LOCUS       sequence-425006-        7381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one AAVi plasmid expressing S. aureus dCas9-KRAB with sgRNA 
            cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7381)
  AUTHORS   Laurette P, Cao C, Ramanujam D, Schwaderer M, Lueneburg T, Kuss S, 
            Weiss L, Dilshat R, Furlong EEM, Rezende F, Engelhardt S, Gilsbach R
  TITLE     In Vivo Silencing of Regulatory Elements Using a Single AAV-CRISPRi 
            Vector.
  JOURNAL   Circ Res. 2023 Dec 22. doi: 10.1161/CIRCRESAHA.123.323854.
  PUBMED    38131200
REFERENCE   2  (bases 1 to 7381)
  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: "Circ Res. 
            2023 Dec 22. doi: 10.1161/CIRCRESAHA.123.323854."
COMMENT     Sequence Label: AAVi U6_gRNA CMV_SadCas9-KRAB
FEATURES             Location/Qualifiers
     source          1..7381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1..18
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     repeat_region   28..157
                     /label=AAV2 ITR
     promoter        211..459
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     211..231
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     382..401
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        601..904
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        906..1108
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1059..1079
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1105..1129
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1133..1142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1145..1165
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4346..4393
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4427..4621
                     /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             4688..4708
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4714..4835
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4751..4770)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4805..4824
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4883..5012
                     /label=AAV2 ITR
     primer_bind     complement(5035..5054)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5036..5054)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      complement(5312..5900)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5392..5411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6071..6862,6863..6931))
                     /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     6694..6713
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6932..7036)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7104..7122
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7160..7182)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7282..7301
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 atttaggtga cactatagaa ctcgagcctg cgcgctcgct cgctcactga ggccgcccgg
       61 gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct cagtgagcga gcgagcgcgc
      121 agagagggag tggccaactc catcactagg ggttccttgt agttaatgat taacccgcca
      181 tgctacttat ctacgtagcc atgctctaga gagggcctat ttcccatgat tccttcatat
      241 ttgcatatac gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca
      301 aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt
      361 ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat
      421 ttcttggctt tatatatctt gtggaaagga cgaaacaccg tcttcgacca ggatgggcac
      481 cacccgaaga cgttatagta ctctgtgctg gaaacagcac agaatctact ataacaaggc
      541 aaaatgccgt gtttatctcg tcaacttgtt ggcgagattt ttttaccggt ccggtgccac
      601 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      661 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      721 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      781 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      841 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      901 catgttgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      961 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
     1021 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
     1081 acggtgggag gtttatataa gcagagctcg tttagtgaac cgtcagatcc ttgccaccat
     1141 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagcca agcggaacta
     1201 catcctgggc ctggccatcg gcatcaccag cgtgggctac ggcatcatcg actacgagac
     1261 acgggacgtg atcgatgccg gcgtgcggct gttcaaagag gccaacgtgg aaaacaacga
     1321 gggcaggcgg agcaagagag gcgccagaag gctgaagcgg cggaggcggc atagaatcca
     1381 gagagtgaag aagctgctgt tcgactacaa cctgctgacc gaccacagcg agctgagcgg
     1441 catcaacccc tacgaggcca gagtgaaggg cctgagccag aagctgagcg aggaagagtt
     1501 ctctgccgcc ctgctgcacc tggccaagag aagaggcgtg cacaacgtga acgaggtgga
     1561 agaggacacc ggcaacgagc tgtccaccaa agagcagatc agccggaaca gcaaggccct
     1621 ggaagagaaa tacgtggccg aactgcagct ggaacggctg aagaaagacg gcgaagtgcg
     1681 gggcagcatc aacagattca agaccagcga ctacgtgaaa gaagccaaac agctgctgaa
     1741 ggtgcagaag gcctaccacc agctggacca gagcttcatc gacacctaca tcgacctgct
     1801 ggaaacccgg cggacctact atgagggacc tggcgagggc agccccttcg gctggaagga
     1861 catcaaagaa tggtacgaga tgctgatggg ccactgcacc tacttccccg aggaactgcg
     1921 gagcgtgaag tacgcctaca acgccgacct gtacaacgcc ctgaacgacc tgaacaatct
     1981 cgtgatcacc agggacgaga acgagaagct ggaatattac gagaagttcc agatcatcga
     2041 gaacgtgttc aagcagaaga agaagcccac cctgaagcag atcgccaaag aaatcctcgt
     2101 gaacgaagag gatattaagg gctacagagt gaccagcacc ggcaagcccg agttcaccaa
     2161 cctgaaggtg taccacgaca tcaaggacat taccgcccgg aaagagatta ttgagaacgc
     2221 cgagctgctg gatcagattg ccaagatcct gaccatctac cagagcagcg aggacatcca
     2281 ggaagaactg accaatctga actccgagct gacccaggaa gagatcgagc agatctctaa
     2341 tctgaagggc tataccggca cccacaacct gagcctgaag gccatcaacc tgatcctgga
     2401 cgagctgtgg cacaccaacg acaaccagat cgctatcttc aaccggctga agctggtgcc
     2461 caagaaggtg gacctgtccc agcagaaaga gatccccacc accctggtgg acgacttcat
     2521 cctgagcccc gtcgtgaaga gaagcttcat ccagagcatc aaagtgatca acgccatcat
     2581 caagaagtac ggcctgccca acgacatcat tatcgagctg gcccgcgaga agaactccaa
     2641 ggacgcccag aaaatgatca acgagatgca gaagcggaac cggcagacca acgagcggat
     2701 cgaggaaatc atccggacca ccggcaaaga gaacgccaag tacctgatcg agaagatcaa
     2761 gctgcacgac atgcaggaag gcaagtgcct gtacagcctg gaagccatcc ctctggaaga
     2821 tctgctgaac aaccccttca actatgaggt ggaccacatc atccccagaa gcgtgtcctt
     2881 cgacaacagc ttcaacaaca aggtgctcgt gaagcaggaa gaagccagca agaagggcaa
     2941 ccggacccca ttccagtacc tgagcagcag cgacagcaag atcagctacg aaaccttcaa
     3001 gaagcacatc ctgaatctgg ccaagggcaa gggcagaatc agcaagacca agaaagagta
     3061 tctgctggaa gaacgggaca tcaacaggtt ctccgtgcag aaagacttca tcaaccggaa
     3121 cctggtggat accagatacg ccaccagagg cctgatgaac ctgctgcgga gctacttcag
     3181 agtgaacaac ctggacgtga aagtgaagtc catcaatggc ggcttcacca gctttctgcg
     3241 gcggaagtgg aagtttaaga aagagcggaa caaggggtac aagcaccacg ccgaggacgc
     3301 cctgatcatt gccaacgccg atttcatctt caaagagtgg aagaaactgg acaaggccaa
     3361 aaaagtgatg gaaaaccaga tgttcgagga aaagcaggcc gagagcatgc ccgagatcga
     3421 aaccgagcag gagtacaaag agatcttcat caccccccac cagatcaagc acattaagga
     3481 cttcaaggac tacaagtaca gccaccgggt ggacaagaag cctaatagag agctgattaa
     3541 cgacaccctg tactccaccc ggaaggacga caagggcaac accctgatcg tgaacaatct
     3601 gaacggcctg tacgacaagg acaatgacaa gctgaaaaag ctgatcaaca agagccccga
     3661 aaagctgctg atgtaccacc acgaccccca gacctaccag aaactgaagc tgattatgga
     3721 acagtacggc gacgagaaga atcccctgta caagtactac gaggaaaccg ggaactacct
     3781 gaccaagtac tccaaaaagg acaacggccc cgtgatcaag aagattaagt attacggcaa
     3841 caaactgaac gcccatctgg acatcaccga cgactacccc aacagcagaa acaaggtcgt
     3901 gaagctgtcc ctgaagccct acagattcga cgtgtacctg gacaatggcg tgtacaagtt
     3961 cgtgaccgtg aagaatctgg atgtgatcaa aaaagaaaac tactacgaag tgaatagcaa
     4021 gtgctatgag gaagctaaga agctgaagaa gatcagcaac caggccgagt ttatcgcctc
     4081 cttctacaac aacgatctga tcaagatcaa cggcgagctg tatagagtga tcggcgtgaa
     4141 caacgacctg ctgaaccgga tcgaagtgaa catgatcgac atcacctacc gcgagtacct
     4201 ggaaaacatg aacgacaaga ggccccccag gatcattaag acaatcgcct ccaagaccca
     4261 gagcattaag aagtacagca cagacattct gggcaacctg tatgaagtga aatctaagaa
     4321 gcaccctcag atcatcaaaa agggcaaaag gccggcggcc acgaaaaagg ccggccaggc
     4381 aaaaaagaaa aagggatccg atgctaagtc actgactgcc tggtcccgga cactggtgac
     4441 cttcaaggat gtgtttgtgg acttcaccag ggaggagtgg aagctgctgg acactgctca
     4501 gcagatcctg tacagaaatg tgatgctgga gaactataag aacctggttt ccttgggtta
     4561 tcagcttact aagccagatg tgatcctccg gttggagaag ggagaagagc cctggctggt
     4621 ggagagagaa attcaccaag agacccatcc tgattcagag actgcatttg aaatcaaatc
     4681 atcagttccg aaaaagaaac gcaaagttta agaaacttgt ttattgcagc ttataatggt
     4741 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     4801 agttgtggtt tgtccaaact catcaatgta tcttagcggt cgaccgtaga taagtagcat
     4861 ggcgggttaa tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg
     4921 cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc
     4981 cgggcggcct cagtgagcga gcgagcgcgc agaagctatc agatctgccg gtctccctat
     5041 agtgagtcgt attaatttcg ataagccagg ttaacctgca ttaatgaatc ggccaacgcg
     5101 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5161 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5221 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5281 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5341 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5401 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5461 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5521 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5581 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5641 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5701 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5761 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5821 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5881 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5941 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6001 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6061 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6121 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6181 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6241 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6301 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6361 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6421 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6481 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6541 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6601 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6661 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6721 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6781 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6841 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6901 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6961 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7021 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     7081 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt ctcgcgcgtt
     7141 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc
     7201 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
     7261 gtcggggctg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgg
     7321 acatattgtc gttagaacgc ggctacaatt aatacataac cttatgtatc atacacatac
     7381 g
//
