LOCUS       sequence-287959-        7470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SauriCas9, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7470)
  AUTHORS   Hu Z, Wang S, Zhang C, Gao N, Li M, Wang D, Wang D, Liu D, Liu H, 
            Ong SG, Wang H, Wang Y
  TITLE     A compact Cas9 ortholog from Staphylococcus Auricularis (SauriCas9) 
            expands the DNA targeting scope.
  JOURNAL   PLoS Biol. 2020 Mar 30;18(3):e3000686. doi: 
            10.1371/journal.pbio.3000686. eCollection 2020 Mar.
  PUBMED    32226015
REFERENCE   2  (bases 1 to 7470)
  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: "PLoS 
            Biol."; date: "2020-03-30"; volume: "18(3):e3000686. doi"; pages: " 
            10.1371/journal.pbio.3000686. eCollection 2020 Mar"
COMMENT     Sequence Label: pAAV-CMV-SauriCas9
FEATURES             Location/Qualifiers
     source          1..7470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        154..533
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        534..737
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     688..708
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      756..765
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             768..788
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3993..4040
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4047..4127
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4155..4172)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4161..4368
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4375..4623
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4375..4395
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4546..4565
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4644..4719
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4733..4873
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4733..4862
                     /label=AAV2 ITR
     rep_origin      4948..5403
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5035..5054)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5245..5266
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5420..5439)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5539..5561
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5599..5617)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5685..5789
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5790..5858,5859..6650)
                     /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(6008..6027)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6821..7409
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7310..7329
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggcctcta gactcgaggc gttgacattg attattgact agttattaat
      181 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
      241 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
      301 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
      361 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
      421 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
      481 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc
      541 ggttttggca gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc
      601 tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa
      661 aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg
      721 tctatataag cagagctctc tggctaacta ccggtgccac catggcccca aagaagaagc
      781 ggaaggtcgg tatccacgga gtcccagcag cccaggagaa ccagcagaag cagaactaca
      841 tcctgggcct ggacatcgga atcaccagcg tcggctacgg actgatcgat agcaagacaa
      901 gagaagtgat cgacgccggc gttagactct ttccagaagc tgatagcgag aacaactcca
      961 accgcagaag caagcggggc gccagacggt taaaacggag aagaatccac cggctgaacc
     1021 gggtcaaaga cctgctcgct gattaccaga tgatcgatct taacaatgtt cctaagagca
     1081 ccgaccccta caccatcaga gtgaagggcc tccgggagcc tctgacaaaa gaagaattcg
     1141 ccatcgccct cctgcatatc gctaagagaa gaggcctgca caacatcagt gtgtccatgg
     1201 gcgacgaaga gcaggacaat gaactgagca ccaagcagca gctgcaaaag aatgcccagc
     1261 aactgcagga caagtatgtg tgcgaactgc agttagaacg gctgaccaac atcaacaagg
     1321 tcagaggcga gaagaacaga tttaagacag aggactttgt gaaagaagtg aaacagctgt
     1381 gcgaaaccca gagacagtac cacaacatcg acgaccaatt catccagcag tacatcgacc
     1441 tggtgtctac aagacgggag tacttcgagg gccccggcaa cggctctcca tacggctggg
     1501 acggcgacct gctgaagtgg tacgagaagc tgatgggcag atgcacctat ttccccgaag
     1561 aactgaggtc cgtgaagtac gcctacagcg ccgacctctt caacgccctg aacgacctga
     1621 acaacctcgt tgtgaccagg gatgacaatc caaagcttga gtactacgag aagtaccaca
     1681 ttattgagaa cgtgttcaag caaaagaaga atcccacact caaacaaatc gccaaagaga
     1741 tcggcgtgca agattacgac atccggggct atagaatcac aaagagcggc aaacctcagt
     1801 tcacctcttt taagctgtat cacgacctga agaacatctt cgagcaggcc aaatacctgg
     1861 aagatgtgga aatgctggac gagatcgcca agatcctgac catctaccag gatgagatta
     1921 gcatcaagaa agccctggac cagctgcccg aactgctgac agagagcgag aaatctcaga
     1981 tcgcacagct caccggctat acaggcaccc acagactgag cctgaagtgc atccacattg
     2041 tgatcgacga gctgtgggag agccccgaga accagatgga aatctttacc agactgaatc
     2101 tgaaacctaa gaaggtggaa atgagcgaga tcgacagcat acccaccacc ctggtcgacg
     2161 agttcatcct ctcacctgtg gtgaagcggg ccttcatcca gagcatcaag gtaatcaacg
     2221 cagtgatcaa tcggttcggc ctgccagagg acatcatcat cgagctggcc agagaaaaga
     2281 atagcaagga tcggagaaag ttcattaaca agctgcagaa acaaaatgag gccacaagaa
     2341 agaaaatcga acagctgctg gccaagtacg gcaacaccaa tgccaagtac atgatcgaga
     2401 agatcaagct gcacgacatg caggagggca agtgcctgta cagcctggag gctattcctc
     2461 tggaagacct gctgagcaac ccgacacact acgaagttga ccacattatc cccagatctg
     2521 tgagctttga caacagcctg aacaacaaag tgctggtgaa acaaagcgaa aacagcaaga
     2581 agggcaatcg caccccttac cagtacctga gcagcaacga gtctaagatt agctacaacc
     2641 agtttaagca gcacatcctg aacctgagca aggccaagga cagaatcagc aagaaaaaaa
     2701 gagatatgct gctggaagag agagatatca acaagttcga agtgcagaag gaattcatta
     2761 accggaacct ggtggataca cggtacgcca ccagagaact gtctaacctg ctgaagacct
     2821 acttcagcac ccatgactac gccgtgaagg tgaagaccat caacggcggc ttcactaacc
     2881 acctgaggaa ggtgtgggat ttcaagaagc acagaaacca cggctacaag caccacgccg
     2941 aagatgccct ggtgatcgcc aacgccgact tcctgtttaa gacacataag gccctgcgga
     3001 gaaccgataa gatcctggaa caacctggcc tggaagtgaa tgatacaacc gtgaaagtgg
     3061 acaccgagga aaaataccag gagctgttcg agacacctaa gcaagtgaag aacatcaagc
     3121 agttccggga cttcaagtac agccaccgag tggacaagaa gcctaaccgg cagcttatca
     3181 acgacacact gtactccacc agagagattg atggcgaaac ctacgtggtg cagaccctta
     3241 aggatctgta cgccaaggac aacgagaaag tgaagaagct gttcaccgaa agacctcaga
     3301 agatcctgat gtaccagcac gaccctaaga ccttcgagaa actgatgaca atcctgaacc
     3361 agtacgctga ggccaagaac cctctggctg cttattacga ggacaaaggc gagtacgtga
     3421 ccaagtacgc caagaaaggc aatggacctg ccatccacaa gatcaagtat atcgataaga
     3481 agcttggatc ttacctggat gttagcaaca agtatcctga gacacagaac aagcttgtga
     3541 agctgtccct gaagagcttt agattcgaca tctacaagtg tgaacagggc tacaagatgg
     3601 tgtccatcgg atacctggac gtgctgaaga aagataacta ctactacatc cctaaggaca
     3661 agtacgaggc cgagaagcag aaaaagaaga tcaaggaatc tgatcttttt gtgggcagct
     3721 tctactacaa cgacctcatc atgtacgagg atgaactgtt cagagtgata ggagtgaaca
     3781 gcgacatcaa caatctggtt gagctaaaca tggtcgacat tacctacaag gacttctgcg
     3841 aggtgaacaa cgtgacaggc gagaaaagaa tcaaaaagac tatcggcaag cgcgtggtcc
     3901 tgatcgagaa gtacaccaca gatattctag gcaacctgta caagactccc ctgcctaaga
     3961 agccccagct tatcttcaag cggggagaac tgaaaaggcc ggcggccacg aaaaaggccg
     4021 gccaggcaaa aaagaaaaag ggatcctacc catacgatgt tccagattac gcttacccat
     4081 acgatgttcc agattacgct tacccatacg atgttccaga ttacgcttaa gaattcctag
     4141 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     4201 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     4261 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     4321 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagg taccgagggc
     4381 ctatttccca tgattccttc atatttgcat atacgataca aggctgttag agagataatt
     4441 ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa
     4501 taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt
     4561 accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac
     4621 accggagacc acggcaggtc tcagttttag tactctggaa acagaatcta ctaaaacaag
     4681 gcaaaatgcc gtgtttatct cgtcaacttg ttggcgagat ttttgcggcc gcaggaaccc
     4741 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga
     4801 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc
     4861 agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     4921 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     4981 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccttagcg cccgctcctt
     5041 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5101 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5161 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5221 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaact
     5281 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggtc tattggttaa
     5341 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     5401 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     5461 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5521 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5581 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5641 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5701 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     5761 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     5821 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     5881 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     5941 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6001 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6061 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6121 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6181 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6241 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6301 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6361 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6421 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6481 ataaatctgg agccggtgag cgtggaagcc gcggtatcat tgcagcactg gggccagatg
     6541 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6601 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6661 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6721 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     6781 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     6841 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     6901 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     6961 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7021 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     7081 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     7141 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7201 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7261 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7321 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7381 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7441 tggccttttg ctggcctttt gctcacatgt
//
