LOCUS       sequence-292681-        7455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Sa-SauriCas9, and cloning backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7455)
  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 7455)
  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-Sa-SauriCas9
FEATURES             Location/Qualifiers
     source          1..7455
                     /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             3978..4025
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4032..4112
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(4140..4157)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4146..4353
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        4360..4608
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     4360..4380
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     4531..4550
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        4629..4704
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     repeat_region   4718..4858
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4718..4847
                     /label=AAV2 ITR
     rep_origin      4933..5388
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5020..5039)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5230..5251
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5405..5424)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5524..5546
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5584..5602)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5670..5774
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5775..5843,5844..6635)
                     /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(5993..6012)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6806..7394
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7295..7314
                     /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 ccaagcggaa ctacatcctg ggcctggaca
      841 tcggcatcac cagcgtgggc tacggcatca tcgactacga gacacgggac gtgatcgatg
      901 ccggcgtgcg gctgttcaaa gaggccaacg tggaaaacaa cgagggcagg cggagcaaga
      961 gaggcgccag aaggctgaag cggcggaggc ggcatagaat ccagagagtg aagaagctgc
     1021 tgttcgacta caacctgctg accgaccaca gcgagctgag cggcatcaac ccctacgagg
     1081 ccagagtgaa gggcctgagc cagaagctga gcgaggaaga gttctctgcc gccctgctgc
     1141 acctggccaa gagaagaggc gtgcacaacg tgaacgaggt ggaagaggac accggcaacg
     1201 agctgtccac caaagagcag atcagccgga acagcaaggc cctggaagag aaatacgtgg
     1261 ccgaactgca gctggaacgg ctgaagaaag acggcgaagt gcggggcagc atcaacagat
     1321 tcaagaccag cgactacgtg aaagaagcca aacagctgct gaaggtgcag aaggcctacc
     1381 accagctgga ccagagcttc atcgacacct acatcgacct gctggaaacc cggcggacct
     1441 actatgaggg acctggcgag ggcagcccct tcggctggaa ggacatcaaa gaatggtacg
     1501 agatgctgat gggccactgc acctacttcc ccgaggaact gcggagcgtg aagtacgcct
     1561 acaacgccga cctgtacaac gccctgaacg acctgaacaa tctcgtgatc accagggacg
     1621 agaacgagaa gctggaatat tacgagaagt tccagatcat cgagaacgtg ttcaagcaga
     1681 agaagaagcc caccctgaag cagatcgcca aagaaatcct cgtgaacgaa gaggatatta
     1741 agggctacag agtgaccagc accggcaagc ccgagttcac caacctgaag gtgtaccacg
     1801 acatcaagga cattaccgcc cggaaagaga ttattgagaa cgccgagctg ctggatcaga
     1861 ttgccaagat cctgaccatc taccagagca gcgaggacat ccaggaagaa ctgaccaatc
     1921 tgaactccga gctgacccag gaagagatcg agcagatctc taatctgaag ggctataccg
     1981 gcacccacaa cctgagcctg aaggccatca acctgatcct ggacgagctg tggcacacca
     2041 acgacaacca gatcgctatc ttcaaccggc tgaagctggt gcccaagaag gtggacctgt
     2101 cccagcagaa agagatcccc accaccctgg tggacgactt catcctgagc cccgtcgtga
     2161 agagaagctt catccagagc atcaaagtga tcaacgccat catcaagaag tacggcctgc
     2221 ccaacgacat cattatcgag ctggcccgcg agaagaactc caaggacgcc cagaaaatga
     2281 tcaacgagat gcagaagcgg aaccggcaga ccaacgagcg gatcgaggaa atcatccgga
     2341 ccaccggcaa agagaacgcc aagtacctga tcgagaagat caagctgcac gacatgcagg
     2401 aaggcaagtg cctgtacagc ctggaagcca tccctctgga agatctgctg aacaacccct
     2461 tcaactatga ggtggaccac atcatcccca gaagcgtgtc cttcgacaac agcttcaaca
     2521 acaaggtgct cgtgaagcag gaagaaaaca gcaagaaggg caaccggacc ccattccagt
     2581 acctgagcag cagcgacagc aagatcagct acgaaacctt caagaagcac atcctgaatc
     2641 tggccaaggg caagggcaga atcagcaaga ccaagaaaga gtatctgctg gaagaacggg
     2701 acatcaacag gttctccgtg cagaaagact tcatcaaccg gaacctggtg gataccagat
     2761 acgccaccag aggcctgatg aacctgctgc ggagctactt cagagtgaac aacctggacg
     2821 tgaaagtgaa gtccatcaat ggcggcttca ccagctttct gcggcggaag tggaagttta
     2881 agaaagagcg gaacaagggg tacaagcacc acgccgagga cgccctgatc attgccaacg
     2941 ccgatttcat cttcaaagag tggaagaaac tggacaaggc caaaaaagtg atggaaaacc
     3001 agatgttcga ggaaaagcag gccgagagca tgcccgagat cgaaaccgag caggagtaca
     3061 aagagatctt catcaccccc caccagatca agcacattaa ggacttcaag gactacaagt
     3121 acagccaccg ggtggacaag aagcctaata gagagctgat taacgacacc ctgtactcca
     3181 cccggaagga cgacaagggc aacaccctga tcgtgaacaa tctgaacggc ctgtacgaca
     3241 aggacaatga caagctgaaa aagctgatca acaagagccc cgaaaagctg ctgatgtacc
     3301 accacgaccc ccagacctac cagaaactga agctgattat ggaacagtac ggcgacgaga
     3361 agaatcccct gtacaagtac tacgaggaaa ccgggaacta cctgaccaag tactccaaaa
     3421 aggacaacgg ccccgtgatc aagaagatta agtattacgg caacaaactg aacgcccatc
     3481 tggacatcac cgacgactac cccaacagca gaaacaaggt cgtgaagctg tccctgaaga
     3541 gcttccgctt cgacatctac aagtgcgagc agggctacaa gatggtgagc atcggctacc
     3601 tggacgtgct gaagaaggac aactactact acatccccaa ggacaagtac gaggccgaga
     3661 agcagaagaa gaagataaag gagagcgacc tgttcgtggg cagcttctac tacaacgacc
     3721 tgataatgta cgaggacgag ctgttccgcg tgataggcgt gaacagcgac atcaacaacc
     3781 tggtggagct gaacatggtg gacatcacct acaaggactt ctgcgaggtg aacaacgtga
     3841 ccggcgagaa gcgcatcaag aagaccatcg gcaagcgcgt ggtgctgata gagaagtaca
     3901 ccaccgacat cctgggcaac ctgtacaaga cccccctgcc caagaagccc cagctgatat
     3961 tcaagcgcgg cgagctgaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga
     4021 aaaagggatc ctacccatac gatgttccag attacgctta cccatacgat gttccagatt
     4081 acgcttaccc atacgatgtt ccagattacg cttaagaatt cctagagctc gctgatcagc
     4141 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     4201 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     4261 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     4321 ggattgggaa gagaatagca ggcatgctgg ggaggtaccg agggcctatt tcccatgatt
     4381 ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat taatttgact
     4441 gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt tcttgggtag
     4501 tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt aacttgaaag
     4561 tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccgg agaccacggc
     4621 aggtctcagt tttagtactc tggaaacaga atctactaaa acaaggcaaa atgccgtgtt
     4681 tatctcgtca acttgttggc gagatttttg cggccgcagg aacccctagt gatggagttg
     4741 gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga
     4801 cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg
     4861 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa
     4921 agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4981 gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt
     5041 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     5101 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt
     5161 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     5221 tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt
     5281 cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt
     5341 aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc
     5401 tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg
     5461 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     5521 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa
     5581 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     5641 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5701 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5761 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5821 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5881 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5941 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     6001 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     6061 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     6121 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6181 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6241 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6301 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6361 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6421 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6481 gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6541 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6601 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6661 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6721 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6781 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6841 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6901 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6961 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     7021 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     7081 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     7141 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7201 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7261 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7321 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7381 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7441 cttttgctca catgt
//
