LOCUS       sequence-416836-        7499 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SaCas9 by the specific cTNT promoter and sgRNA targeted 
            the mouse Camk2d gene by U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7499)
  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 7499)
  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-msCAMK2d-sgRNA-cTNT-SaCas9-HA-OLLAS
FEATURES             Location/Qualifiers
     source          1..7499
                     /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"
     CDS             2194..2235
                     /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    2253..2301
                     /note="synthetic polyadenylation signal"
     repeat_region   2349..2478
                     /label=AAV2 ITR
     rep_origin      complement(2715..3303)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2795..2814)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3474..4265,4266..4334))
                     /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     4097..4116
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4335..4439)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4465..4920)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4602..4623)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4814..4833
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   4992..5121
                     /label=AAV2 ITR
     promoter        5243..5491
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     5243..5263
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     5414..5433
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        5513..5588
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     intron          6159..6291
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     6274..6293
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        6336..6354
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             6411..6431
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(6456..7499,1..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 gctagcggct tcgccaacga gcttggaccc
     2221 aggttgatgg gaaagtaaga attcctagag cgaataaaag atctttattt tcattagatc
     2281 tgtgtgttgg ttttttgtgt gatgcagcta cgtagataag tagcatggcg ggttaatcat
     2341 taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     2401 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     2461 gagcgagcga gcgcgcagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2521 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2581 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2641 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2701 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2761 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2821 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2881 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     2941 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     3001 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     3061 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     3121 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     3181 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     3241 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     3301 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     3361 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3421 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3481 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3541 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3601 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3661 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3721 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3781 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3841 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3901 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3961 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     4021 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     4081 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     4141 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     4201 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     4261 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     4321 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     4381 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     4441 acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta aaattcgcgt
     4501 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     4561 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     4621 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     4681 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     4741 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     4801 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     4861 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     4921 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
     4981 tattacgcca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
     5041 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
     5101 actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacgt
     5161 agccatgctc tggtgtacaa aaaagcaggc tttaaaggaa ccaattcagt cgactggatc
     5221 cggtaccaag gtcgggcagg aagagggcct atttcccatg attccttcat atttgcatat
     5281 acgatacaag gctgttagag agataattag aattaatttg actgtaaaca caaagatatt
     5341 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     5401 atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc
     5461 tttatatatc ttgtggaaag gacgaaacac cgtatgaaaa tccctactgg acgttttagt
     5521 actctggaaa cagaatctac taaaacaagg caaaatgccg tgtttatctc gtcaacttgt
     5581 tggcgagatt tttttctaga cgggcccccc ctcgaggtcg ggataaaagc agtctgggct
     5641 ttcacatgac agcatctggg gctgcggcag agggtcgggt ccgaagcgct gccttatcag
     5701 cgtccccagc cctgggaggt gacagctggc tggcttgtgt cagcccctcg ggcactcacg
     5761 tatctccgtc cgacgggttt aaaatagcaa aactctgagg ccacacaata gcttgggctt
     5821 atatgggctc ctgtggggga agggggagca cggagggggc cggggccgct gctgccaaaa
     5881 tagcagctca caagtgttgc attcctctct gggcgccggg cacattcctg ctggctctgc
     5941 ccgccccggg gtgggcgccg gggggacctt aaagcctctg ccccccaagg agcccttccc
     6001 agacagccgc cggcacccac cgctccgtgg gacgatcccc gaagctctag agctttattg
     6061 cggtagttta tcacagttaa attgctaacg cagtcagtgc ttctgacaca acagtctcga
     6121 acttaagctg cagaagttgg tcgtgaggca ctgggcaggt aagtatcaag gttacaagac
     6181 aggtttaagg agaccaatag aaactgggct tgtcgagaca gagaagactc ttgcgtttct
     6241 gataggcacc tattggtctt actgacatcc actttgcctt tctctccaca ggtgtccact
     6301 cccagttcaa ttacagctct taaggctaga gtacttaata cgactcacta taggctagcc
     6361 ctcgagaagc ggccgcacta ctccgcggac tactagagct caccatggcc ccaaagaaga
     6421 agcggaaggt cggtatccac ggagtcccag cagccaagcg gaactacatc ctgggcctgg
     6481 acatcggcat caccagcgtg ggctacggca tcatcgacta cgagacacgg gacgtgatcg
     6541 atgccggcgt gcggctgttc aaagaggcca acgtggaaaa caacgagggc aggcggagca
     6601 agagaggcgc cagaaggctg aagcggcgga ggcggcatag aatccagaga gtgaagaagc
     6661 tgctgttcga ctacaacctg ctgaccgacc acagcgagct gagcggcatc aacccctacg
     6721 aggccagagt gaagggcctg agccagaagc tgagcgagga agagttctct gccgccctgc
     6781 tgcacctggc caagagaaga ggcgtgcaca acgtgaacga ggtggaagag gacaccggca
     6841 acgagctgtc caccaaagag cagatcagcc ggaacagcaa ggccctggaa gagaaatacg
     6901 tggccgaact gcagctggaa cggctgaaga aagacggcga agtgcggggc agcatcaaca
     6961 gattcaagac cagcgactac gtgaaagaag ccaaacagct gctgaaggtg cagaaggcct
     7021 accaccagct ggaccagagc ttcatcgaca cctacatcga cctgctggaa acccggcgga
     7081 cctactatga gggacctggc gagggcagcc ccttcggctg gaaggacatc aaagaatggt
     7141 acgagatgct gatgggccac tgcacctact tccccgagga actgcggagc gtgaagtacg
     7201 cctacaacgc cgacctgtac aacgccctga acgacctgaa caatctcgtg atcaccaggg
     7261 acgagaacga gaagctggaa tattacgaga agttccagat catcgagaac gtgttcaagc
     7321 agaagaagaa gcccaccctg aagcagatcg ccaaagaaat cctcgtgaac gaagaggata
     7381 ttaagggcta cagagtgacc agcaccggca agcccgagtt caccaacctg aaggtgtacc
     7441 acgacatcaa ggacattacc gcccggaaag agattattga gaacgccgag ctgctggat
//
