LOCUS       sequence-207996-        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   5..134
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     regulatory      667..676
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             682..702
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             727..4086
                     /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             4099..4119
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    4150..4198
                     /note="synthetic polyadenylation signal"
     promoter        complement(4314..4562)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(4372..4391)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(4542..4562)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     repeat_region   4591..4731
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4591..4720
                     /label=AAV2 ITR
     primer_bind     complement(4893..4912)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4962..5066
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5067..5135,5136..5927)
                     /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(5285..5304)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6098..6686
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6587..6606
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg ccgcccgggc gtcgggcgac
       61 ctttggtcgc ccggcctcag tgagcgagcg agcgcgcaga gagggagtgg ccaactccat
      121 cactaggggt tcctgcggcc tctagactcg agccctaaaa tgggcaaaca ttgcaagcag
      181 caaacagcaa acacacagcc ctccctgcct gctgaccttg gagctggggc agaggtcaga
      241 gacctctctg ggcccatgcc acctccaaca tccactcgac cccttggaat ttcggtggag
      301 aggagcagag gttgtcctgg cgtggtttag gtagtgtgag agggtggaca caggacgctg
      361 tggtttctga gccagggggc gactcagatc ccagccagtg gacttagccc ctgtttgctc
      421 ctccgataac tggggtgacc ttggttaata ttcaccagca gcctcccccg ttgcccctct
      481 ggatccactg cttaaatacg gacgaggaca gggccctgtc tcctcagctt caggcaccac
      541 cactgacctg ggacagtgaa tccggactct aaggtaaata taaaattttt aagtgtataa
      601 tgtgttaaac tactgattct aattgtttct ctcttttaga ttccaacctt tggaactgaa
      661 ccggtcgcca ccatgggcgc cccaaagaag aagcggaagg tcggtatcca cggagtccca
      721 gcagccagcg acctggtgct gggcctggac atcggcatcg gcagcgtggg cgtgggcatc
      781 ctgaacaagg tgaccggcga gatcatccac aagaacagtc gcatcttccc tgctgctcag
      841 gctgagaaca acctggtgcg ccgcaccaac cgccagggtc gccggcttgc tcgccgcaag
      901 aagcaccggc gcgtgcgcct gaaccgcctg ttcgaggaga gcggcctgat caccgacttc
      961 accaagatca gcatcaacct gaacccctac cagctgcgcg tgaagggcct gaccgacgag
     1021 ctgagcaacg aggagctgtt catcgccctg aagaacatgg tgaagcaccg cggcatcagc
     1081 tacctggacg acgccagcga cgacggcaac agcagcgtgg gcgactacgc ccagatcgtg
     1141 aaggagaaca gcaagcagct ggagaccaag acccccggcc agatccagct ggagcgctac
     1201 cagacctacg gccagctgcg cggcgacttc accgtggaga aggacggcaa gaagcaccgc
     1261 ctgatcaacg tgttccccac cagcgcctac cgcagcgagg ccctgcgcat cctgcagacc
     1321 cagcaggagt tcaaccccca gatcaccgac gagttcatca accgctacct ggagatcctg
     1381 accggcaagc gcaagtacta ccacggcccc ggcaacgaga agagccgcac cgactacggc
     1441 cgctaccgca ccagcggcga gaccctggac aacatcttcg gcatcctgat cggcaagtgc
     1501 accttctacc ccgacgagtt ccgcgccgcc aaggccagct acaccgccca ggagttcaat
     1561 ctgctgaacg acctgaacaa cctgaccgtg cccaccgaga ccaagaagct gagcaaggag
     1621 cagaagaacc agatcatcaa ctacgtgaag aacgagaagg ctatgggccc cgccaagctg
     1681 ttcaagtaca tcgccaagct gctgagctgc gacgtggccg acatcaaggg ctaccgcatc
     1741 gacaagagcg gcaaggccga gatccacacc ttcgaggcct accgcaagat gaagaccctg
     1801 gagaccctgg acatcgagca gatggaccga gagaccctgg acaagctggc ctacgtgctg
     1861 accctgaaca ccgagcgcga gggcatccag gaggccctgg agcacgagtt cgccgacggc
     1921 agcttcagcc agaaacaggt ggacgagctg gtgcagttcc gcaaggccaa cagcagcatc
     1981 ttcggcaagg gctggcacaa cttcagcgtg aagctgatga tggagctgat ccccgagctg
     2041 tacgagacca gcgaggagca gatgaccatc ctgacccgcc tgggcaagca gaagaccacc
     2101 agcagcagca acaagaccaa gtacatcgac gagaagctgc tgaccgagga gatctacaac
     2161 cccgtggtgg ccaagagcgt gcgccaggcc atcaagatcg tgaacgccgc catcaaggag
     2221 tacggcgact tcgacaacat cgtgatcgag atggcccgcg agaccaacga ggacgacgag
     2281 aagaaggcca tccagaagat ccagaaggcc aacaaggacg agaaggacgc cgccatgctg
     2341 aaggccgcca accagtacaa cggcaaggcc gagctgcccc acagcgtgtt ccacggccac
     2401 aagcagctgg ccaccaagat ccgcctgtgg caccagcagg gcgagcgctg cctgtacacc
     2461 ggcaagacca tcagcatcca cgacctgatc aacaacagca accagttcga ggtggaccac
     2521 atcctgcccc tgagcatcac cttcgacgac agcctggcca acaaggtgct ggtgtacgcc
     2581 accgccaacc aggagaaggg ccagcgcacc ccctaccagg ccctggacag catggacgac
     2641 gcctggagct tccgcgagct gaaggccttc gtgcgcgaga gcaagaccct gagcaacaag
     2701 aagaaggagt atctgctgac cgaggaggac atcagcaagt tcgacgtgcg caagaagttc
     2761 atcgagcgca acctggtgga cacccgctac gccagccgcg tggtgctgaa cgccctgcag
     2821 gagcacttcc gcgcccacaa gatcgacacc aaggtgagcg tggtgcgcgg ccagttcacc
     2881 agccagctgc gccgccactg gggcatcgag aagacccgcg acacctacca ccaccacgcc
     2941 gtggacgccc tgatcattgc ggcttctagc cagctgaacc tgtggaagaa gcagaagaac
     3001 accctggtga gctacagcga ggaccagctg ctggacatcg agaccggcga gctgatcagc
     3061 gacgacgagt acaaggagag cgtgttcaag gccccctacc agcacttcgt ggacaccctg
     3121 aagagcaagg agttcgagga cagcatcctg ttcagctacc aggtggacag caagttcaac
     3181 cgcaagatca gcgacgccac catctacgcc acccgccagg ccaaggtggg caaggacaag
     3241 gccgacgaga cctacgtgct gggcaagatc aaggacatct acacccagga cggctacgac
     3301 gccttcatga agatctacaa gaaggacaag agcaagttcc tgatgtaccg ccacgacccc
     3361 cagaccttcg agaaggtgat cgagcccatc ctggagaact accccaacaa gcagatcaac
     3421 gagaaaggca aggaggtgcc ctgcaacccc ttcctgaagt acaaggagga gcacggctac
     3481 atccgcaagt acagcaagaa gggcaacggc cccgagatca agagcctgaa gtactacgac
     3541 agcaagctgg gcaaccacat cgacatcacc cccaaggaca gcaacaacaa ggtggtgctg
     3601 cagagcgtga gcccctggcg cgccgacgtg tacttcaaca agaccaccgg caagtacgag
     3661 atcctggggc tgaagtacgc cgatctgcag tttgagaaag gcacaggcac ctacaagatc
     3721 agccaggaga agtacaacga catcaagaag aaggagggcg tggacagcga cagcgagttc
     3781 aagttcaccc tgtacaagaa cgaccttctg ctggtgaagg acaccgagac caaggagcaa
     3841 cagctgttcc gcttcctgag ccgcaccatg cccaagcaga agcactacgt ggagctgaag
     3901 ccctacgaca agcagaagtt cgagggcggc gaggccctga tcaaggtgct gggcaacgtg
     3961 gccaacagcg gccagtgcaa gaagggcctg ggcaagagca acatcagcat ctacaaggtg
     4021 cgcaccgacg tgctgggcaa ccagcacatc atcaagaacg agggcgacaa gcccaagttg
     4081 gacttcagca gggctgaccc caagaagaag aggaaggtgg gatcctgata agaattcgtc
     4141 gacaagctta ataaaagatc tttattttca ttagatctgt gtgttggttt tttgtgtgcg
     4201 tacgaaaaaa aacaccctgc cataaaatga cagggtgttg atttcggcat gaagccttat
     4261 ctttgtagct tctggtacca gagtacaaag accgagacgc attaatcgtc tcgggtgttt
     4321 cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc
     4381 atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac
     4441 tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt
     4501 atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc
     4561 tcttcctgcc cgacctacta gtgcggccgc aggaacccct agtgatggag ttggccactc
     4621 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg
     4681 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga
     4741 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc
     4801 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     4861 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     4921 cgccacgttc gccggcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4981 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     5041 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     5101 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     5161 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     5221 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     5281 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     5341 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     5401 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     5461 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5521 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5581 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     5641 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5701 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5761 aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta
     5821 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5881 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     5941 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     6001 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6061 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6121 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6181 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6241 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6301 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6361 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6421 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6481 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6541 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6601 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6661 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6721 ccttttgctg gccttttgct cac
//
