LOCUS       sequence-362043-        7447 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A single vector containing Cas9 from Staphylococcus aureus (SaCas9) 
            and its sgRNA targeting Rxfp3.
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  JOURNAL   Unpublished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            "Unpublished"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-Rxfp3
FEATURES             Location/Qualifiers
     source          1..7447
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..175
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(329..348)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     448..470
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(508..526)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        594..698
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(699..767,768..1559)
                     /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(917..936)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1730..2318
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2219..2238
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2380..2509
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        2533..2912
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2913..3116
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3067..3087
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3135..3144
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3147..3167
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3192..6347
                     /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             6348..6395
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6402..6482
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(6510..6527)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6516..6723
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        6730..6978
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6730..6750
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6901..6920
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7000..7075
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   7089..7229
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7089..7218
                     /label=AAV2 ITR
     rep_origin      join(7304..7447,1..312)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7391..7410)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
       61 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
      121 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
      181 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
      241 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
      301 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
      361 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
      421 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
      481 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
      541 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
      601 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      661 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
      721 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
      781 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
      841 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
      901 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
      961 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     1021 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     1081 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     1141 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     1201 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     1261 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     1321 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     1381 ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt gcagcactgg
     1441 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     1501 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     1561 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1621 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1681 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1741 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1801 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1861 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1921 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     1981 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     2041 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     2101 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     2161 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     2221 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     2281 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     2341 tacggttcct ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc
     2401 gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
     2461 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg cggcctctag
     2521 actcgaggcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     2581 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     2641 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     2701 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     2761 tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta
     2821 aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt
     2881 acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
     2941 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     3001 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc
     3061 cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct
     3121 ggctaactac cggtgccacc atggccccaa agaagaagcg gaaggtcggt atccacggag
     3181 tcccagcagc caagcggaac tacatcctgg gcctggacat cggcatcacc agcgtgggct
     3241 acggcatcat cgactacgag acacgggacg tgatcgatgc cggcgtgcgg ctgttcaaag
     3301 aggccaacgt ggaaaacaac gagggcaggc ggagcaagag aggcgccaga aggctgaagc
     3361 ggcggaggcg gcatagaatc cagagagtga agaagctgct gttcgactac aacctgctga
     3421 ccgaccacag cgagctgagc ggcatcaacc cctacgaggc cagagtgaag ggcctgagcc
     3481 agaagctgag cgaggaagag ttctctgccg ccctgctgca cctggccaag agaagaggcg
     3541 tgcacaacgt gaacgaggtg gaagaggaca ccggcaacga gctgtccacc aaagagcaga
     3601 tcagccggaa cagcaaggcc ctggaagaga aatacgtggc cgaactgcag ctggaacggc
     3661 tgaagaaaga cggcgaagtg cggggcagca tcaacagatt caagaccagc gactacgtga
     3721 aagaagccaa acagctgctg aaggtgcaga aggcctacca ccagctggac cagagcttca
     3781 tcgacaccta catcgacctg ctggaaaccc ggcggaccta ctatgaggga cctggcgagg
     3841 gcagcccctt cggctggaag gacatcaaag aatggtacga gatgctgatg ggccactgca
     3901 cctacttccc cgaggaactg cggagcgtga agtacgccta caacgccgac ctgtacaacg
     3961 ccctgaacga cctgaacaat ctcgtgatca ccagggacga gaacgagaag ctggaatatt
     4021 acgagaagtt ccagatcatc gagaacgtgt tcaagcagaa gaagaagccc accctgaagc
     4081 agatcgccaa agaaatcctc gtgaacgaag aggatattaa gggctacaga gtgaccagca
     4141 ccggcaagcc cgagttcacc aacctgaagg tgtaccacga catcaaggac attaccgccc
     4201 ggaaagagat tattgagaac gccgagctgc tggatcagat tgccaagatc ctgaccatct
     4261 accagagcag cgaggacatc caggaagaac tgaccaatct gaactccgag ctgacccagg
     4321 aagagatcga gcagatctct aatctgaagg gctataccgg cacccacaac ctgagcctga
     4381 aggccatcaa cctgatcctg gacgagctgt ggcacaccaa cgacaaccag atcgctatct
     4441 tcaaccggct gaagctggtg cccaagaagg tggacctgtc ccagcagaaa gagatcccca
     4501 ccaccctggt ggacgacttc atcctgagcc ccgtcgtgaa gagaagcttc atccagagca
     4561 tcaaagtgat caacgccatc atcaagaagt acggcctgcc caacgacatc attatcgagc
     4621 tggcccgcga gaagaactcc aaggacgccc agaaaatgat caacgagatg cagaagcgga
     4681 accggcagac caacgagcgg atcgaggaaa tcatccggac caccggcaaa gagaacgcca
     4741 agtacctgat cgagaagatc aagctgcacg acatgcagga aggcaagtgc ctgtacagcc
     4801 tggaagccat ccctctggaa gatctgctga acaacccctt caactatgag gtggaccaca
     4861 tcatccccag aagcgtgtcc ttcgacaaca gcttcaacaa caaggtgctc gtgaagcagg
     4921 aagaaaacag caagaagggc aaccggaccc cattccagta cctgagcagc agcgacagca
     4981 agatcagcta cgaaaccttc aagaagcaca tcctgaatct ggccaagggc aagggcagaa
     5041 tcagcaagac caagaaagag tatctgctgg aagaacggga catcaacagg ttctccgtgc
     5101 agaaagactt catcaaccgg aacctggtgg ataccagata cgccaccaga ggcctgatga
     5161 acctgctgcg gagctacttc agagtgaaca acctggacgt gaaagtgaag tccatcaatg
     5221 gcggcttcac cagctttctg cggcggaagt ggaagtttaa gaaagagcgg aacaaggggt
     5281 acaagcacca cgccgaggac gccctgatca ttgccaacgc cgatttcatc ttcaaagagt
     5341 ggaagaaact ggacaaggcc aaaaaagtga tggaaaacca gatgttcgag gaaaagcagg
     5401 ccgagagcat gcccgagatc gaaaccgagc aggagtacaa agagatcttc atcacccccc
     5461 accagatcaa gcacattaag gacttcaagg actacaagta cagccaccgg gtggacaaga
     5521 agcctaatag agagctgatt aacgacaccc tgtactccac ccggaaggac gacaagggca
     5581 acaccctgat cgtgaacaat ctgaacggcc tgtacgacaa ggacaatgac aagctgaaaa
     5641 agctgatcaa caagagcccc gaaaagctgc tgatgtacca ccacgacccc cagacctacc
     5701 agaaactgaa gctgattatg gaacagtacg gcgacgagaa gaatcccctg tacaagtact
     5761 acgaggaaac cgggaactac ctgaccaagt actccaaaaa ggacaacggc cccgtgatca
     5821 agaagattaa gtattacggc aacaaactga acgcccatct ggacatcacc gacgactacc
     5881 ccaacagcag aaacaaggtc gtgaagctgt ccctgaagcc ctacagattc gacgtgtacc
     5941 tggacaatgg cgtgtacaag ttcgtgaccg tgaagaatct ggatgtgatc aaaaaagaaa
     6001 actactacga agtgaatagc aagtgctatg aggaagctaa gaagctgaag aagatcagca
     6061 accaggccga gtttatcgcc tccttctaca acaacgatct gatcaagatc aacggcgagc
     6121 tgtatagagt gatcggcgtg aacaacgacc tgctgaaccg gatcgaagtg aacatgatcg
     6181 acatcaccta ccgcgagtac ctggaaaaca tgaacgacaa gaggcccccc aggatcatta
     6241 agacaatcgc ctccaagacc cagagcatta agaagtacag cacagacatt ctgggcaacc
     6301 tgtatgaagt gaaatctaag aagcaccctc agatcatcaa aaagggcaaa aggccggcgg
     6361 ccacgaaaaa ggccggccag gcaaaaaaga aaaagggatc ctacccatac gatgttccag
     6421 attacgctta cccatacgat gttccagatt acgcttaccc atacgatgtt ccagattacg
     6481 cttaagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     6541 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     6601 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     6661 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg
     6721 ggaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     6781 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     6841 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     6901 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     6961 tggaaaggac gaaacaccgt tggccacttc cagcaagtcg ttttagtact ctggaaacag
     7021 aatctactaa aacaaggcaa aatgccgtgt ttatctcgtc aacttgttgg cgagattttt
     7081 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     7141 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     7201 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     7261 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     7321 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     7381 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     7441 cgtcaag
//
