LOCUS       sequence-416838-        7256 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SpRY cas9C by the constitutive CASI promoter and sgRNA 
            targeting the oxidative activation sites of Camk2d by U6 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7256)
  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 7256)
  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-CASI-InteinC-SpRY C aa714-1368-U6-Camk2d ...
FEATURES             Location/Qualifiers
     source          1..7256
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   12..141
                     /label=AAV2 ITR
     enhancer        217..520
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        522..799
                     /label=chicken beta-actin promoter
     regulatory      1273..1282
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1277..1286
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3442..3462
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    3477..3532
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3531..3550)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     misc_RNA        complement(3637..3712)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(3734..3982)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3792..3811)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3962..3982)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4317..4446
                     /label=AAV2 ITR
     primer_bind     complement(4465..4482)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4465..4481)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4474..4496)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4623..5078
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4710..4729)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4920..4941
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5104..5208
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5209..5277,5278..6069)
                     /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(5427..5446)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6240..6828
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6729..6748
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    7116..7137
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7152..7157,7158..7175,7176..7182)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7190..7206
                     /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     7195..7217
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7214..7230
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7214..7230
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ccttaattag gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacgt
      181 agccatgctc taggaagatc ggaattcgga gttccgcgtt acataactta cggtaaatgg
      241 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      301 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      361 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      421 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      481 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtcgaggtga gccccacgtt
      541 ctgcttcact ctccccatct cccccccctc cccaccccca attttgtatt tatttatttt
      601 ttaattattt tgtgcagcga tgggggcggg gggggggggg gggcgcgcgc crgggggggg
      661 ggggsggggs grggggsggg gsggggsgrg gcggagaggt gcggcggcag ccaatcagag
      721 cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg cggcggcggc cctataaaaa
      781 gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc
      841 cgccgcctcg cgccgcccgc cccggctctg actgaccgcg ttactaaaac aggtaagtcc
      901 ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg
      961 ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag
     1021 cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag
     1081 gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg
     1141 aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat
     1201 gatgcctcta ctaaccatgt tcatgttttc tttttttttc tacaggtcct gggtgacgaa
     1261 caggctagcg ccgccaccat ggcggcggcg tgcccggaac tgcgtcagct ggcgcagagc
     1321 gatgtgtatt gggatccgat tgtgagcatt gaaccggatg gcgtggaaga agtgtttgat
     1381 ctgaccgtgc cgggcccgca taactttgtg gcgaacgata ttattgcgca taactctggc
     1441 cagggcgata gcctgcacga gcacattgcc aatctggccg gcagccccgc cattaagaag
     1501 ggcatcctgc agacagtgaa ggtggtggac gagctcgtga aagtgatggg ccggcacaag
     1561 cccgagaaca tcgtgatcga aatggccaga gagaaccaga ccacccagaa gggacagaag
     1621 aacagccgcg agagaatgaa gcggatcgaa gagggcatca aagagctggg cagccagatc
     1681 ctgaaagaac accccgtgga aaacacccag ctgcagaacg agaagctgta cctgtactac
     1741 ctgcagaatg ggcgggatat gtacgtggac caggaactgg acatcaaccg gctgtccgac
     1801 tacgatgtgg accatatcgt gcctcagagc tttctgaagg acgactccat cgacaacaag
     1861 gtgctgacca gaagcgacaa gaaccggggc aagagcgaca acgtgccctc cgaagaggtc
     1921 gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg ccaagctgat tacccagaga
     1981 aagttcgaca atctgaccaa ggccgagaga ggcggcctga gcgaactgga taaggccggc
     2041 ttcatcaaga gacagctggt ggaaacccgg cagatcacaa agcacgtggc acagatcctg
     2101 gactcccgga tgaacactaa gtacgacgag aatgacaagc tgatccggga agtgaaagtg
     2161 atcaccctga agtccaagct ggtgtccgat ttccggaagg atttccagtt ttacaaagtg
     2221 cgcgagatca acaactacca ccacgcccac gacgcctacc tgaacgccgt cgtgggaacc
     2281 gccctgatca aaaagtaccc taagctggaa agcgagttcg tgtacggcga ctacaaggtg
     2341 tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa tcggcaaggc taccgccaag
     2401 tacttcttct acagcaacat catgaacttt ttcaagaccg agattaccct ggccaacggc
     2461 gagatccgga agcggcctct gatcgagaca aacggcgaaa ccggggagat cgtgtgggat
     2521 aagggccggg attttgccac cgtgcggaaa gtgctgagca tgccccaagt gaatatcgtg
     2581 aaaaagaccg aggtgcagac aggcggcttc agcaaagagt ctatcagacc caagaggaac
     2641 agcgataagc tgatcgccag aaagaaggac tgggacccta agaagtacgg cggcttcctg
     2701 tggcccaccg tggcctattc tgtgctggtg gtggccaaag tggaaaaggg caagtccaag
     2761 aaactgaaga gtgtgaaaga gctgctgggg atcaccatca tggaaagaag cagcttcgag
     2821 aagaatccca tcgactttct ggaagccaag ggctacaaag aagtgaaaaa ggacctgatc
     2881 atcaagctgc ctaagtactc cctgttcgag ctggaaaacg gccggaagag aatgctggcc
     2941 tctgccaagc agctgcagaa gggaaacgaa ctggccctgc cctccaaata tgtgaacttc
     3001 ctgtacctgg ccagccacta tgagaagctg aagggctccc ccgaggataa tgagcagaaa
     3061 cagctgtttg tggaacagca caagcactac ctggacgaga tcatcgagca gatcagcgag
     3121 ttctccaaga gagtgatcct ggccgacgct aatctggaca aagtgctgtc cgcctacaac
     3181 aagcaccggg ataagcccat cagagagcag gccgagaata tcatccacct gtttaccctg
     3241 accagactgg gagcccctag agccttcaag tactttgaca ccaccatcga ccccaagcag
     3301 tacagaagca ccaaagaggt gctggacgcc accctgatcc accagagcat caccggcctg
     3361 tacgagacac ggatcgacct gtctcagctg ggaggtgact ctggcggctc aaaaagaacc
     3421 gccgacggca gcgaattcga gcccaagaag aagaggaaag tctaaaccgg ttggctaata
     3481 aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag
     3541 gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc
     3601 aacatatgcc catatgaggc cctcacgcgt aaaaaagcac cgactcggtg ccactttttc
     3661 aagttgataa cggactagcc ttattttaac ttgctatttc tagctctaaa actcctgcct
     3721 gtgcatcatg gacggtgttt cgtcctttcc acaagatata taaagccaag aaatcgaaat
     3781 actttcaagt tacggtaagc atatgatagt ccattttaaa acataatttt aaaactgcaa
     3841 actacccaag aaattattac tttctacgtc acgtattttg tactaatatc tttgtgttta
     3901 cagtcaaatt aattccaatt atctctctaa cagccttgta tcgtatatgc aaatatgaag
     3961 gaatcatggg aaataggccc tcggtaccgg cgatgctggc tgccatgaac aaaggttggc
     4021 tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa
     4081 aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa
     4141 catccctaaa attttcctta catgttttac tagccagatt tttcctcctc tcctgactac
     4201 tcccagtcat agctgtccct cttctcttat ggagatcgga tccgaattcc cgataaggat
     4261 cttcctagag catggctacg tagataagta gcatggcggg ttaatcatta actacaagga
     4321 acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg
     4381 gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc
     4441 gcgcagcctt aattaaccta attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     4501 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     4561 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     4621 ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     4681 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     4741 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     4801 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     4861 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     4921 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     4981 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     5041 taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat
     5101 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     5161 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     5221 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     5281 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     5341 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     5401 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc
     5461 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     5521 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     5581 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     5641 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     5701 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg
     5761 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     5821 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     5881 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     5941 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     6001 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     6061 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     6121 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     6181 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     6241 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     6301 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     6361 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     6421 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     6481 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     6541 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     6601 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6661 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6721 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6781 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6841 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6901 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     6961 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     7021 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     7081 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     7141 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     7201 taacaatttc acacaggaaa cagctatgac catgattacg ccagatttaa ttaagg
//
