccctacg aggccagagt gaagggcctg
     6781 agccagaagc tgagcgagga agagttctct gccgccctgc tgcacctggc caagagaaga
     6841 ggcgtgcaca acgtgaacga ggtggaagag gacaccggca acgagctgtc caccaaagag
     6901 cagatcagcc ggaacagcaa ggccctggaa gagaaatacg tggccgaact gcagctggaa
     6961 cggctgaaga aagacggcga agtgcggggc agcatcaaca gattcaagac cagcgactac
     7021 gtgaaagaag ccaaacagct gctgaaggtg cagaaggcct accaccagct ggaccagagc
     7081 ttcatcgaca cctacatcga cctgctggaa acccggcgga cctactatga gggacctggc
     7141 gagggcagcc ccttcggctg gaaggacatc aaagaatggt acgagatgct gatgggccac
     7201 tgcacctact tccccgagga actgcggagc gtgaagtacg cctacaacgc cgacctgtac
     7261 aacgccctga acgacctgaa caatctcgtg atcaccaggg acgagaacga gaagctggaa
     7321 tattacgaga agttccagat catcgagaac gtgttcaagc agaagaagaa gcccaccctg
     7381 aagcagatcg ccaaagaaat cctcgtgaac gaagaggata ttaagggcta cagagtgacc
     7441 agcaccggca agcccgagtt caccaacctg aaggtgtacc acgacatcaa ggacattacc
     7501 gcccggaaag agattattga gaacgccgag ctgctggat
//
