LOCUS       sequence-362036-        7448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A single vector containing Cas9 from Staphylococcus aureus (SaCas9) 
            and its sgRNA targeting Adra1b.
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  JOURNAL   Unpublished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            "Unpublished"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-Adra1b
FEATURES             Location/Qualifiers
     source          1..7448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   6..146
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6..135
                     /label=AAV2 ITR
     rep_origin      221..676
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(308..327)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     518..539
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(693..712)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     812..834
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(872..890)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        958..1062
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1063..1131,1132..1923)
                     /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(1281..1300)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2094..2682
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2583..2602
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2744..2873
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        2897..3276
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3277..3480
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3431..3451
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3499..3508
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3511..3531
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3556..6711
                     /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             6712..6759
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6766..6846
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(6874..6891)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6880..7087
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        7094..7342
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     7094..7114
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     7265..7284
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        7365..7440
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
ORIGIN
        1 gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc gctcgctcac
       61 tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag
      121 cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc ttacgcatct
      181 gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca
      241 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta
      301 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
      361 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
      421 cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt
      481 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
      541 acaacactca actctatctc gggctattct tttgatttat aagggatttt gccgatttcg
      601 gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
      661 ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
      721 agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg
      781 gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca
      841 ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt
      901 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
      961 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1021 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1081 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1141 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1201 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1261 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     1321 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1381 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1441 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1501 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     1561 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     1621 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     1681 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     1741 tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca
     1801 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     1861 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     1921 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     1981 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2041 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2101 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2161 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2221 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     2281 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2341 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2401 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2461 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     2521 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     2581 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     2641 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     2701 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag gcagctgcgc
     2761 gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
     2821 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt cctgcggcct
     2881 ctagactcga ggcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     2941 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     3001 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     3061 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     3121 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     3181 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     3241 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     3301 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     3361 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     3421 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     3481 ctctggctaa ctaccggtgc caccatggcc ccaaagaaga agcggaaggt cggtatccac
     3541 ggagtcccag cagccaagcg gaactacatc ctgggcctgg acatcggcat caccagcgtg
     3601 ggctacggca tcatcgacta cgagacacgg gacgtgatcg atgccggcgt gcggctgttc
     3661 aaagaggcca acgtggaaaa caacgagggc aggcggagca agagaggcgc cagaaggctg
     3721 aagcggcgga ggcggcatag aatccagaga gtgaagaagc tgctgttcga ctacaacctg
     3781 ctgaccgacc acagcgagct gagcggcatc aacccctacg aggccagagt gaagggcctg
     3841 agccagaagc tgagcgagga agagttctct gccgccctgc tgcacctggc caagagaaga
     3901 ggcgtgcaca acgtgaacga ggtggaagag gacaccggca acgagctgtc caccaaagag
     3961 cagatcagcc ggaacagcaa ggccctggaa gagaaatacg tggccgaact gcagctggaa
     4021 cggctgaaga aagacggcga agtgcggggc agcatcaaca gattcaagac cagcgactac
     4081 gtgaaagaag ccaaacagct gctgaaggtg cagaaggcct accaccagct ggaccagagc
     4141 ttcatcgaca cctacatcga cctgctggaa acccggcgga cctactatga gggacctggc
     4201 gagggcagcc ccttcggctg gaaggacatc aaagaatggt acgagatgct gatgggccac
     4261 tgcacctact tccccgagga actgcggagc gtgaagtacg cctacaacgc cgacctgtac
     4321 aacgccctga acgacctgaa caatctcgtg atcaccaggg acgagaacga gaagctggaa
     4381 tattacgaga agttccagat catcgagaac gtgttcaagc agaagaagaa gcccaccctg
     4441 aagcagatcg ccaaagaaat cctcgtgaac gaagaggata ttaagggcta cagagtgacc
     4501 agcaccggca agcccgagtt caccaacctg aaggtgtacc acgacatcaa ggacattacc
     4561 gcccggaaag agattattga gaacgccgag ctgctggatc agattgccaa gatcctgacc
     4621 atctaccaga gcagcgagga catccaggaa gaactgacca atctgaactc cgagctgacc
     4681 caggaagaga tcgagcagat ctctaatctg aagggctata ccggcaccca caacctgagc
     4741 ctgaaggcca tcaacctgat cctggacgag ctgtggcaca ccaacgacaa ccagatcgct
     4801 atcttcaacc ggctgaagct ggtgcccaag aaggtggacc tgtcccagca gaaagagatc
     4861 cccaccaccc tggtggacga cttcatcctg agccccgtcg tgaagagaag cttcatccag
     4921 agcatcaaag tgatcaacgc catcatcaag aagtacggcc tgcccaacga catcattatc
     4981 gagctggccc gcgagaagaa ctccaaggac gcccagaaaa tgatcaacga gatgcagaag
     5041 cggaaccggc agaccaacga gcggatcgag gaaatcatcc ggaccaccgg caaagagaac
     5101 gccaagtacc tgatcgagaa gatcaagctg cacgacatgc aggaaggcaa gtgcctgtac
     5161 agcctggaag ccatccctct ggaagatctg ctgaacaacc ccttcaacta tgaggtggac
     5221 cacatcatcc ccagaagcgt gtccttcgac aacagcttca acaacaaggt gctcgtgaag
     5281 caggaagaaa acagcaagaa gggcaaccgg accccattcc agtacctgag cagcagcgac
     5341 agcaagatca gctacgaaac cttcaagaag cacatcctga atctggccaa gggcaagggc
     5401 agaatcagca agaccaagaa agagtatctg ctggaagaac gggacatcaa caggttctcc
     5461 gtgcagaaag acttcatcaa ccggaacctg gtggatacca gatacgccac cagaggcctg
     5521 atgaacctgc tgcggagcta cttcagagtg aacaacctgg acgtgaaagt gaagtccatc
     5581 aatggcggct tcaccagctt tctgcggcgg aagtggaagt ttaagaaaga gcggaacaag
     5641 gggtacaagc accacgccga ggacgccctg atcattgcca acgccgattt catcttcaaa
     5701 gagtggaaga aactggacaa ggccaaaaaa gtgatggaaa accagatgtt cgaggaaaag
     5761 caggccgaga gcatgcccga gatcgaaacc gagcaggagt acaaagagat cttcatcacc
     5821 ccccaccaga tcaagcacat taaggacttc aaggactaca agtacagcca ccgggtggac
     5881 aagaagccta atagagagct gattaacgac accctgtact ccacccggaa ggacgacaag
     5941 ggcaacaccc tgatcgtgaa caatctgaac ggcctgtacg acaaggacaa tgacaagctg
     6001 aaaaagctga tcaacaagag ccccgaaaag ctgctgatgt accaccacga cccccagacc
     6061 taccagaaac tgaagctgat tatggaacag tacggcgacg agaagaatcc cctgtacaag
     6121 tactacgagg aaaccgggaa ctacctgacc aagtactcca aaaaggacaa cggccccgtg
     6181 atcaagaaga ttaagtatta cggcaacaaa ctgaacgccc atctggacat caccgacgac
     6241 taccccaaca gcagaaacaa ggtcgtgaag ctgtccctga agccctacag attcgacgtg
     6301 tacctggaca atggcgtgta caagttcgtg accgtgaaga atctggatgt gatcaaaaaa
     6361 gaaaactact acgaagtgaa tagcaagtgc tatgaggaag ctaagaagct gaagaagatc
     6421 agcaaccagg ccgagtttat cgcctccttc tacaacaacg atctgatcaa gatcaacggc
     6481 gagctgtata gagtgatcgg cgtgaacaac gacctgctga accggatcga agtgaacatg
     6541 atcgacatca cctaccgcga gtacctggaa aacatgaacg acaagaggcc ccccaggatc
     6601 attaagacaa tcgcctccaa gacccagagc attaagaagt acagcacaga cattctgggc
     6661 aacctgtatg aagtgaaatc taagaagcac cctcagatca tcaaaaaggg caaaaggccg
     6721 gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg gatcctaccc atacgatgtt
     6781 ccagattacg cttacccata cgatgttcca gattacgctt acccatacga tgttccagat
     6841 tacgcttaag aattcctaga gctcgctgat cagcctcgac tgtgccttct agttgccagc
     6901 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     6961 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     7021 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat agcaggcatg
     7081 ctggggaggt accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     7141 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     7201 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     7261 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     7321 cttgtggaaa ggacgaaaca ccgcatcagc acctgcccac tggggtttta gtactctgga
     7381 aacagaatct actaaaacaa ggcaaaatgc cgtgtttatc tcgtcaactt gttggcgaga
     7441 tttttgcg
//
