LOCUS       sequence-199067-        7089 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A human codon-optimized Cas9 from Staphylococcus aureus (SaCas9)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7089)
  AUTHORS   Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B,
            Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F
  TITLE     In vivo genome editing using Staphylococcus aureus Cas9.
  JOURNAL   Nature. 2015 Apr 1. doi: 10.1038/nature14299.
  PUBMED    25830891
REFERENCE   2  (bases 1 to 7089)
  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: "Nature. 
            2015 Apr 1. doi: 10.1038/nature14299."
COMMENT     Sequence Label: pX600-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA
FEATURES             Location/Qualifiers
     source          1..7089
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   16..156
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   16..145
                     /label=AAV2 ITR
     rep_origin      231..686
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(318..337)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     528..549
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(703..722)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     822..844
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(882..900)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        968..1072
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1073..1141,1142..1933)
                     /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(1291..1310)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2104..2692
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2593..2612
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   2754..2883
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        2907..3286
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3287..3490
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3441..3461
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3509..3518
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3521..3541
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3566..6721
                     /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             6722..6769
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6776..6856
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(6884..6901)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    join(6890..7089,1..8)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 gctggggagg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
       61 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
      121 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
      181 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
      241 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
      301 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
      361 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
      421 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
      481 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
      541 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
      601 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
      661 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
      721 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
      781 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
      841 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
      901 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
      961 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     1021 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     1081 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     1141 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     1201 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     1261 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     1321 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     1381 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     1441 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     1501 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     1561 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     1621 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     1681 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     1741 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtggaa gccgcggtat
     1801 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     1861 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     1921 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     1981 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     2041 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2101 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2161 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2221 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2281 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2341 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2401 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     2461 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     2521 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     2581 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     2641 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     2701 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtcctgcag
     2761 gcagctgcgc gctcgctcgc tcactgaggc cgcccgggcg tcgggcgacc tttggtcgcc
     2821 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt
     2881 cctgcggcct ctagactcga ggcgttgaca ttgattattg actagttatt aatagtaatc
     2941 aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt
     3001 aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta
     3061 tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg
     3121 gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga
     3181 cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt
     3241 tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg
     3301 gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc
     3361 cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg
     3421 taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat
     3481 aagcagagct ctctggctaa ctaccggtgc caccatggcc ccaaagaaga agcggaaggt
     3541 cggtatccac ggagtcccag cagccaagcg gaactacatc ctgggcctgg acatcggcat
     3601 caccagcgtg ggctacggca tcatcgacta cgagacacgg gacgtgatcg atgccggcgt
     3661 gcggctgttc aaagaggcca acgtggaaaa caacgagggc aggcggagca agagaggcgc
     3721 cagaaggctg aagcggcgga ggcggcatag aatccagaga gtgaagaagc tgctgttcga
     3781 ctacaacctg ctgaccgacc acagcgagct gagcggcatc aacccctacg aggccagagt
     3841 gaagggcctg agccagaagc tgagcgagga agagttctct gccgccctgc tgcacctggc
     3901 caagagaaga ggcgtgcaca acgtgaacga ggtggaagag gacaccggca acgagctgtc
     3961 caccaaagag cagatcagcc ggaacagcaa ggccctggaa gagaaatacg tggccgaact
     4021 gcagctggaa cggctgaaga aagacggcga agtgcggggc agcatcaaca gattcaagac
     4081 cagcgactac gtgaaagaag ccaaacagct gctgaaggtg cagaaggcct accaccagct
     4141 ggaccagagc ttcatcgaca cctacatcga cctgctggaa acccggcgga cctactatga
     4201 gggacctggc gagggcagcc ccttcggctg gaaggacatc aaagaatggt acgagatgct
     4261 gatgggccac tgcacctact tccccgagga actgcggagc gtgaagtacg cctacaacgc
     4321 cgacctgtac aacgccctga acgacctgaa caatctcgtg atcaccaggg acgagaacga
     4381 gaagctggaa tattacgaga agttccagat catcgagaac gtgttcaagc agaagaagaa
     4441 gcccaccctg aagcagatcg ccaaagaaat cctcgtgaac gaagaggata ttaagggcta
     4501 cagagtgacc agcaccggca agcccgagtt caccaacctg aaggtgtacc acgacatcaa
     4561 ggacattacc gcccggaaag agattattga gaacgccgag ctgctggatc agattgccaa
     4621 gatcctgacc atctaccaga gcagcgagga catccaggaa gaactgacca atctgaactc
     4681 cgagctgacc caggaagaga tcgagcagat ctctaatctg aagggctata ccggcaccca
     4741 caacctgagc ctgaaggcca tcaacctgat cctggacgag ctgtggcaca ccaacgacaa
     4801 ccagatcgct atcttcaacc ggctgaagct ggtgcccaag aaggtggacc tgtcccagca
     4861 gaaagagatc cccaccaccc tggtggacga cttcatcctg agccccgtcg tgaagagaag
     4921 cttcatccag agcatcaaag tgatcaacgc catcatcaag aagtacggcc tgcccaacga
     4981 catcattatc gagctggccc gcgagaagaa ctccaaggac gcccagaaaa tgatcaacga
     5041 gatgcagaag cggaaccggc agaccaacga gcggatcgag gaaatcatcc ggaccaccgg
     5101 caaagagaac gccaagtacc tgatcgagaa gatcaagctg cacgacatgc aggaaggcaa
     5161 gtgcctgtac agcctggaag ccatccctct ggaagatctg ctgaacaacc ccttcaacta
     5221 tgaggtggac cacatcatcc ccagaagcgt gtccttcgac aacagcttca acaacaaggt
     5281 gctcgtgaag caggaagaaa acagcaagaa gggcaaccgg accccattcc agtacctgag
     5341 cagcagcgac agcaagatca gctacgaaac cttcaagaag cacatcctga atctggccaa
     5401 gggcaagggc agaatcagca agaccaagaa agagtatctg ctggaagaac gggacatcaa
     5461 caggttctcc gtgcagaaag acttcatcaa ccggaacctg gtggatacca gatacgccac
     5521 cagaggcctg atgaacctgc tgcggagcta cttcagagtg aacaacctgg acgtgaaagt
     5581 gaagtccatc aatggcggct tcaccagctt tctgcggcgg aagtggaagt ttaagaaaga
     5641 gcggaacaag gggtacaagc accacgccga ggacgccctg atcattgcca acgccgattt
     5701 catcttcaaa gagtggaaga aactggacaa ggccaaaaaa gtgatggaaa accagatgtt
     5761 cgaggaaaag caggccgaga gcatgcccga gatcgaaacc gagcaggagt acaaagagat
     5821 cttcatcacc ccccaccaga tcaagcacat taaggacttc aaggactaca agtacagcca
     5881 ccgggtggac aagaagccta atagagagct gattaacgac accctgtact ccacccggaa
     5941 ggacgacaag ggcaacaccc tgatcgtgaa caatctgaac ggcctgtacg acaaggacaa
     6001 tgacaagctg aaaaagctga tcaacaagag ccccgaaaag ctgctgatgt accaccacga
     6061 cccccagacc taccagaaac tgaagctgat tatggaacag tacggcgacg agaagaatcc
     6121 cctgtacaag tactacgagg aaaccgggaa ctacctgacc aagtactcca aaaaggacaa
     6181 cggccccgtg atcaagaaga ttaagtatta cggcaacaaa ctgaacgccc atctggacat
     6241 caccgacgac taccccaaca gcagaaacaa ggtcgtgaag ctgtccctga agccctacag
     6301 attcgacgtg tacctggaca atggcgtgta caagttcgtg accgtgaaga atctggatgt
     6361 gatcaaaaaa gaaaactact acgaagtgaa tagcaagtgc tatgaggaag ctaagaagct
     6421 gaagaagatc agcaaccagg ccgagtttat cgcctccttc tacaacaacg atctgatcaa
     6481 gatcaacggc gagctgtata gagtgatcgg cgtgaacaac gacctgctga accggatcga
     6541 agtgaacatg atcgacatca cctaccgcga gtacctggaa aacatgaacg acaagaggcc
     6601 ccccaggatc attaagacaa tcgcctccaa gacccagagc attaagaagt acagcacaga
     6661 cattctgggc aacctgtatg aagtgaaatc taagaagcac cctcagatca tcaaaaaggg
     6721 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg gatcctaccc
     6781 atacgatgtt ccagattacg cttacccata cgatgttcca gattacgctt acccatacga
     6841 tgttccagat tacgcttaag aattcctaga gctcgctgat cagcctcgac tgtgccttct
     6901 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     6961 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     7021 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     7081 agcaggcat
//
