LOCUS       sequence-100380-        7101 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A catalytically inactive SaCas9 (dSaCas9) with the D10A and N580A 
            mutations. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7101)
  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 7101)
  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: 
            pX603-AAV-CMV::NLS-dSaCas9(D10A,N580A)-NLS-3xHA-bGHpA
FEATURES             Location/Qualifiers
     source          1..7101
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(62..650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(142..161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(821..1612,1613..1681))
                     /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     1444..1463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1682..1786)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1854..1872
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(1910..1932)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2032..2051
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(2068..2523)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2205..2226)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2417..2436
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   2598..2738
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2609..2738
                     /label=AAV2 ITR
     enhancer        2758..3137
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3138..3341
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3292..3312
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      3360..3369
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3372..3392
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6573..6620
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             6627..6707
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(6735..6752)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6741..6948
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   6961..7101
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6961..7090
                     /label=AAV2 ITR
ORIGIN
        1 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
       61 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
      121 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
      181 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
      241 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
      301 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
      361 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
      421 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
      481 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
      541 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
      601 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
      661 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
      721 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
      781 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
      841 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
      901 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
      961 ggcttccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     1021 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     1081 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     1141 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     1201 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     1261 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     1321 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     1381 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     1441 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     1501 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     1561 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     1621 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     1681 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     1741 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     1801 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     1861 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     1921 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     1981 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     2041 agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg
     2101 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     2161 cttataaatc aaaagaatag cccgagatag ggttgagtgt tgttccagtt tggaacaaga
     2221 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     2281 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag
     2341 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     2401 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     2461 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     2521 cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa
     2581 ataccgcatc aggcgcccct gcaggcagct gcgcgctcgc tcgctcactg aggccgcccg
     2641 ggcaaagccc gggcgtcggg cgacctttgg tcgcccggcc tcagtgagcg agcgagcgcg
     2701 cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcctcg aggcgttgac
     2761 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
     2821 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
     2881 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
     2941 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
     3001 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
     3061 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
     3121 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
     3181 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
     3241 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
     3301 gcggtaggcg tgtacggtgg gaggtctata taagcagagc tctctggcta actaccggtg
     3361 ccaccatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccaagc
     3421 ggaactacat cctgggcctg gccatcggca tcaccagcgt gggctacggc atcatcgact
     3481 acgagacacg ggacgtgatc gatgccggcg tgcggctgtt caaagaggcc aacgtggaaa
     3541 acaacgaggg caggcggagc aagagaggcg ccagaaggct gaagcggcgg aggcggcata
     3601 gaatccagag agtgaagaag ctgctgttcg actacaacct gctgaccgac cacagcgagc
     3661 tgagcggcat caacccctac gaggccagag tgaagggcct gagccagaag ctgagcgagg
     3721 aagagttctc tgccgccctg ctgcacctgg ccaagagaag aggcgtgcac aacgtgaacg
     3781 aggtggaaga ggacaccggc aacgagctgt ccaccaaaga gcagatcagc cggaacagca
     3841 aggccctgga agagaaatac gtggccgaac tgcagctgga acggctgaag aaagacggcg
     3901 aagtgcgggg cagcatcaac agattcaaga ccagcgacta cgtgaaagaa gccaaacagc
     3961 tgctgaaggt gcagaaggcc taccaccagc tggaccagag cttcatcgac acctacatcg
     4021 acctgctgga aacccggcgg acctactatg agggacctgg cgagggcagc cccttcggct
     4081 ggaaggacat caaagaatgg tacgagatgc tgatgggcca ctgcacctac ttccccgagg
     4141 aactgcggag cgtgaagtac gcctacaacg ccgacctgta caacgccctg aacgacctga
     4201 acaatctcgt gatcaccagg gacgagaacg agaagctgga atattacgag aagttccaga
     4261 tcatcgagaa cgtgttcaag cagaagaaga agcccaccct gaagcagatc gccaaagaaa
     4321 tcctcgtgaa cgaagaggat attaagggct acagagtgac cagcaccggc aagcccgagt
     4381 tcaccaacct gaaggtgtac cacgacatca aggacattac cgcccggaaa gagattattg
     4441 agaacgccga gctgctggat cagattgcca agatcctgac catctaccag agcagcgagg
     4501 acatccagga agaactgacc aatctgaact ccgagctgac ccaggaagag atcgagcaga
     4561 tctctaatct gaagggctat accggcaccc acaacctgag cctgaaggcc atcaacctga
     4621 tcctggacga gctgtggcac accaacgaca accagatcgc tatcttcaac cggctgaagc
     4681 tggtgcccaa gaaggtggac ctgtcccagc agaaagagat ccccaccacc ctggtggacg
     4741 acttcatcct gagccccgtc gtgaagagaa gcttcatcca gagcatcaaa gtgatcaacg
     4801 ccatcatcaa gaagtacggc ctgcccaacg acatcattat cgagctggcc cgcgagaaga
     4861 actccaagga cgcccagaaa atgatcaacg agatgcagaa gcggaaccgg cagaccaacg
     4921 agcggatcga ggaaatcatc cggaccaccg gcaaagagaa cgccaagtac ctgatcgaga
     4981 agatcaagct gcacgacatg caggaaggca agtgcctgta cagcctggaa gccatccctc
     5041 tggaagatct gctgaacaac cccttcaact atgaggtgga ccacatcatc cccagaagcg
     5101 tgtccttcga caacagcttc aacaacaagg tgctcgtgaa gcaggaagaa gccagcaaga
     5161 agggcaaccg gaccccattc cagtacctga gcagcagcga cagcaagatc agctacgaaa
     5221 ccttcaagaa gcacatcctg aatctggcca agggcaaggg cagaatcagc aagaccaaga
     5281 aagagtatct gctggaagaa cgggacatca acaggttctc cgtgcagaaa gacttcatca
     5341 accggaacct ggtggatacc agatacgcca ccagaggcct gatgaacctg ctgcggagct
     5401 acttcagagt gaacaacctg gacgtgaaag tgaagtccat caatggcggc ttcaccagct
     5461 ttctgcggcg gaagtggaag tttaagaaag agcggaacaa ggggtacaag caccacgccg
     5521 aggacgccct gatcattgcc aacgccgatt tcatcttcaa agagtggaag aaactggaca
     5581 aggccaaaaa agtgatggaa aaccagatgt tcgaggaaaa gcaggccgag agcatgcccg
     5641 agatcgaaac cgagcaggag tacaaagaga tcttcatcac cccccaccag atcaagcaca
     5701 ttaaggactt caaggactac aagtacagcc accgggtgga caagaagcct aatagagagc
     5761 tgattaacga caccctgtac tccacccgga aggacgacaa gggcaacacc ctgatcgtga
     5821 acaatctgaa cggcctgtac gacaaggaca atgacaagct gaaaaagctg atcaacaaga
     5881 gccccgaaaa gctgctgatg taccaccacg acccccagac ctaccagaaa ctgaagctga
     5941 ttatggaaca gtacggcgac gagaagaatc ccctgtacaa gtactacgag gaaaccggga
     6001 actacctgac caagtactcc aaaaaggaca acggccccgt gatcaagaag attaagtatt
     6061 acggcaacaa actgaacgcc catctggaca tcaccgacga ctaccccaac agcagaaaca
     6121 aggtcgtgaa gctgtccctg aagccctaca gattcgacgt gtacctggac aatggcgtgt
     6181 acaagttcgt gaccgtgaag aatctggatg tgatcaaaaa agaaaactac tacgaagtga
     6241 atagcaagtg ctatgaggaa gctaagaagc tgaagaagat cagcaaccag gccgagttta
     6301 tcgcctcctt ctacaacaac gatctgatca agatcaacgg cgagctgtat agagtgatcg
     6361 gcgtgaacaa cgacctgctg aaccggatcg aagtgaacat gatcgacatc acctaccgcg
     6421 agtacctgga aaacatgaac gacaagaggc cccccaggat cattaagaca atcgcctcca
     6481 agacccagag cattaagaag tacagcacag acattctggg caacctgtat gaagtgaaat
     6541 ctaagaagca ccctcagatc atcaaaaagg gcaaaaggcc ggcggccacg aaaaaggccg
     6601 gccaggcaaa aaagaaaaag ggatcctacc catacgatgt tccagattac gcttatccct
     6661 acgacgtgcc tgattatgca tacccatatg atgtccccga ctatgcctaa gaattcctag
     6721 agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     6781 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     6841 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     6901 ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagc tagaggccgc
     6961 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     7021 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     7081 gagcgcgcag ctgcctgcag g
//
