LOCUS       sequence-362044-        7447 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A single vector containing Cas9 from Staphylococcus aureus (SaCas9) 
            and its sgRNA targeting Oprl1.
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  JOURNAL   Unpublished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            "Unpublished"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-Oprl1
FEATURES             Location/Qualifiers
     source          1..7447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   341..470
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(532..1120)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(612..631)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1291..2082,2083..2151))
                     /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     1914..1933
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2152..2256)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2324..2342
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2380..2402)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2502..2521
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2538..2993)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2675..2696)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2887..2906
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   3068..3208
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3079..3208
                     /label=AAV2 ITR
     misc_RNA        complement(3222..3297)
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     promoter        complement(3319..3567)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3377..3396)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3547..3567)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     polyA_signal    3574..3781
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3770..3787
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3815..3895)
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     CDS             complement(3902..3949)
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             complement(3950..7105)
                     /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(7130..7150)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      7153..7162
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7181..7384)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(7210..7230)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        join(7385..7447,1..317)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtactgggca taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact
       61 tggcatatga tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca
      121 ttgacgtcaa tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt
      181 caatgggcgg gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac
      241 gcggaactcc atatatgggc tatgaactaa tgaccccgta attgattact attaataact
      301 agtcaataat caatgtcaac gcctcgagtc tagaggccgc aggaacccct agtgatggag
      361 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
      421 cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg acatgtgagc
      481 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
      541 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
      601 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
      661 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
      721 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
      781 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
      841 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
      901 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
      961 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     1021 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     1081 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     1141 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     1201 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     1261 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     1321 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     1381 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc ggcttccacg
     1441 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     1501 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     1561 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     1621 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     1681 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     1741 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     1801 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     1861 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     1921 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     1981 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     2041 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     2101 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     2161 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     2221 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     2281 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     2341 gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     2401 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     2461 gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag agcagattgt
     2521 actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg cgttaaattt
     2581 ttgttaaatc agctcatttt ttaaccaata gaccgaaatc ggcaaaatcc cttataaatc
     2641 aaaagaatag cccgagatag agttgagtgt tgttccagtt tggaacaaga gtccactatt
     2701 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     2761 acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     2821 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     2881 aaaggaaggg aagaaagcga aaggagcggg cgctaaggcg ctggcaagtg tagcggtcac
     2941 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtactatgg
     3001 ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3061 aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg ggcaaagccc
     3121 gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga
     3181 gtggccaact ccatcactag gggttcctgc ggccgcaaaa atctcgccaa caagttgacg
     3241 agataaacac ggcattttgc cttgttttag tagattctgt ttccagagta ctaaaacgtc
     3301 ttgctgacac tgcccttcgg tgtttcgtcc tttccacaag atatataaag ccaagaaatc
     3361 gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac
     3421 tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt
     3481 gtttacagtc aaattaattc caattatctc tctaacagcc ttgtatcgta tatgcaaata
     3541 tgaaggaatc atgggaaata ggccctcggt acctccccag catgcctgct attctcttcc
     3601 caatcctccc ccttgctgtc ctgccccacc ccacccccca gaatagaatg acacctactc
     3661 agacaatgcg atgcaatttc ctcattttat taggaaagga cagtgggagt ggcaccttcc
     3721 agggtcaagg aaggcacggg ggaggggcaa acaacagatg gctggcaact agaaggcaca
     3781 gtcgaggctg atcagcgagc tctaggaatt cttaagcgta atctggaaca tcgtatgggt
     3841 aagcgtaatc tggaacatcg tatgggtaag cgtaatctgg aacatcgtat gggtaggatc
     3901 cctttttctt ttttgcctgg ccggcctttt tcgtggccgc cggccttttg ccctttttga
     3961 tgatctgagg gtgcttctta gatttcactt catacaggtt gcccagaatg tctgtgctgt
     4021 acttcttaat gctctgggtc ttggaggcga ttgtcttaat gatcctgggg ggcctcttgt
     4081 cgttcatgtt ttccaggtac tcgcggtagg tgatgtcgat catgttcact tcgatccggt
     4141 tcagcaggtc gttgttcacg ccgatcactc tatacagctc gccgttgatc ttgatcagat
     4201 cgttgttgta gaaggaggcg ataaactcgg cctggttgct gatcttcttc agcttcttag
     4261 cttcctcata gcacttgcta ttcacttcgt agtagttttc ttttttgatc acatccagat
     4321 tcttcacggt cacgaacttg tacacgccat tgtccaggta cacgtcgaat ctgtagggct
     4381 tcagggacag cttcacgacc ttgtttctgc tgttggggta gtcgtcggtg atgtccagat
     4441 gggcgttcag tttgttgccg taatacttaa tcttcttgat cacggggccg ttgtcctttt
     4501 tggagtactt ggtcaggtag ttcccggttt cctcgtagta cttgtacagg ggattcttct
     4561 cgtcgccgta ctgttccata atcagcttca gtttctggta ggtctggggg tcgtggtggt
     4621 acatcagcag cttttcgggg ctcttgttga tcagcttttt cagcttgtca ttgtccttgt
     4681 cgtacaggcc gttcagattg ttcacgatca gggtgttgcc cttgtcgtcc ttccgggtgg
     4741 agtacagggt gtcgttaatc agctctctat taggcttctt gtccacccgg tggctgtact
     4801 tgtagtcctt gaagtcctta atgtgcttga tctggtgggg ggtgatgaag atctctttgt
     4861 actcctgctc ggtttcgatc tcgggcatgc tctcggcctg cttttcctcg aacatctggt
     4921 tttccatcac ttttttggcc ttgtccagtt tcttccactc tttgaagatg aaatcggcgt
     4981 tggcaatgat cagggcgtcc tcggcgtggt gcttgtaccc cttgttccgc tctttcttaa
     5041 acttccactt ccgccgcaga aagctggtga agccgccatt gatggacttc actttcacgt
     5101 ccaggttgtt cactctgaag tagctccgca gcaggttcat caggcctctg gtggcgtatc
     5161 tggtatccac caggttccgg ttgatgaagt ctttctgcac ggagaacctg ttgatgtccc
     5221 gttcttccag cagatactct ttcttggtct tgctgattct gcccttgccc ttggccagat
     5281 tcaggatgtg cttcttgaag gtttcgtagc tgatcttgct gtcgctgctg ctcaggtact
     5341 ggaatggggt ccggttgccc ttcttgctgt tttcttcctg cttcacgagc accttgttgt
     5401 tgaagctgtt gtcgaaggac acgcttctgg ggatgatgtg gtccacctca tagttgaagg
     5461 ggttgttcag cagatcttcc agagggatgg cttccaggct gtacaggcac ttgccttcct
     5521 gcatgtcgtg cagcttgatc ttctcgatca ggtacttggc gttctctttg ccggtggtcc
     5581 ggatgatttc ctcgatccgc tcgttggtct gccggttccg cttctgcatc tcgttgatca
     5641 ttttctgggc gtccttggag ttcttctcgc gggccagctc gataatgatg tcgttgggca
     5701 ggccgtactt cttgatgatg gcgttgatca ctttgatgct ctggatgaag cttctcttca
     5761 cgacggggct caggatgaag tcgtccacca gggtggtggg gatctctttc tgctgggaca
     5821 ggtccacctt cttgggcacc agcttcagcc ggttgaagat agcgatctgg ttgtcgttgg
     5881 tgtgccacag ctcgtccagg atcaggttga tggccttcag gctcaggttg tgggtgccgg
     5941 tatagccctt cagattagag atctgctcga tctcttcctg ggtcagctcg gagttcagat
     6001 tggtcagttc ttcctggatg tcctcgctgc tctggtagat ggtcaggatc ttggcaatct
     6061 gatccagcag ctcggcgttc tcaataatct ctttccgggc ggtaatgtcc ttgatgtcgt
     6121 ggtacacctt caggttggtg aactcgggct tgccggtgct ggtcactctg tagcccttaa
     6181 tatcctcttc gttcacgagg atttctttgg cgatctgctt cagggtgggc ttcttcttct
     6241 gcttgaacac gttctcgatg atctggaact tctcgtaata ttccagcttc tcgttctcgt
     6301 ccctggtgat cacgagattg ttcaggtcgt tcagggcgtt gtacaggtcg gcgttgtagg
     6361 cgtacttcac gctccgcagt tcctcgggga agtaggtgca gtggcccatc agcatctcgt
     6421 accattcttt gatgtccttc cagccgaagg ggctgccctc gccaggtccc tcatagtagg
     6481 tccgccgggt ttccagcagg tcgatgtagg tgtcgatgaa gctctggtcc agctggtggt
     6541 aggccttctg caccttcagc agctgtttgg cttctttcac gtagtcgctg gtcttgaatc
     6601 tgttgatgct gccccgcact tcgccgtctt tcttcagccg ttccagctgc agttcggcca
     6661 cgtatttctc ttccagggcc ttgctgttcc ggctgatctg ctctttggtg gacagctcgt
     6721 tgccggtgtc ctcttccacc tcgttcacgt tgtgcacgcc tcttctcttg gccaggtgca
     6781 gcagggcggc agagaactct tcctcgctca gcttctggct caggcccttc actctggcct
     6841 cgtaggggtt gatgccgctc agctcgctgt ggtcggtcag caggttgtag tcgaacagca
     6901 gcttcttcac tctctggatt ctatgccgcc tccgccgctt cagccttctg gcgcctctct
     6961 tgctccgcct gccctcgttg ttttccacgt tggcctcttt gaacagccgc acgccggcat
     7021 cgatcacgtc ccgtgtctcg tagtcgatga tgccgtagcc cacgctggtg atgccgatgt
     7081 ccaggcccag gatgtagttc cgcttggctg ctgggactcc gtggataccg accttccgct
     7141 tcttctttgg ggccatggtg gcaccggtag ttagccagag agctctgctt atatagacct
     7201 cccaccgtac acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg
     7261 gaaagtcccg ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac
     7321 ttggaaatcc ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca
     7381 tcaccatggt aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata
     7441 aggtcat
//
