LOCUS       sequence-201560-        7348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV expressing deactivated S. aureus Cas9 fused to a KRAB repressor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7348)
  AUTHORS   Thakore PI, Kwon JB, Nelson CE, Rouse DC, Gemberling MP, Oliver ML, 
            Gersbach CA
  TITLE     RNA-guided transcriptional silencing in vivo with S. aureus 
            CRISPR-Cas9 repressors.
  JOURNAL   Nat Commun. 2018 Apr 26;9(1):1674. doi: 10.1038/s41467-018-04048-4.
  PUBMED    29700298
REFERENCE   2  (bases 1 to 7348)
  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"; journalName: "Nat 
            Commun."; date: "2018-04-26"; volume: "9(1):1674. doi"; pages: " 
            10.1038/s41467-018-04048-4"
COMMENT     Sequence Label: AAV CMV-dSaCas9-KRAB-bGHpA
FEATURES             Location/Qualifiers
     source          1..7348
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   7..147
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7..136
                     /label=AAV2 ITR
     rep_origin      222..677
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(309..328)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     519..540
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(694..713)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     813..835
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(873..891)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        959..1063
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1064..1132,1133..1924)
                     /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(1282..1301)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2095..2683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2584..2603
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2745..2874
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        2898..3277
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3278..3481
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3432..3452
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             3509..3535
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3539..3559
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6740..6787
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6821..7015
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQILYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             7082..7102
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(7130..7147)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7136..7343
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca
       61 ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga
      121 gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc
      181 tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc
      241 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
      301 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
      361 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
      421 ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt
      481 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
      541 aacaacactc aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc
      601 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
      661 attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
      721 aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
      781 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
      841 accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt
      901 taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg
      961 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     1021 ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt
     1081 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     1141 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     1201 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     1261 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca
     1321 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     1381 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     1441 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     1501 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     1561 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac
     1621 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     1681 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     1741 ctggtttatt gctgataaat ctggagccgg tgagcgtgga agccgcggta tcattgcagc
     1801 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     1861 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     1921 gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta
     1981 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     2041 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     2101 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     2161 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     2221 agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa
     2281 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     2341 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     2401 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     2461 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     2521 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     2581 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     2641 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     2701 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgtcctgca ggcagctgcg
     2761 cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac ctttggtcgc ccggcctcag
     2821 tgagcgagcg agcgcgcaga gagggagtgg ccaactccat cactaggggt tcctgcggcc
     2881 tctagactcg aggcgttgac attgattatt gactagttat taatagtaat caattacggg
     2941 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     3001 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     3061 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     3121 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
     3181 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
     3241 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
     3301 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
     3361 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
     3421 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
     3481 tctctggcta actaccggtg ccaccatgta cccatacgat gttccagatt acgctgcccc
     3541 aaagaagaag cggaaggtcg gtatccacgg agtcccagca gccaagcgga actacatcct
     3601 gggcctggcc atcggcatca ccagcgtggg ctacggcatc atcgactacg agacacggga
     3661 cgtgatcgat gccggcgtgc ggctgttcaa agaggccaac gtggaaaaca acgagggcag
     3721 gcggagcaag agaggcgcca gaaggctgaa gcggcggagg cggcatagaa tccagagagt
     3781 gaagaagctg ctgttcgact acaacctgct gaccgaccac agcgagctga gcggcatcaa
     3841 cccctacgag gccagagtga agggcctgag ccagaagctg agcgaggaag agttctctgc
     3901 cgccctgctg cacctggcca agagaagagg cgtgcacaac gtgaacgagg tggaagagga
     3961 caccggcaac gagctgtcca ccaaagagca gatcagccgg aacagcaagg ccctggaaga
     4021 gaaatacgtg gccgaactgc agctggaacg gctgaagaaa gacggcgaag tgcggggcag
     4081 catcaacaga ttcaagacca gcgactacgt gaaagaagcc aaacagctgc tgaaggtgca
     4141 gaaggcctac caccagctgg accagagctt catcgacacc tacatcgacc tgctggaaac
     4201 ccggcggacc tactatgagg gacctggcga gggcagcccc ttcggctgga aggacatcaa
     4261 agaatggtac gagatgctga tgggccactg cacctacttc cccgaggaac tgcggagcgt
     4321 gaagtacgcc tacaacgccg acctgtacaa cgccctgaac gacctgaaca atctcgtgat
     4381 caccagggac gagaacgaga agctggaata ttacgagaag ttccagatca tcgagaacgt
     4441 gttcaagcag aagaagaagc ccaccctgaa gcagatcgcc aaagaaatcc tcgtgaacga
     4501 agaggatatt aagggctaca gagtgaccag caccggcaag cccgagttca ccaacctgaa
     4561 ggtgtaccac gacatcaagg acattaccgc ccggaaagag attattgaga acgccgagct
     4621 gctggatcag attgccaaga tcctgaccat ctaccagagc agcgaggaca tccaggaaga
     4681 actgaccaat ctgaactccg agctgaccca ggaagagatc gagcagatct ctaatctgaa
     4741 gggctatacc ggcacccaca acctgagcct gaaggccatc aacctgatcc tggacgagct
     4801 gtggcacacc aacgacaacc agatcgctat cttcaaccgg ctgaagctgg tgcccaagaa
     4861 ggtggacctg tcccagcaga aagagatccc caccaccctg gtggacgact tcatcctgag
     4921 ccccgtcgtg aagagaagct tcatccagag catcaaagtg atcaacgcca tcatcaagaa
     4981 gtacggcctg cccaacgaca tcattatcga gctggcccgc gagaagaact ccaaggacgc
     5041 ccagaaaatg atcaacgaga tgcagaagcg gaaccggcag accaacgagc ggatcgagga
     5101 aatcatccgg accaccggca aagagaacgc caagtacctg atcgagaaga tcaagctgca
     5161 cgacatgcag gaaggcaagt gcctgtacag cctggaagcc atccctctgg aagatctgct
     5221 gaacaacccc ttcaactatg aggtggacca catcatcccc agaagcgtgt ccttcgacaa
     5281 cagcttcaac aacaaggtgc tcgtgaagca ggaagaagcc agcaagaagg gcaaccggac
     5341 cccattccag tacctgagca gcagcgacag caagatcagc tacgaaacct tcaagaagca
     5401 catcctgaat ctggccaagg gcaagggcag aatcagcaag accaagaaag agtatctgct
     5461 ggaagaacgg gacatcaaca ggttctccgt gcagaaagac ttcatcaacc ggaacctggt
     5521 ggataccaga tacgccacca gaggcctgat gaacctgctg cggagctact tcagagtgaa
     5581 caacctggac gtgaaagtga agtccatcaa tggcggcttc accagctttc tgcggcggaa
     5641 gtggaagttt aagaaagagc ggaacaaggg gtacaagcac cacgccgagg acgccctgat
     5701 cattgccaac gccgatttca tcttcaaaga gtggaagaaa ctggacaagg ccaaaaaagt
     5761 gatggaaaac cagatgttcg aggaaaagca ggccgagagc atgcccgaga tcgaaaccga
     5821 gcaggagtac aaagagatct tcatcacccc ccaccagatc aagcacatta aggacttcaa
     5881 ggactacaag tacagccacc gggtggacaa gaagcctaat agagagctga ttaacgacac
     5941 cctgtactcc acccggaagg acgacaaggg caacaccctg atcgtgaaca atctgaacgg
     6001 cctgtacgac aaggacaatg acaagctgaa aaagctgatc aacaagagcc ccgaaaagct
     6061 gctgatgtac caccacgacc cccagaccta ccagaaactg aagctgatta tggaacagta
     6121 cggcgacgag aagaatcccc tgtacaagta ctacgaggaa accgggaact acctgaccaa
     6181 gtactccaaa aaggacaacg gccccgtgat caagaagatt aagtattacg gcaacaaact
     6241 gaacgcccat ctggacatca ccgacgacta ccccaacagc agaaacaagg tcgtgaagct
     6301 gtccctgaag ccctacagat tcgacgtgta cctggacaat ggcgtgtaca agttcgtgac
     6361 cgtgaagaat ctggatgtga tcaaaaaaga aaactactac gaagtgaata gcaagtgcta
     6421 tgaggaagct aagaagctga agaagatcag caaccaggcc gagtttatcg cctccttcta
     6481 caacaacgat ctgatcaaga tcaacggcga gctgtataga gtgatcggcg tgaacaacga
     6541 cctgctgaac cggatcgaag tgaacatgat cgacatcacc taccgcgagt acctggaaaa
     6601 catgaacgac aagaggcccc ccaggatcat taagacaatc gcctccaaga cccagagcat
     6661 taagaagtac agcacagaca ttctgggcaa cctgtatgaa gtgaaatcta agaagcaccc
     6721 tcagatcatc aaaaagggca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa
     6781 gaaaaaggga tccgatgcta agtcactgac tgcctggtcc cggacactgg tgaccttcaa
     6841 ggatgtgttt gtggacttca ccagggagga gtggaagctg ctggacactg ctcagcagat
     6901 cctgtacaga aatgtgatgc tggagaacta taagaacctg gtttccttgg gttatcagct
     6961 tactaagcca gatgtgatcc tccggttgga gaagggagaa gagccctggc tggtggagag
     7021 agaaattcac caagagaccc atcctgattc agagactgca tttgaaatca aatcatcagt
     7081 tccgaaaaag aaacgcaaag tttaagaatt cctagagctc gctgatcagc ctcgactgtg
     7141 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     7201 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     7261 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     7321 gagaatagca ggcatgctgg ggaggtac
//
