LOCUS       sequence-391385-        8384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression construct encoding the S. aureus Cas9.D10A nickase 
            (SaCas9.D10A).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8384)
  AUTHORS   Wang Q, Liu J, Janssen JM, Le Bouteiller M, Frock RL, Goncalves MAFV
  TITLE     Precise and broad scope genome editing based on high-specificity 
            Cas9 nickases.
  JOURNAL   Nucleic Acids Res. 2021 Jan 25;49(2):1173-1198. doi: 
            10.1093/nar/gkaa1236.
  PUBMED    33398349
REFERENCE   2  (bases 1 to 8384)
  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"; doi: "10"; journalName: 
            "Nucleic Acids Res"; date: "2021-01-25- 25"; volume: "49"; issue: 
            "2"; pages: "1173-1198"
COMMENT     Sequence Label: BA31_pU.CAG.SaCas9-D10A.rBGpA
FEATURES             Location/Qualifiers
     source          1..8384
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        33..409
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        411..686
                     /label=chicken beta-actin promoter
     intron          687..1703
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1627..1649
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1711..1730
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1789..1798
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1801..1821
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5002..5049
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5056..5136
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(5251..5270)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5316..5371
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5370..5389)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5750..5767)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5750..5766)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5759..5781)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5975..5994)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6094..6116
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6154..6172)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6240..6344
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6345..6413,6414..7205)
                     /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(6563..6582)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7376..7964
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7865..7884
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8118..8135
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8252..8273
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8288..8293,8294..8311,8312..8318)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8326..8342
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     8331..8353
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8350..8366
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8350..8366
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gtttaaacat ttaaatctcg agccatggat tcgacattga ttattgacta gttattaata
       61 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
      121 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
      181 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
      241 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
      301 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
      361 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tcgaggtgag
      421 ccccacgttc tgcttcactc tccccatctc ccccccctcc ccacccccaa ttttgtattt
      481 atttattttt taattatttt gtgcagcgat gggggcgggg gggggggggg sgcgcgccag
      541 gcggggcggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca
      601 atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct
      661 ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc
      721 gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg
      781 tgagcgggcg ggacggccct tctcctccgg gctgtaatta gcgcttggtt taatgacggc
      841 ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct ccgggagggc cctttgtgcg
      901 gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt ggggagcgcc gcgtgcggct
      961 ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc ggggctttgt gcgctccgca
     1021 gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg gtgcgggggg ggctgcgagg
     1081 ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg agcagggggt gtgggcgcgt
     1141 cggtcgggct gcaacccccc ctgcaccccc ctccccgagt tgctgagcac ggcccggctt
     1201 cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc cgtgccgggc ggggggtggc
     1261 ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc cggggagggc tcgggggagg
     1321 ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc ggcgagccgc agccattgcc
     1381 ttttatggta atcgtgcgag agggcgcagg gacttccttt gtcccaaatc tgtgcggagc
     1441 cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cggggcgaag cggtgcggcg
     1501 ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc gccgcgccgc cgtccccttc
     1561 tccctctcca gcctcggggc tgtccgcggg gggacggctg ccttcggggg ggacggggca
     1621 gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct aaccatgttc
     1681 atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttattgtg ctgtctcatc
     1741 attttggcaa agaattatcg catgcctgca gagctctaga gtcccggtgc caccatggcc
     1801 ccaaagaaga agcggaaggt cggtatccac ggagtcccag cagccaagcg gaactacatc
     1861 ctgggcctgg ccatcggcat caccagcgtg ggctacggca tcatcgacta cgagacacgg
     1921 gacgtgatcg atgccggcgt gcggctgttc aaagaggcca acgtggaaaa caacgagggc
     1981 aggcggagca agagaggcgc cagaaggctg aagcggcgga ggcggcatag aatccagaga
     2041 gtgaagaagc tgctgttcga ctacaacctg ctgaccgacc acagcgagct gagcggcatc
     2101 aacccctacg aggccagagt gaagggcctg agccagaagc tgagcgagga agagttctct
     2161 gccgccctgc tgcacctggc caagagaaga ggcgtgcaca acgtgaacga ggtggaagag
     2221 gacaccggca acgagctgtc caccaaagag cagatcagcc ggaacagcaa ggccctggaa
     2281 gagaaatacg tggccgaact gcagctggaa cggctgaaga aagacggcga agtgcggggc
     2341 agcatcaaca gattcaagac cagcgactac gtgaaagaag ccaaacagct gctgaaggtg
     2401 cagaaggcct accaccagct ggaccagagc ttcatcgaca cctacatcga cctgctggaa
     2461 acccggcgga cctactatga gggacctggc gagggcagcc ccttcggctg gaaggacatc
     2521 aaagaatggt acgagatgct gatgggccac tgcacctact tccccgagga actgcggagc
     2581 gtgaagtacg cctacaacgc cgacctgtac aacgccctga acgacctgaa caatctcgtg
     2641 atcaccaggg acgagaacga gaagctggaa tattacgaga agttccagat catcgagaac
     2701 gtgttcaagc agaagaagaa gcccaccctg aagcagatcg ccaaagaaat cctcgtgaac
     2761 gaagaggata ttaagggcta cagagtgacc agcaccggca agcccgagtt caccaacctg
     2821 aaggtgtacc acgacatcaa ggacattacc gcccggaaag agattattga gaacgccgag
     2881 ctgctggatc agattgccaa gatcctgacc atctaccaga gcagcgagga catccaggaa
     2941 gaactgacca atctgaactc cgagctgacc caggaagaga tcgagcagat ctctaatctg
     3001 aagggctata ccggcaccca caacctgagc ctgaaggcca tcaacctgat cctggacgag
     3061 ctgtggcaca ccaacgacaa ccagatcgct atcttcaacc ggctgaagct ggtgcccaag
     3121 aaggtggacc tgtcccagca gaaagagatc cccaccaccc tggtggacga cttcatcctg
     3181 agccccgtcg tgaagagaag cttcatccag agcatcaaag tgatcaacgc catcatcaag
     3241 aagtacggcc tgcccaacga catcattatc gagctggccc gcgagaagaa ctccaaggac
     3301 gcccagaaaa tgatcaacga gatgcagaag cggaaccggc agaccaacga gcggatcgag
     3361 gaaatcatcc ggaccaccgg caaagagaac gccaagtacc tgatcgagaa gatcaagctg
     3421 cacgacatgc aggaaggcaa gtgcctgtac agcctggaag ccatccctct ggaagatctg
     3481 ctgaacaacc ccttcaacta tgaggtggac cacatcatcc ccagaagcgt gtccttcgac
     3541 aacagcttca acaacaaggt gctcgtgaag caggaagaaa acagcaagaa gggcaaccgg
     3601 accccattcc agtacctgag cagcagcgac agcaagatca gctacgaaac cttcaagaag
     3661 cacatcctga atctggccaa gggcaagggc agaatcagca agaccaagaa agagtatctg
     3721 ctggaagaac gggacatcaa caggttctcc gtgcagaaag acttcatcaa ccggaacctg
     3781 gtggatacca gatacgccac cagaggcctg atgaacctgc tgcggagcta cttcagagtg
     3841 aacaacctgg acgtgaaagt gaagtccatc aatggcggct tcaccagctt tctgcggcgg
     3901 aagtggaagt ttaagaaaga gcggaacaag gggtacaagc accacgccga ggacgccctg
     3961 atcattgcca acgccgattt catcttcaaa gagtggaaga aactggacaa ggccaaaaaa
     4021 gtgatggaaa accagatgtt cgaggaaaag caggccgaga gcatgcccga gatcgaaacc
     4081 gagcaggagt acaaagagat cttcatcacc ccccaccaga tcaagcacat taaggacttc
     4141 aaggactaca agtacagcca ccgggtggac aagaagccta atagagagct gattaacgac
     4201 accctgtact ccacccggaa ggacgacaag ggcaacaccc tgatcgtgaa caatctgaac
     4261 ggcctgtacg acaaggacaa tgacaagctg aaaaagctga tcaacaagag ccccgaaaag
     4321 ctgctgatgt accaccacga cccccagacc taccagaaac tgaagctgat tatggaacag
     4381 tacggcgacg agaagaatcc cctgtacaag tactacgagg aaaccgggaa ctacctgacc
     4441 aagtactcca aaaaggacaa cggccccgtg atcaagaaga ttaagtatta cggcaacaaa
     4501 ctgaacgccc atctggacat caccgacgac taccccaaca gcagaaacaa ggtcgtgaag
     4561 ctgtccctga agccctacag attcgacgtg tacctggaca atggcgtgta caagttcgtg
     4621 accgtgaaga atctggatgt gatcaaaaaa gaaaactact acgaagtgaa tagcaagtgc
     4681 tatgaggaag ctaagaagct gaagaagatc agcaaccagg ccgagtttat cgcctccttc
     4741 tacaacaacg atctgatcaa gatcaacggc gagctgtata gagtgatcgg cgtgaacaac
     4801 gacctgctga accggatcga agtgaacatg atcgacatca cctaccgcga gtacctggaa
     4861 aacatgaacg acaagaggcc ccccaggatc attaagacaa tcgcctccaa gacccagagc
     4921 attaagaagt acagcacaga cattctgggc aacctgtatg aagtgaaatc taagaagcac
     4981 cctcagatca tcaaaaaggg caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa
     5041 aagaaaaagg gatcctaccc atacgatgtt ccagattacg cttacccata cgatgttcca
     5101 gattacgctt acccatacga tgttccagat tacgcttaag aattggccgc acttaagtta
     5161 cgcgtggaaa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc
     5221 caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga
     5281 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     5341 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     5401 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc
     5461 tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     5521 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     5581 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     5641 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct
     5701 cgacctgcag cccaagctga tcccgggatt taaatgttta aacgaattca ctggccgtcg
     5761 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     5821 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     5881 agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt
     5941 gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     6001 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     6061 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     6121 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     6181 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     6241 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6301 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6361 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6421 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6481 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6541 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6601 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     6661 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     6721 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     6781 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     6841 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     6901 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     6961 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     7021 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     7081 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     7141 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     7201 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     7261 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7321 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7381 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7441 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7501 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     7561 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7621 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     7681 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     7741 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     7801 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     7861 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     7921 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     7981 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     8041 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     8101 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     8161 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     8221 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     8281 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     8341 aatttcacac aggaaacagc tatgaccatg attacgccaa gctt
//
