LOCUS       sequence-152874-        7010 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Cas9N in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7010)
  AUTHORS   Chew WL, Tabebordbar M, Cheng JK, Mali P, Wu EY, Ng AH, Zhu K, 
            Wagers AJ, Church GM
  TITLE     A multifunctional AAV-CRISPR-Cas9 and its host response.
  JOURNAL   Nat Methods. 2016 Sep 5. doi: 10.1038/nmeth.3993.
  PUBMED    27595405
REFERENCE   2  (bases 1 to 7010)
  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: "Nat 
            Methods. 2016 Sep 5. doi: 10.1038/nmeth.3993."
COMMENT     Sequence Label: pAAV-SMVP-Cas9N
FEATURES             Location/Qualifiers
     source          1..7010
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     84..103
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    471..492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(507..512,513..530,531..537)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    545..561
                     /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     550..572
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     569..585
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     569..585
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   612..741
                     /label=AAV2 ITR
     enhancer        1039..1418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1419..1622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1573..1593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          1714..1846
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1876..1885
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1888..1908
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1933..3648
                     /codon_start=1
                     /product="N-terminal portion of Streptococcus pyogenes Cas9
                     (Zetsche et al., 2015)"
                     /label=Cas9(N)
                     /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE"
     primer_bind     complement(4388..4407)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    4417..4498
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4442..4461
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4681..4810
                     /label=AAV2 ITR
     primer_bind     complement(4829..4846)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4829..4845)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4838..4860)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4987..5442
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5074..5093)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5284..5305
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5469..5573
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5574..5642,5643..6434)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVVMVTTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5792..5811)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6605..7010,1..183)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
       61 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      121 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      181 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctgcgg ttttgctcac
      241 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
      301 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      361 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
      421 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
      481 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
      541 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaga
      601 tttaattaag gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
      661 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      721 actccatcac taggggttcc ttgtagttaa tgattaaccc gccatgctac ttatctacct
      781 gcggccgcgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
      841 aagtatgcaa agcatgcatc tcaattagtc agcaaccagt cccggtctcc tcccatgcat
      901 gtcaatattg gccattagcc atattattca ttggttatat agcataaatc aatattggct
      961 attggccatt gcatacgttg tatctatatc ataatatgta catttatatt ggctcatgtc
     1021 caatatgacc gccatgttgg cattgattat tgactagtta ttaatagtaa tcaattacgg
     1081 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     1141 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     1201 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     1261 cccacttggc agtacatcaa gtgtatcata tgccaagtcc gccccctatt gacgtcaatg
     1321 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttacgggac tttcctactt
     1381 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
     1441 ccaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
     1501 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaataacc
     1561 ccgccccgtt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
     1621 gtcgtttagt gaaccgtcag atcactagta gctttattgc ggtagtttat cacagttaaa
     1681 ttgctaacgc agtcagtgct cgactgatca caggtaagta tcaaggttac aagacaggtt
     1741 taaggaggcc aatagaaact gggcttgtcg agacagagaa gattcttgcg tttctgatag
     1801 gcacctattg gtcttactga catccacttt gcctttctct ccacaggggt accgaagccg
     1861 ctagcgctac cggtcgccac catggcccca aagaagaagc ggaaggtcgg tatccacgga
     1921 gtcccagcag ccgacaagaa gtactccatt gggctcgata tcggcacaaa cagcgtcggc
     1981 tgggccgtca ttacggacga gtacaaggtg ccgagcaaaa aattcaaagt tctgggcaat
     2041 accgatcgcc acagcataaa gaagaacctc attggcgccc tcctgttcga ctccggggaa
     2101 acggccgaag ccacgcggct caaaagaaca gcacggcgca gatatacccg cagaaagaat
     2161 cggatctgct acctgcagga gatctttagt aatgagatgg ctaaggtgga tgactctttc
     2221 ttccataggc tggaggagtc ctttttggtg gaggaggata aaaagcacga gcgccaccca
     2281 atctttggca atatcgtgga cgaggtggcg taccatgaaa agtacccaac catatatcat
     2341 ctgaggaaga agcttgtaga cagtactgat aaggctgact tgcggttgat ctatctcgcg
     2401 ctggcgcata tgatcaaatt tcggggacac ttcctcatcg agggggacct gaacccagac
     2461 aacagcgatg tcgacaaact ctttatccaa ctggttcaga cttacaatca gcttttcgaa
     2521 gagaacccga tcaacgcatc cggagttgac gccaaagcaa tcctgagcgc taggctgtcc
     2581 aaatcccggc ggctcgaaaa cctcatcgca cagctccctg gggagaagaa gaacggcctg
     2641 tttggtaatc ttatcgccct gtcactcggg ctgaccccca actttaaatc taacttcgac
     2701 ctggccgaag atgccaagct tcaactgagc aaagacacct acgatgatga tctcgacaat
     2761 ctgctggccc agatcggcga ccagtacgca gacctttttt tggcggcaaa gaacctgtca
     2821 gacgccattc tgctgagtga tattctgcga gtgaacacgg agatcaccaa agctccgctg
     2881 agcgctagta tgatcaagcg ctatgatgag caccaccaag acttgacttt gctgaaggcc
     2941 cttgtcagac agcaactgcc tgagaagtac aaggaaattt tcttcgatca gtctaaaaat
     3001 ggctacgccg gatacattga cggcggagca agccaggagg aattttacaa atttattaag
     3061 cccatcttgg aaaaaatgga cggcaccgag gagctgctgg taaagcttaa cagagaagat
     3121 ctgttgcgca aacagcgcac tttcgacaat ggaagcatcc cccaccagat tcacctgggc
     3181 gaactgcacg ctatcctcag gcggcaagag gatttctacc cctttttgaa agataacagg
     3241 gaaaagattg agaaaatcct cacatttcgg ataccctact atgtaggccc cctcgcccgg
     3301 ggaaattcca gattcgcgtg gatgactcgc aaatcagaag agaccatcac tccctggaac
     3361 ttcgaggaag tcgtggataa gggggcctct gcccagtcct tcatcgaaag gatgactaac
     3421 tttgataaaa atctgcctaa cgaaaaggtg cttcctaaac actctctgct gtacgagtac
     3481 ttcacagttt ataacgagct caccaaggtc aaatacgtca cagaagggat gagaaagcca
     3541 gcattcctgt ctggagagca gaagaaagct atcgtggacc tcctcttcaa gacgaaccgg
     3601 aaagttaccg tgaaacagct caaagaagac tatttcaaaa agattgaatg tttcgactct
     3661 gttgaaatca gcggagtgga ggatcgcttc aacgcatccc tgggaacgta tcacgatctc
     3721 ctgaaaatca ttaaagacaa ggacttcctg gacaatgagg agaacgagga cattcttgag
     3781 gacattgtcc tcacccttac gttgtttgaa gatagggaga tgattgaaga acgcttgaaa
     3841 acttacgctc atctcttcga cgacaaagtc atgaaacagc tcaagaggcg ccgatataca
     3901 ggatgggggc ggctgtcaag aaaactgatc aatgggatcc gagacaagca gagtggaaag
     3961 acaatcctgg attttcttaa gtccgatgga tttgccaacc ggaacttcat gcagttgatc
     4021 catgatgact ctctcacctt taaggaggac atccagaaag cacaagtttg tctggctggc
     4081 gatactctca ttaccctggc cgatggacga cgagtgccta ttagagaact ggtgtcacag
     4141 cagaattttt ccgtgtgggc tctgaatcct cagacttacc gcctggagag ggctagagtg
     4201 agtagagctt tctgtaccgg catcaaacct gtgtaccgcc tcaccactag actggggaga
     4261 tccattaggg ccactgccaa ccaccgattt ctcacacctc agggctggaa acgagtcgat
     4321 gaactccagc ctggagatta cctggctctg cctaggagaa tccctactgc ctcctgatct
     4381 cgagctcgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     4441 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     4501 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     4561 aagcaagtaa aacctctaca aatgtggtag cggccgcgga atgtgtgtca gtaaaacctc
     4621 tacaaatgtg gtagcggccg caggtagata agtagcatgg cgggttaatc attaactaca
     4681 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
     4741 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
     4801 gagcgcgcag ccttaattaa cctaattcac tggccgtcgt tttacaacgt cgtgactggg
     4861 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
     4921 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
     4981 aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     5041 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     5101 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     5161 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     5221 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     5281 atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg
     5341 atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa
     5401 aatttaacgc gaattttaac aaaatattaa cgtttataat ttcaggtggc atctttcggg
     5461 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5521 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5581 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5641 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5701 gttacatcga actggatctc aatagtggta agatccttga gagttttcgc cccgaagaac
     5761 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5821 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5881 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5941 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     6001 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     6061 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     6121 taatggtaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     6181 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     6241 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     6301 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     6361 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6421 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6481 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6541 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6601 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6661 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6721 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     6781 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6841 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6901 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6961 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
//
