LOCUS       sequence-211314-        6743 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A single vector AAV-Cas9 system containing a liver-specific promoter
            with Cas9 from Streptococcus thermophilus CRISPR1 (St1Cas9 LMD-9) 
            and its U6-driven sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6743)
  AUTHORS   Agudelo D, Carter S, Velimirovic M, Duringer A, Rivest JF, Levesque 
            S, Loehr J, Mouchiroud M, Cyr D, Waters PJ, Laplante M, Moineau S, 
            Goulet A, Doyon Y
  TITLE     Versatile and robust genome editing with Streptococcus thermophilus 
            CRISPR1-Cas9.
  JOURNAL   Genome Res. 2020 Jan;30(1):107-117. doi: 10.1101/gr.255414.119. Epub
            2020 Jan 3.
  PUBMED    31900288
REFERENCE   2  (bases 1 to 6743)
  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: 
            "10.1101/gr.255414.119"; journalName: "Genome Res"; date: "2020-01";
            volume: "30"; issue: "1"; pages: "107-117"
COMMENT     Sequence Label: pAAV_LP1B_St1Cas9_LMD-9_SpA_U6_sgRNA
FEATURES             Location/Qualifiers
     source          1..6743
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   61..190
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      723..732
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             738..758
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             783..4142
                     /codon_start=1
                     /product="Cas9 endonuclease from the Streptococcus 
                     thermophilus Type II CRISPR/Cas system (Esvelt et al., 
                     2013)"
                     /label=St1Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="SDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVR
                     RTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEEL
                     FIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYGQ
                     LRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGKR
                     KYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNLLN
                     DLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRIDK
                     SGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS
                     FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTT
                     SSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDD
                     EKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCL
                     YTGKTISIHDLINNSNQFEVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDS
                     MDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVL
                     NALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLW
                     KKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQ
                     VDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKF
                     LMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPE
                     IKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQF
                     EKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTM
                     PKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQH
                     IIKNEGDKPKLDF"
     CDS             4155..4175
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4206..4254
                     /note="synthetic polyadenylation signal"
     promoter        complement(4370..4618)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4428..4447)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4598..4618)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4647..4787
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4647..4776
                     /label=AAV2 ITR
     primer_bind     complement(4949..4968)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5018..5122
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5123..5191,5192..5983)
                     /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(5341..5360)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6154..6742
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6643..6662
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
       61 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
      121 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      181 aggggttcct gcggcctcta gactcgagcc ctaaaatggg caaacattgc aagcagcaaa
      241 cagcaaacac acagccctcc ctgcctgctg accttggagc tggggcagag gtcagagacc
      301 tctctgggcc catgccacct ccaacatcca ctcgacccct tggaatttcg gtggagagga
      361 gcagaggttg tcctggcgtg gtttaggtag tgtgagaggg tggacacagg acgctgtggt
      421 ttctgagcca gggggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc
      481 gataactggg gtgaccttgg ttaatattca ccagcagcct cccccgttgc ccctctggat
      541 ccactgctta aatacggacg aggacagggc cctgtctcct cagcttcagg caccaccact
      601 gacctgggac agtgaatccg gactctaagg taaatataaa atttttaagt gtataatgtg
      661 ttaaactact gattctaatt gtttctctct tttagattcc aacctttgga actgaaccgg
      721 tcgccaccat gggcgcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag
      781 ccagcgacct ggtgctgggc ctggacatcg gcatcggcag cgtgggcgtg ggcatcctga
      841 acaaggtgac cggcgagatc atccacaaga acagtcgcat cttccctgct gctcaggctg
      901 agaacaacct ggtgcgccgc accaaccgcc agggtcgccg gcttgctcgc cgcaagaagc
      961 accggcgcgt gcgcctgaac cgcctgttcg aggagagcgg cctgatcacc gacttcacca
     1021 agatcagcat caacctgaac ccctaccagc tgcgcgtgaa gggcctgacc gacgagctga
     1081 gcaacgagga gctgttcatc gccctgaaga acatggtgaa gcaccgcggc atcagctacc
     1141 tggacgacgc cagcgacgac ggcaacagca gcgtgggcga ctacgcccag atcgtgaagg
     1201 agaacagcaa gcagctggag accaagaccc ccggccagat ccagctggag cgctaccaga
     1261 cctacggcca gctgcgcggc gacttcaccg tggagaagga cggcaagaag caccgcctga
     1321 tcaacgtgtt ccccaccagc gcctaccgca gcgaggccct gcgcatcctg cagacccagc
     1381 aggagttcaa cccccagatc accgacgagt tcatcaaccg ctacctggag atcctgaccg
     1441 gcaagcgcaa gtactaccac ggccccggca acgagaagag ccgcaccgac tacggccgct
     1501 accgcaccag cggcgagacc ctggacaaca tcttcggcat cctgatcggc aagtgcacct
     1561 tctaccccga cgagttccgc gccgccaagg ccagctacac cgcccaggag ttcaatctgc
     1621 tgaacgacct gaacaacctg accgtgccca ccgagaccaa gaagctgagc aaggagcaga
     1681 agaaccagat catcaactac gtgaagaacg agaaggctat gggccccgcc aagctgttca
     1741 agtacatcgc caagctgctg agctgcgacg tggccgacat caagggctac cgcatcgaca
     1801 agagcggcaa ggccgagatc cacaccttcg aggcctaccg caagatgaag accctggaga
     1861 ccctggacat cgagcagatg gaccgagaga ccctggacaa gctggcctac gtgctgaccc
     1921 tgaacaccga gcgcgagggc atccaggagg ccctggagca cgagttcgcc gacggcagct
     1981 tcagccagaa acaggtggac gagctggtgc agttccgcaa ggccaacagc agcatcttcg
     2041 gcaagggctg gcacaacttc agcgtgaagc tgatgatgga gctgatcccc gagctgtacg
     2101 agaccagcga ggagcagatg accatcctga cccgcctggg caagcagaag accaccagca
     2161 gcagcaacaa gaccaagtac atcgacgaga agctgctgac cgaggagatc tacaaccccg
     2221 tggtggccaa gagcgtgcgc caggccatca agatcgtgaa cgccgccatc aaggagtacg
     2281 gcgacttcga caacatcgtg atcgagatgg cccgcgagac caacgaggac gacgagaaga
     2341 aggccatcca gaagatccag aaggccaaca aggacgagaa ggacgccgcc atgctgaagg
     2401 ccgccaacca gtacaacggc aaggccgagc tgccccacag cgtgttccac ggccacaagc
     2461 agctggccac caagatccgc ctgtggcacc agcagggcga gcgctgcctg tacaccggca
     2521 agaccatcag catccacgac ctgatcaaca acagcaacca gttcgaggtg gaccacatcc
     2581 tgcccctgag catcaccttc gacgacagcc tggccaacaa ggtgctggtg tacgccaccg
     2641 ccaaccagga gaagggccag cgcaccccct accaggccct ggacagcatg gacgacgcct
     2701 ggagcttccg cgagctgaag gccttcgtgc gcgagagcaa gaccctgagc aacaagaaga
     2761 aggagtatct gctgaccgag gaggacatca gcaagttcga cgtgcgcaag aagttcatcg
     2821 agcgcaacct ggtggacacc cgctacgcca gccgcgtggt gctgaacgcc ctgcaggagc
     2881 acttccgcgc ccacaagatc gacaccaagg tgagcgtggt gcgcggccag ttcaccagcc
     2941 agctgcgccg ccactggggc atcgagaaga cccgcgacac ctaccaccac cacgccgtgg
     3001 acgccctgat cattgcggct tctagccagc tgaacctgtg gaagaagcag aagaacaccc
     3061 tggtgagcta cagcgaggac cagctgctgg acatcgagac cggcgagctg atcagcgacg
     3121 acgagtacaa ggagagcgtg ttcaaggccc cctaccagca cttcgtggac accctgaaga
     3181 gcaaggagtt cgaggacagc atcctgttca gctaccaggt ggacagcaag ttcaaccgca
     3241 agatcagcga cgccaccatc tacgccaccc gccaggccaa ggtgggcaag gacaaggccg
     3301 acgagaccta cgtgctgggc aagatcaagg acatctacac ccaggacggc tacgacgcct
     3361 tcatgaagat ctacaagaag gacaagagca agttcctgat gtaccgccac gacccccaga
     3421 ccttcgagaa ggtgatcgag cccatcctgg agaactaccc caacaagcag atcaacgaga
     3481 aaggcaagga ggtgccctgc aaccccttcc tgaagtacaa ggaggagcac ggctacatcc
     3541 gcaagtacag caagaagggc aacggccccg agatcaagag cctgaagtac tacgacagca
     3601 agctgggcaa ccacatcgac atcaccccca aggacagcaa caacaaggtg gtgctgcaga
     3661 gcgtgagccc ctggcgcgcc gacgtgtact tcaacaagac caccggcaag tacgagatcc
     3721 tggggctgaa gtacgccgat ctgcagtttg agaaaggcac aggcacctac aagatcagcc
     3781 aggagaagta caacgacatc aagaagaagg agggcgtgga cagcgacagc gagttcaagt
     3841 tcaccctgta caagaacgac cttctgctgg tgaaggacac cgagaccaag gagcaacagc
     3901 tgttccgctt cctgagccgc accatgccca agcagaagca ctacgtggag ctgaagccct
     3961 acgacaagca gaagttcgag ggcggcgagg ccctgatcaa ggtgctgggc aacgtggcca
     4021 acagcggcca gtgcaagaag ggcctgggca agagcaacat cagcatctac aaggtgcgca
     4081 ccgacgtgct gggcaaccag cacatcatca agaacgaggg cgacaagccc aagttggact
     4141 tcagcagggc tgaccccaag aagaagagga aggtgggatc ctgataagaa ttcgtcgaca
     4201 agcttaataa aagatcttta ttttcattag atctgtgtgt tggttttttg tgtgcgtacg
     4261 aaaaaaaaca ccctgccata aaatgacagg gtgttgattt cggcatgaag ccttatcttt
     4321 gtagcttctg gtaccagagt acaaagaccg agacgcatta atcgtctcgg gtgtttcgtc
     4381 ctttccacaa gatatataaa gccaagaaat cgaaatactt tcaagttacg gtaagcatat
     4441 gatagtccat tttaaaacat aattttaaaa ctgcaaacta cccaagaaat tattactttc
     4501 tacgtcacgt attttgtact aatatctttg tgtttacagt caaattaatt ctaattatct
     4561 ctctaacagc cttgtatcgt atatgcaaat atgaaggaat catgggaaat aggccctctt
     4621 cctgcccgac ctactagtgc ggccgcagga acccctagtg atggagttgg ccactccctc
     4681 tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt
     4741 tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg
     4801 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag
     4861 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     4921 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     4981 acgttcgccg gcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     5041 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     5101 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     5161 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     5221 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     5281 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     5341 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     5401 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     5461 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5521 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5581 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5641 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     5701 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5761 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5821 tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag atggtaagcc
     5881 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5941 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     6001 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     6061 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     6121 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     6181 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     6241 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     6301 tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     6361 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     6421 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6481 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6541 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6601 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6661 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6721 aggggggcgg agcctatgga aaa
//
