LOCUS       sequence-362038-        7447 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 7447)
  TITLE     Various Crispr/Cas9 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7447)
  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            "Unpublished"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..7447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   134..263
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        287..666
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        667..870
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     821..841
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      889..898
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             901..921
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             946..4101
                     /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             4102..4149
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4156..4236
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4264..4281)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4270..4477
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4484..4732
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4484..4504
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4655..4674
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4754..4829
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4843..4983
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4843..4972
                     /label=AAV2 ITR
     rep_origin      5058..5513
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5145..5164)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5355..5376
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5530..5549)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5649..5671
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5709..5727)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5795..5899
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5900..5968,5969..6760)
                     /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(6118..6137)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6931..7447,1..72)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7420..7439
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
       61 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      121 tttgctcaca tgtcctgcag gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg
      181 tcgggcgacc tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc
      241 caactccatc actaggggtt cctgcggcct ctagactcga ggcgttgaca ttgattattg
      301 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      361 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      421 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      481 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      541 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      601 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      661 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      721 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      781 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      841 gtacggtggg aggtctatat aagcagagct ctctggctaa ctaccggtgc caccatggcc
      901 ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccaagcg gaactacatc
      961 ctgggcctgg acatcggcat caccagcgtg ggctacggca tcatcgacta cgagacacgg
     1021 gacgtgatcg atgccggcgt gcggctgttc aaagaggcca acgtggaaaa caacgagggc
     1081 aggcggagca agagaggcgc cagaaggctg aagcggcgga ggcggcatag aatccagaga
     1141 gtgaagaagc tgctgttcga ctacaacctg ctgaccgacc acagcgagct gagcggcatc
     1201 aacccctacg aggccagagt gaagggcctg agccagaagc tgagcgagga agagttctct
     1261 gccgccctgc tgcacctggc caagagaaga ggcgtgcaca acgtgaacga ggtggaagag
     1321 gacaccggca acgagctgtc caccaaagag cagatcagcc ggaacagcaa ggccctggaa
     1381 gagaaatacg tggccgaact gcagctggaa cggctgaaga aagacggcga agtgcggggc
     1441 agcatcaaca gattcaagac cagcgactac gtgaaagaag ccaaacagct gctgaaggtg
     1501 cagaaggcct accaccagct ggaccagagc ttcatcgaca cctacatcga cctgctggaa
     1561 acccggcgga cctactatga gggacctggc gagggcagcc ccttcggctg gaaggacatc
     1621 aaagaatggt acgagatgct gatgggccac tgcacctact tccccgagga actgcggagc
     1681 gtgaagtacg cctacaacgc cgacctgtac aacgccctga acgacctgaa caatctcgtg
     1741 atcaccaggg acgagaacga gaagctggaa tattacgaga agttccagat catcgagaac
     1801 gtgttcaagc agaagaagaa gcccaccctg aagcagatcg ccaaagaaat cctcgtgaac
     1861 gaagaggata ttaagggcta cagagtgacc agcaccggca agcccgagtt caccaacctg
     1921 aaggtgtacc acgacatcaa ggacattacc gcccggaaag agattattga gaacgccgag
     1981 ctgctggatc agattgccaa gatcctgacc atctaccaga gcagcgagga catccaggaa
     2041 gaactgacca atctgaactc cgagctgacc caggaagaga tcgagcagat ctctaatctg
     2101 aagggctata ccggcaccca caacctgagc ctgaaggcca tcaacctgat cctggacgag
     2161 ctgtggcaca ccaacgacaa ccagatcgct atcttcaacc ggctgaagct ggtgcccaag
     2221 aaggtggacc tgtcccagca gaaagagatc cccaccaccc tggtggacga cttcatcctg
     2281 agccccgtcg tgaagagaag cttcatccag agcatcaaag tgatcaacgc catcatcaag
     2341 aagtacggcc tgcccaacga catcattatc gagctggccc gcgagaagaa ctccaaggac
     2401 gcccagaaaa tgatcaacga gatgcagaag cggaaccggc agaccaacga gcggatcgag
     2461 gaaatcatcc ggaccaccgg caaagagaac gccaagtacc tgatcgagaa gatcaagctg
     2521 cacgacatgc aggaaggcaa gtgcctgtac agcctggaag ccatccctct ggaagatctg
     2581 ctgaacaacc ccttcaacta tgaggtggac cacatcatcc ccagaagcgt gtccttcgac
     2641 aacagcttca acaacaaggt gctcgtgaag caggaagaaa acagcaagaa gggcaaccgg
     2701 accccattcc agtacctgag cagcagcgac agcaagatca gctacgaaac cttcaagaag
     2761 cacatcctga atctggccaa gggcaagggc agaatcagca agaccaagaa agagtatctg
     2821 ctggaagaac gggacatcaa caggttctcc gtgcagaaag acttcatcaa ccggaacctg
     2881 gtggatacca gatacgccac cagaggcctg atgaacctgc tgcggagcta cttcagagtg
     2941 aacaacctgg acgtgaaagt gaagtccatc aatggcggct tcaccagctt tctgcggcgg
     3001 aagtggaagt ttaagaaaga gcggaacaag gggtacaagc accacgccga ggacgccctg
     3061 atcattgcca acgccgattt catcttcaaa gagtggaaga aactggacaa ggccaaaaaa
     3121 gtgatggaaa accagatgtt cgaggaaaag caggccgaga gcatgcccga gatcgaaacc
     3181 gagcaggagt acaaagagat cttcatcacc ccccaccaga tcaagcacat taaggacttc
     3241 aaggactaca agtacagcca ccgggtggac aagaagccta atagagagct gattaacgac
     3301 accctgtact ccacccggaa ggacgacaag ggcaacaccc tgatcgtgaa caatctgaac
     3361 ggcctgtacg acaaggacaa tgacaagctg aaaaagctga tcaacaagag ccccgaaaag
     3421 ctgctgatgt accaccacga cccccagacc taccagaaac tgaagctgat tatggaacag
     3481 tacggcgacg agaagaatcc cctgtacaag tactacgagg aaaccgggaa ctacctgacc
     3541 aagtactcca aaaaggacaa cggccccgtg atcaagaaga ttaagtatta cggcaacaaa
     3601 ctgaacgccc atctggacat caccgacgac taccccaaca gcagaaacaa ggtcgtgaag
     3661 ctgtccctga agccctacag attcgacgtg tacctggaca atggcgtgta caagttcgtg
     3721 accgtgaaga atctggatgt gatcaaaaaa gaaaactact acgaagtgaa tagcaagtgc
     3781 tatgaggaag ctaagaagct gaagaagatc agcaaccagg ccgagtttat cgcctccttc
     3841 tacaacaacg atctgatcaa gatcaacggc gagctgtata gagtgatcgg cgtgaacaac
     3901 gacctgctga accggatcga agtgaacatg atcgacatca cctaccgcga gtacctggaa
     3961 aacatgaacg acaagaggcc ccccaggatc attaagacaa tcgcctccaa gacccagagc
     4021 attaagaagt acagcacaga cattctgggc aacctgtatg aagtgaaatc taagaagcac
     4081 cctcagatca tcaaaaaggg caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa
     4141 aagaaaaagg gatcctaccc atacgatgtt ccagattacg cttacccata cgatgttcca
     4201 gattacgctt acccatacga tgttccagat tacgcttaag aattcctaga gctcgctgat
     4261 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     4321 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     4381 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     4441 gggaggattg ggaagagaat agcaggcatg ctggggaggt accgagggcc tatttcccat
     4501 gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt
     4561 gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg
     4621 gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg
     4681 aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggcgcggc
     4741 ggacgacagg ttggttttag tactctggaa acagaatcta ctaaaacaag gcaaaatgcc
     4801 gtgtttatct cgtcaacttg ttggcgagat ttttgcggcc gcaggaaccc ctagtgatgg
     4861 agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg
     4921 cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc
     4981 aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac
     5041 gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5101 tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt tcgctttctt
     5161 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5221 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     5281 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5341 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact ctatctcggg
     5401 ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa aaaatgagct
     5461 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg
     5521 cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac
     5581 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     5641 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     5701 acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc
     5761 ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     5821 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     5881 atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt
     5941 tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc
     6001 tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat
     6061 ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct
     6121 atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca
     6181 ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg
     6241 catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa
     6301 cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg
     6361 ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga
     6421 cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg
     6481 cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt
     6541 tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg
     6601 agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg gtaagccctc
     6661 ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca
     6721 gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc
     6781 atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
     6841 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     6901 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     6961 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     7021 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     7081 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     7141 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     7201 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     7261 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     7321 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     7381 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     7441 tagtcct
//
