LOCUS       sequence-362042-        7448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A single vector containing Cas9 from Staphylococcus aureus (SaCas9) 
            and its sgRNA targeting Adrb1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7448)
  TITLE     Various Crispr/Cas9 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7448)
  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: 
            "Unpublished"
COMMENT     Sequence Label: 
            pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA-Adrb1
FEATURES             Location/Qualifiers
     source          1..7448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   109..249
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   120..249
                     /label=AAV2 ITR
     misc_RNA        complement(263..338)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(361..609)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(419..438)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(589..609)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     polyA_signal    616..823
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     812..829
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(857..937)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(944..991)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(992..4147)
                     /codon_start=1
                     /product="Cas9 endonuclease from the Staphylococcus aureus 
                     Type II CRISPR/Cas system"
                     /label=SaCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEG
                     RRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEF
                     SAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEV
                     RGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGW
                     KDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQ
                     IIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEI
                     IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI
                     NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIK
                     VINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAK
                     YLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQ
                     EENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFS
                     VQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERN
                     KGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF
                     ITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDN
                     DKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKD
                     NGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNL
                     DVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLN
                     RIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQI
                     IKKG"
     CDS             complement(4172..4192)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      4195..4204
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4223..4426)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4252..4272)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4427..4806
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   4830..4959
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(5021..5609)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5101..5120)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5780..6571,6572..6640))
                     /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     6403..6422
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6641..6745)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6813..6831
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6869..6891)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6991..7010
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(join(7027..7448,1..34))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7164..7185)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7376..7395
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccgccgcgc ttaatgcgcc gctacagggc gcgtactatg gttgctttga cgtatgcggt
       61 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgcccc tgcaggcagc
      121 tgcgcgctcg ctcgctcact gaggccgccc gggcaaagcc cgggcgtcgg gcgacctttg
      181 gtcgcccggc ctcagtgagc gagcgagcgc gcagagaggg agtggccaac tccatcacta
      241 ggggttcctg cggccgcaaa aatctcgcca acaagttgac gagataaaca cggcattttg
      301 ccttgtttta gtagattctg tttccagagt actaaaacgg tccactgctg cgacagcggc
      361 ggtgtttcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
      421 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
      481 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
      541 tccaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
      601 taggccctcg gtacctcccc agcatgcctg ctattctctt cccaatcctc ccccttgctg
      661 tcctgcccca ccccaccccc cagaatagaa tgacacctac tcagacaatg cgatgcaatt
      721 tcctcatttt attaggaaag gacagtggga gtggcacctt ccagggtcaa ggaaggcacg
      781 ggggaggggc aaacaacaga tggctggcaa ctagaaggca cagtcgaggc tgatcagcga
      841 gctctaggaa ttcttaagcg taatctggaa catcgtatgg gtaagcgtaa tctggaacat
      901 cgtatgggta agcgtaatct ggaacatcgt atgggtagga tccctttttc ttttttgcct
      961 ggccggcctt tttcgtggcc gccggccttt tgcccttttt gatgatctga gggtgcttct
     1021 tagatttcac ttcatacagg ttgcccagaa tgtctgtgct gtacttctta atgctctggg
     1081 tcttggaggc gattgtctta atgatcctgg ggggcctctt gtcgttcatg ttttccaggt
     1141 actcgcggta ggtgatgtcg atcatgttca cttcgatccg gttcagcagg tcgttgttca
     1201 cgccgatcac tctatacagc tcgccgttga tcttgatcag atcgttgttg tagaaggagg
     1261 cgataaactc ggcctggttg ctgatcttct tcagcttctt agcttcctca tagcacttgc
     1321 tattcacttc gtagtagttt tcttttttga tcacatccag attcttcacg gtcacgaact
     1381 tgtacacgcc attgtccagg tacacgtcga atctgtaggg cttcagggac agcttcacga
     1441 ccttgtttct gctgttgggg tagtcgtcgg tgatgtccag atgggcgttc agtttgttgc
     1501 cgtaatactt aatcttcttg atcacggggc cgttgtcctt tttggagtac ttggtcaggt
     1561 agttcccggt ttcctcgtag tacttgtaca ggggattctt ctcgtcgccg tactgttcca
     1621 taatcagctt cagtttctgg taggtctggg ggtcgtggtg gtacatcagc agcttttcgg
     1681 ggctcttgtt gatcagcttt ttcagcttgt cattgtcctt gtcgtacagg ccgttcagat
     1741 tgttcacgat cagggtgttg cccttgtcgt ccttccgggt ggagtacagg gtgtcgttaa
     1801 tcagctctct attaggcttc ttgtccaccc ggtggctgta cttgtagtcc ttgaagtcct
     1861 taatgtgctt gatctggtgg ggggtgatga agatctcttt gtactcctgc tcggtttcga
     1921 tctcgggcat gctctcggcc tgcttttcct cgaacatctg gttttccatc acttttttgg
     1981 ccttgtccag tttcttccac tctttgaaga tgaaatcggc gttggcaatg atcagggcgt
     2041 cctcggcgtg gtgcttgtac cccttgttcc gctctttctt aaacttccac ttccgccgca
     2101 gaaagctggt gaagccgcca ttgatggact tcactttcac gtccaggttg ttcactctga
     2161 agtagctccg cagcaggttc atcaggcctc tggtggcgta tctggtatcc accaggttcc
     2221 ggttgatgaa gtctttctgc acggagaacc tgttgatgtc ccgttcttcc agcagatact
     2281 ctttcttggt cttgctgatt ctgcccttgc ccttggccag attcaggatg tgcttcttga
     2341 aggtttcgta gctgatcttg ctgtcgctgc tgctcaggta ctggaatggg gtccggttgc
     2401 ccttcttgct gttttcttcc tgcttcacga gcaccttgtt gttgaagctg ttgtcgaagg
     2461 acacgcttct ggggatgatg tggtccacct catagttgaa ggggttgttc agcagatctt
     2521 ccagagggat ggcttccagg ctgtacaggc acttgccttc ctgcatgtcg tgcagcttga
     2581 tcttctcgat caggtacttg gcgttctctt tgccggtggt ccggatgatt tcctcgatcc
     2641 gctcgttggt ctgccggttc cgcttctgca tctcgttgat cattttctgg gcgtccttgg
     2701 agttcttctc gcgggccagc tcgataatga tgtcgttggg caggccgtac ttcttgatga
     2761 tggcgttgat cactttgatg ctctggatga agcttctctt cacgacgggg ctcaggatga
     2821 agtcgtccac cagggtggtg gggatctctt tctgctggga caggtccacc ttcttgggca
     2881 ccagcttcag ccggttgaag atagcgatct ggttgtcgtt ggtgtgccac agctcgtcca
     2941 ggatcaggtt gatggccttc aggctcaggt tgtgggtgcc ggtatagccc ttcagattag
     3001 agatctgctc gatctcttcc tgggtcagct cggagttcag attggtcagt tcttcctgga
     3061 tgtcctcgct gctctggtag atggtcagga tcttggcaat ctgatccagc agctcggcgt
     3121 tctcaataat ctctttccgg gcggtaatgt ccttgatgtc gtggtacacc ttcaggttgg
     3181 tgaactcggg cttgccggtg ctggtcactc tgtagccctt aatatcctct tcgttcacga
     3241 ggatttcttt ggcgatctgc ttcagggtgg gcttcttctt ctgcttgaac acgttctcga
     3301 tgatctggaa cttctcgtaa tattccagct tctcgttctc gtccctggtg atcacgagat
     3361 tgttcaggtc gttcagggcg ttgtacaggt cggcgttgta ggcgtacttc acgctccgca
     3421 gttcctcggg gaagtaggtg cagtggccca tcagcatctc gtaccattct ttgatgtcct
     3481 tccagccgaa ggggctgccc tcgccaggtc cctcatagta ggtccgccgg gtttccagca
     3541 ggtcgatgta ggtgtcgatg aagctctggt ccagctggtg gtaggccttc tgcaccttca
     3601 gcagctgttt ggcttctttc acgtagtcgc tggtcttgaa tctgttgatg ctgccccgca
     3661 cttcgccgtc tttcttcagc cgttccagct gcagttcggc cacgtatttc tcttccaggg
     3721 ccttgctgtt ccggctgatc tgctctttgg tggacagctc gttgccggtg tcctcttcca
     3781 cctcgttcac gttgtgcacg cctcttctct tggccaggtg cagcagggcg gcagagaact
     3841 cttcctcgct cagcttctgg ctcaggccct tcactctggc ctcgtagggg ttgatgccgc
     3901 tcagctcgct gtggtcggtc agcaggttgt agtcgaacag cagcttcttc actctctgga
     3961 ttctatgccg cctccgccgc ttcagccttc tggcgcctct cttgctccgc ctgccctcgt
     4021 tgttttccac gttggcctct ttgaacagcc gcacgccggc atcgatcacg tcccgtgtct
     4081 cgtagtcgat gatgccgtag cccacgctgg tgatgccgat gtccaggccc aggatgtagt
     4141 tccgcttggc tgctgggact ccgtggatac cgaccttccg cttcttcttt ggggccatgg
     4201 tggcaccggt agttagccag agagctctgc ttatatagac ctcccaccgt acacgcctac
     4261 cgcccatttg cgtcaatggg gcggagttgt tacgacattt tggaaagtcc cgttgatttt
     4321 ggtgccaaaa caaactccca ttgacgtcaa tggggtggag acttggaaat ccccgtgagt
     4381 caaaccgcta tccacgccca ttgatgtact gccaaaaccg catcaccatg gtaatagcga
     4441 tgactaatac gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat
     4501 aatgccaggc gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat
     4561 acacttgatg tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat
     4621 ggaaagtccc tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg
     4681 ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca
     4741 tatatgggct atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc
     4801 aatgtcaacg cctcgagtct agaggccgca ggaaccccta gtgatggagt tggccactcc
     4861 ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg
     4921 cggcctcagt gagcgagcga gcgcgcagct gcctgcagga catgtgagca aaaggccagc
     4981 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5041 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5101 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5161 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5221 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5281 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5341 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5401 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     5461 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     5521 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     5581 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     5641 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     5701 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     5761 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     5821 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     5881 agggcttacc atctggcccc agtgctgcaa tgataccgcg gcttccacgc tcaccggctc
     5941 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6001 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6061 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6121 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6181 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6241 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6301 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6361 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6421 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     6481 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     6541 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     6601 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     6661 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     6721 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     6781 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
     6841 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     6901 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     6961 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     7021 accataaaat tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     7081 gctcattttt taaccaatag accgaaatcg gcaaaatccc ttataaatca aaagaatagc
     7141 ccgagataga gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     7201 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     7261 cacccaaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     7321 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     7381 agaaagcgaa aggagcgggc gctaaggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     7441 ccaccaca
//
