LOCUS       sequence-68412-e        8501 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid encode NmCas9 with two NLS and a HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8501)
  AUTHORS   Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, 
            Thomson JA
  TITLE     Efficient genome engineering in human pluripotent stem cells using 
            Cas9 from Neisseria meningitidis.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Aug 12.
  PUBMED    23940360
REFERENCE   2  (bases 1 to 8501)
  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: "Proc Natl 
            Acad Sci U S A. 2013 Aug 12."
COMMENT     Sequence Label: pSimpleII-NLS-NmCas9-HA-NLS(s)
FEATURES             Location/Qualifiers
     source          1..8501
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        236..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          467..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1415..1424
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1427..1447
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1448..4690
                     /codon_start=1
                     /product="Cas9 endonuclease from the Neisseria meningitidis
                     Type II CRISPR/Cas system (Esvelt et al., 2013)"
                     /label=NmCas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="AAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVF
                     ERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKS
                     LPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAG
                     NAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFG
                     NPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTK
                     LNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDN
                     AEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK
                     DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE
                     EKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKE
                     IEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGR
                     LNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFK
                     ARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRV
                     FASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFD
                     GKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAEK
                     LSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDL
                     EKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKT
                     GVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQL
                     IDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEG
                     IGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     CDS             4691..4717
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4724..4750
                     /codon_start=1
                     /product="nuclear localization signal (Makkerh et al., 
                     1996)"
                     /label=NLS
                     /translation="PAAKKKKLD"
     primer_bind     complement(4766..4783)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4772..4996
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5185..6298
                     /label=mini-oriP
                     /note="internally truncated variant of the Epstein-Barr 
                     virus oriP replication origin (Tanaka et al., 1999)"
     primer_bind     complement(6344..6360)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6344..6360)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6357..6379)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6368..6384
                     /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)."
     promoter        complement(join(6392..6398,6399..6416,6417..6422))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6437..6458
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6575..6592)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6746..7334)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6826..6845)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7505..8296,8297..8365))
                     /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     8128..8147
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8366..8470)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcggg ggtgaggctc cggtgcccgt cgtgaggctc
      241 cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg
      301 ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt
      361 cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt
      421 cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg
      481 tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt gaattacttc
      541 cacctggctc cagtacgtga ttcttgatcc cgagctggag ccaggggcgg gccttgcgct
      601 ttaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
      661 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
      721 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
      781 ggccaggatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt
      841 gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga
      901 cgggggtcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctc cagggggctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaagg ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctgga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgaa caccgccacc atggtgccta agaagaagag
     1441 aaaggtggct gccttcaaac ctaattcaat caactacatc ctcggcctcg atatcggcat
     1501 cgcatccgtc ggctgggcga tggtagaaat tgacgaagaa gaaaacccca tccgcctgat
     1561 tgatttgggc gtgcgcgtat ttgagcgtgc cgaagtaccg aaaacaggcg actcccttgc
     1621 catggcaagg cgtttggcgc gcagtgttcg ccgcctgacc cgccgtcgcg cccaccgcct
     1681 gcttcggacc cgccgcctat tgaaacgcga aggcgtatta caagccgcca attttgacga
     1741 aaacggcttg attaaatcct taccgaatac accatggcaa cttcgcgcag ccgcattaga
     1801 ccgcaaactg acgcctttag agtggtcggc agtcttgttg catttaatca aacatcgcgg
     1861 ctatttatcg caacggaaaa acgagggcga aactgccgat aaggagcttg gcgctttgct
     1921 taaaggcgta gccggcaatg cccatgcctt acagacaggc gatttccgca caccggccga
     1981 attggcttta aataaatttg agaaagaaag cggccatatc cgcaatcagc gcagcgatta
     2041 ttcgcatacg ttcagccgca aagatttaca ggcggagctg attttgctgt ttgaaaaaca
     2101 aaaagaattt ggcaatccgc atgtttcagg cggccttaaa gaaggtattg aaaccctact
     2161 gatgacgcaa cgccctgccc tgtccggcga tgccgttcaa aaaatgttgg ggcattgcac
     2221 cttcgaaccg gcagagccga aagccgctaa aaacacctac acagccgaac gtttcatctg
     2281 gctgaccaag ctgaacaacc tgcgtatttt agagcaaggc agcgagcggc cattgaccga
     2341 taccgaacgc gccacgctta tggacgagcc atacagaaaa tccaaactga cttacgcaca
     2401 agcccgtaag ctgctgggtt tagaagatac cgcctttttc aaaggcttgc gctatggtaa
     2461 agacaatgcc gaagcctcaa cattgatgga aatgaaggcc taccatgcca tcagccgtgc
     2521 actggaaaaa gaaggattga aagacaaaaa atccccatta aacctttctc ccgaattaca
     2581 agacgaaatc ggcacggcat tctccctgtt caaaaccgat gaagacatta caggccgtct
     2641 gaaagaccgt atacagcccg aaatcttaga agcgctgttg aaacacatca gcttcgataa
     2701 gttcgtccaa atttccttga aagcattgcg ccgaattgtg cctctaatgg aacaaggcaa
     2761 acgttacgat gaagcctgcg ccgaaatcta cggagaccat tacggcaaga agaatacgga
     2821 agaaaagatt tatctgccgc cgattcccgc cgacgaaatc cgcaaccccg tcgtcttgcg
     2881 cgccttatct caagcacgta aggtcattaa cggcgtggta cgccgttacg gctccccagc
     2941 tcgtatccat attgaaactg caagggaagt aggtaaatcg tttaaagacc gcaaagaaat
     3001 tgagaaacgc caagaagaaa accgcaaaga ccgggaaaaa gccgccgcca aattccgaga
     3061 gtatttcccc aattttgtcg gagaacccaa atccaaagat attctgaaac tgcgcctgta
     3121 cgagcaacaa cacggcaaat gcctgtattc gggcaaagaa atcaacttag gccgtctgaa
     3181 cgaaaaaggc tatgtcgaaa tcgaccatgc cctgccgttc tcgcgcacat gggacgacag
     3241 tttcaacaat aaagtactgg tattgggcag cgaaaaccaa aacaaaggca atcaaacccc
     3301 ttacgaatac ttcaacggca aagacaacag ccgcgaatgg caggaattta aagcgcgtgt
     3361 cgaaaccagc cgtttcccgc gcagtaaaaa acaacggatt ctgctgcaaa aattcgatga
     3421 agacggcttt aaagaacgca atctgaacga cacgcgctac gtcaaccgtt tcctgtgtca
     3481 atttgttgcc gaccgtatgc ggctgacagg taaaggcaag aaacgtgtct ttgcatccaa
     3541 cggacaaatt accaatctgt tgcgcggctt ttggggattg cgcaaagtgc gtgcggaaaa
     3601 cgaccgccat cacgccttgg acgccgtcgt cgttgcctgc tcgaccgttg ccatgcagca
     3661 gaaaattacc cgttttgtac gctataaaga gatgaacgcg tttgacggta aaaccataga
     3721 caaagaaaca ggagaagtgc tgcatcaaaa aacacacttc ccacaacctt gggaattttt
     3781 cgcacaagaa gtcatgattc gcgtcttcgg caaaccggac ggcaaacccg aattcgaaga
     3841 agccgatacc ctagaaaaac tgcgcacgtt gcttgccgaa aaattatcat ctcgccccga
     3901 agccgtacac gaatacgtta cgccactgtt tgtttcacgc gcgcccaatc ggaagatgag
     3961 cgggcaaggg catatggaga ccgtcaaatc cgccaaacga ctggacgaag gcgtcagcgt
     4021 gttgcgcgta ccgctgacac agttaaaact gaaagacttg gaaaaaatgg tcaatcggga
     4081 gcgcgaacct aagctatacg aagcactgaa agcacggctg gaagcacata aagacgatcc
     4141 tgccaaagcc tttgccgagc cgttttacaa atacgataaa gcaggcaacc gcacccaaca
     4201 ggtaaaagcc gtacgcgtag agcaagtaca gaaaaccggc gtatgggtgc gcaaccataa
     4261 cggtattgcc gacaacgcaa ccatggtgcg cgtagatgtg tttgagaaag gcgacaagta
     4321 ttatctggta ccgatttaca gttggcaggt agcgaaaggg attttgccgg atagggctgt
     4381 tgtacaagga aaagatgaag aagattggca acttattgat gatagtttca actttaaatt
     4441 ctcattacac cctaatgatt tagtcgaggt tataacaaaa aaagctagaa tgtttggtta
     4501 ctttgccagc tgccatcgag gcacaggtaa tatcaatata cgcattcatg atcttgatca
     4561 taaaattggc aaaaatggaa tactggaagg tatcggcgtc aaaaccgccc tttcattcca
     4621 aaaataccaa attgacgaac tgggcaaaga aatcagacca tgccgtctga aaaaacgccc
     4681 gcctgtccgt tacccatacg atgttccaga ttacgctgca gctccagcag cgaagaaaaa
     4741 gaagctggat taactcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     4801 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     4861 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     4921 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     4981 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgac cgatgccctt
     5041 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     5101 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgcctggcc ggggtccccc
     5161 ggaaactcgg ccgtggtgac catgcaggaa aaggacaagc agcgaaaatt cacgccccct
     5221 tgggaggtgg cggcatatgc aaaggatagc actcccactc tactactggg tatcatatgc
     5281 tgactgtata tgcatgagga tagcatatgc tacccggata cagattagga tagcatatac
     5341 tacccagata tagattagga tagcatatgc tacccagata tagattagga tagcctatgc
     5401 tacccagata taaattagga tagcatatac tacccagata tagattagga tagcatatgc
     5461 tacccagata tagattagga tagcctatgc tacccagata tagattagga tagcatatgc
     5521 tacccagata tagattagga tagcatatgc tatccagata tttgggtagt atatgctacc
     5581 cagatataaa ttaggatagc atatactacc ctaatctcta ttaggatagc atatgctacc
     5641 cggatacaga ttaggatagc atatactacc cagatataga ttaggatagc atatgctacc
     5701 cagatataga ttaggatagc ctatgctacc cagatataaa ttaggatagc atatactacc
     5761 cagatataga ttaggatagc atatgctacc cagatataga ttaggatagc ctatgctacc
     5821 cagatataga ttaggatagc atatgctatc cagatatttg ggtagtatat gctacccatg
     5881 gcaacattag cccaccgtgc tctcagcgac ctcgtgaata tgaggaccaa caaccctgtg
     5941 cttggcgctc aggcgcaagt gtgtgtaatt tgtcctccag atcgcagcaa tcgcgcccct
     6001 atcttggccc gcccacctac ttatgcaggt attccccggg gtgccattag tggttttgtg
     6061 ggcaagtggt ttgaccgcag tggttagcgg ggttacaatc agccaagtta ttacaccctt
     6121 attttacagt ccaaaaccgc agggcggcgt gtgggggctg acgcgatcgc tgttccttag
     6181 gaccctttta ctaaccctaa ttcgatagca tatgcttccc gttgggtaac atatgctatt
     6241 gaattagggt tagtctggat agtatatact actacccggg aagcatatgc tacccgttta
     6301 gggttatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     6361 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     6421 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     6481 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     6541 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     6601 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     6661 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     6721 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6781 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6841 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6901 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     6961 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     7021 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     7081 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     7141 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     7201 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     7261 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     7321 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     7381 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     7441 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     7501 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     7561 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     7621 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     7681 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     7741 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7801 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7861 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7921 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7981 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     8041 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     8101 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     8161 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     8221 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     8281 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     8341 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     8401 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     8461 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
