LOCUS       sequence-155832-        5409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid that expresses Cas9N in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5409)
  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 5409)
  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: pSMVP-Cas9N
FEATURES             Location/Qualifiers
     source          1..5409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(39..59)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     enhancer        289..668
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        669..872
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     823..843
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          964..1096
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     regulatory      1126..1135
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1138..1158
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1183..2898
                     /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(3638..3657)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     polyA_signal    3667..3748
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3692..3711
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      complement(3946..4534)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4026..4045)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4615..5409)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(4615..5409)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4726..4745)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5336..5355
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 aatattattg aagcatttat cagggttcgt ctcgctgtgg aatgtgtgtc agttagggtg
       61 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
      121 agcaaccagt cccggtctcc tcccatgcat gtcaatattg gccattagcc atattattca
      181 ttggttatat agcataaatc aatattggct attggccatt gcatacgttg tatctatatc
      241 ataatatgta catttatatt ggctcatgtc caatatgacc gccatgttgg cattgattat
      301 tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      361 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      421 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      481 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      541 tgccaagtcc gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      601 agtacatgac cttacgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      661 ttaccatggt gatgcggttt tggcagtaca ccaatgggcg tggatagcgg tttgactcac
      721 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      781 aacgggactt tccaaaatgt cgtaataacc ccgccccgtt gacgcaaatg ggcggtaggc
      841 gtgtacggtg ggaggtctat ataagcagag gtcgtttagt gaaccgtcag atcactagta
      901 gctttattgc ggtagtttat cacagttaaa ttgctaacgc agtcagtgct cgactgatca
      961 caggtaagta tcaaggttac aagacaggtt taaggaggcc aatagaaact gggcttgtcg
     1021 agacagagaa gattcttgcg tttctgatag gcacctattg gtcttactga catccacttt
     1081 gcctttctct ccacaggggt accgaagccg ctagcgctac cggtcgccac catggcccca
     1141 aagaagaagc ggaaggtcgg tatccacgga gtcccagcag ccgacaagaa gtactccatt
     1201 gggctcgata tcggcacaaa cagcgtcggc tgggccgtca ttacggacga gtacaaggtg
     1261 ccgagcaaaa aattcaaagt tctgggcaat accgatcgcc acagcataaa gaagaacctc
     1321 attggcgccc tcctgttcga ctccggggaa acggccgaag ccacgcggct caaaagaaca
     1381 gcacggcgca gatatacccg cagaaagaat cggatctgct acctgcagga gatctttagt
     1441 aatgagatgg ctaaggtgga tgactctttc ttccataggc tggaggagtc ctttttggtg
     1501 gaggaggata aaaagcacga gcgccaccca atctttggca atatcgtgga cgaggtggcg
     1561 taccatgaaa agtacccaac catatatcat ctgaggaaga agcttgtaga cagtactgat
     1621 aaggctgact tgcggttgat ctatctcgcg ctggcgcata tgatcaaatt tcggggacac
     1681 ttcctcatcg agggggacct gaacccagac aacagcgatg tcgacaaact ctttatccaa
     1741 ctggttcaga cttacaatca gcttttcgaa gagaacccga tcaacgcatc cggagttgac
     1801 gccaaagcaa tcctgagcgc taggctgtcc aaatcccggc ggctcgaaaa cctcatcgca
     1861 cagctccctg gggagaagaa gaacggcctg tttggtaatc ttatcgccct gtcactcggg
     1921 ctgaccccca actttaaatc taacttcgac ctggccgaag atgccaagct tcaactgagc
     1981 aaagacacct acgatgatga tctcgacaat ctgctggccc agatcggcga ccagtacgca
     2041 gacctttttt tggcggcaaa gaacctgtca gacgccattc tgctgagtga tattctgcga
     2101 gtgaacacgg agatcaccaa agctccgctg agcgctagta tgatcaagcg ctatgatgag
     2161 caccaccaag acttgacttt gctgaaggcc cttgtcagac agcaactgcc tgagaagtac
     2221 aaggaaattt tcttcgatca gtctaaaaat ggctacgccg gatacattga cggcggagca
     2281 agccaggagg aattttacaa atttattaag cccatcttgg aaaaaatgga cggcaccgag
     2341 gagctgctgg taaagcttaa cagagaagat ctgttgcgca aacagcgcac tttcgacaat
     2401 ggaagcatcc cccaccagat tcacctgggc gaactgcacg ctatcctcag gcggcaagag
     2461 gatttctacc cctttttgaa agataacagg gaaaagattg agaaaatcct cacatttcgg
     2521 ataccctact atgtaggccc cctcgcccgg ggaaattcca gattcgcgtg gatgactcgc
     2581 aaatcagaag agaccatcac tccctggaac ttcgaggaag tcgtggataa gggggcctct
     2641 gcccagtcct tcatcgaaag gatgactaac tttgataaaa atctgcctaa cgaaaaggtg
     2701 cttcctaaac actctctgct gtacgagtac ttcacagttt ataacgagct caccaaggtc
     2761 aaatacgtca cagaagggat gagaaagcca gcattcctgt ctggagagca gaagaaagct
     2821 atcgtggacc tcctcttcaa gacgaaccgg aaagttaccg tgaaacagct caaagaagac
     2881 tatttcaaaa agattgaatg tttcgactct gttgaaatca gcggagtgga ggatcgcttc
     2941 aacgcatccc tgggaacgta tcacgatctc ctgaaaatca ttaaagacaa ggacttcctg
     3001 gacaatgagg agaacgagga cattcttgag gacattgtcc tcacccttac gttgtttgaa
     3061 gatagggaga tgattgaaga acgcttgaaa acttacgctc atctcttcga cgacaaagtc
     3121 atgaaacagc tcaagaggcg ccgatataca ggatgggggc ggctgtcaag aaaactgatc
     3181 aatgggatcc gagacaagca gagtggaaag acaatcctgg attttcttaa gtccgatgga
     3241 tttgccaacc ggaacttcat gcagttgatc catgatgact ctctcacctt taaggaggac
     3301 atccagaaag cacaagtttg tctggctggc gatactctca ttaccctggc cgatggacga
     3361 cgagtgccta ttagagaact ggtgtcacag cagaattttt ccgtgtgggc tctgaatcct
     3421 cagacttacc gcctggagag ggctagagtg agtagagctt tctgtaccgg catcaaacct
     3481 gtgtaccgcc tcaccactag actggggaga tccattaggg ccactgccaa ccaccgattt
     3541 ctcacacctc agggctggaa acgagtcgat gaactccagc ctggagatta cctggctctg
     3601 cctaggagaa tccctactgc ctcctgatct cgagctcgat gagtttggac aaaccacaac
     3661 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     3721 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     3781 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtac
     3841 ttaagagggg gagaccaaag ggcgagacgt taaggcctca cgtgacatgt gagcaaaagg
     3901 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     3961 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     4021 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     4081 cctgccgctt acgggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     4141 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     4201 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     4261 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     4321 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     4381 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     4441 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     4501 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     4561 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatgc cgtctcagaa
     4621 gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta
     4681 aagcacgagg aagcggtcag cccattcgcc gccaagctct tcagcaatat cacgggtagc
     4741 caacgctatg tcctgatagc ggtccgccac acccagccgg ccacagtcga tgaatccaga
     4801 aaagcggcca ttttccacca tgatattcgg caagcaggca tcgccatggg tcacgacgag
     4861 atcctcgccg tcgggcatgc tcgccttgag cctggcgaac agttcggctg gcgcgagccc
     4921 ctgatgctct tcgtccagat catcctgatc gacaagaccg gcttccatcc gagtacgtgc
     4981 tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag gtagccggat caagcgtatg
     5041 cagccgccgc attgcatcag ccatgatgga tactttctcg gcaggagcaa ggtgagatga
     5101 caggagatcc tgccccggca cttcgcccaa tagcagccag tcccttcccg cttcagtgac
     5161 aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc
     5221 ctcgtcttgc agttcattca gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg
     5281 cccctgcgct gacagccgga acacggcggc atcagagcag ccgattgtct gttgtgccca
     5341 gtcatagccg aatagcctct ccacccaagc ggccggagaa cctgcgtgca atccatcttg
     5401 ttcaatcat
//
