LOCUS       sequence-68855-e        8441 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLAG tagged NmCas9 with no NLS..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8441)
  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 8441)
  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-NmCas9-FLAG
FEATURES             Location/Qualifiers
     source          1..8441
                     /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             1421..4666
                     /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="MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRV
                     FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIK
                     SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA
                     GNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEF
                     GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT
                     KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD
                     NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL
                     KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT
                     EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK
                     EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
                     RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF
                     KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR
                     VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF
                     DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE
                     KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD
                     LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK
                     TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ
                     LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE
                     GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     CDS             4667..4690
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4706..4723)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4712..4936
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      5125..6238
                     /label=mini-oriP
                     /note="internally truncated variant of the Epstein-Barr 
                     virus oriP replication origin (Tanaka et al., 1999)"
     primer_bind     complement(6284..6300)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6284..6300)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6297..6319)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6308..6324
                     /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(6332..6338,6339..6356,6357..6362))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6377..6398
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6515..6532)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6686..7274)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6766..6785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7445..8236,8237..8305))
                     /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     8068..8087
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8306..8410)
                     /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 atggctgcct tcaaacctaa
     1441 ttcaatcaac tacatcctcg gcctcgatat cggcatcgca tccgtcggct gggcgatggt
     1501 agaaattgac gaagaagaaa accccatccg cctgattgat ttgggcgtgc gcgtatttga
     1561 gcgtgccgaa gtaccgaaaa caggcgactc ccttgccatg gcaaggcgtt tggcgcgcag
     1621 tgttcgccgc ctgacccgcc gtcgcgccca ccgcctgctt cggacccgcc gcctattgaa
     1681 acgcgaaggc gtattacaag ccgccaattt tgacgaaaac ggcttgatta aatccttacc
     1741 gaatacacca tggcaacttc gcgcagccgc attagaccgc aaactgacgc ctttagagtg
     1801 gtcggcagtc ttgttgcatt taatcaaaca tcgcggctat ttatcgcaac ggaaaaacga
     1861 gggcgaaact gccgataagg agcttggcgc tttgcttaaa ggcgtagccg gcaatgccca
     1921 tgccttacag acaggcgatt tccgcacacc ggccgaattg gctttaaata aatttgagaa
     1981 agaaagcggc catatccgca atcagcgcag cgattattcg catacgttca gccgcaaaga
     2041 tttacaggcg gagctgattt tgctgtttga aaaacaaaaa gaatttggca atccgcatgt
     2101 ttcaggcggc cttaaagaag gtattgaaac cctactgatg acgcaacgcc ctgccctgtc
     2161 cggcgatgcc gttcaaaaaa tgttggggca ttgcaccttc gaaccggcag agccgaaagc
     2221 cgctaaaaac acctacacag ccgaacgttt catctggctg accaagctga acaacctgcg
     2281 tattttagag caaggcagcg agcggccatt gaccgatacc gaacgcgcca cgcttatgga
     2341 cgagccatac agaaaatcca aactgactta cgcacaagcc cgtaagctgc tgggtttaga
     2401 agataccgcc tttttcaaag gcttgcgcta tggtaaagac aatgccgaag cctcaacatt
     2461 gatggaaatg aaggcctacc atgccatcag ccgtgcactg gaaaaagaag gattgaaaga
     2521 caaaaaatcc ccattaaacc tttctcccga attacaagac gaaatcggca cggcattctc
     2581 cctgttcaaa accgatgaag acattacagg ccgtctgaaa gaccgtatac agcccgaaat
     2641 cttagaagcg ctgttgaaac acatcagctt cgataagttc gtccaaattt ccttgaaagc
     2701 attgcgccga attgtgcctc taatggaaca aggcaaacgt tacgatgaag cctgcgccga
     2761 aatctacgga gaccattacg gcaagaagaa tacggaagaa aagatttatc tgccgccgat
     2821 tcccgccgac gaaatccgca accccgtcgt cttgcgcgcc ttatctcaag cacgtaaggt
     2881 cattaacggc gtggtacgcc gttacggctc cccagctcgt atccatattg aaactgcaag
     2941 ggaagtaggt aaatcgttta aagaccgcaa agaaattgag aaacgccaag aagaaaaccg
     3001 caaagaccgg gaaaaagccg ccgccaaatt ccgagagtat ttccccaatt ttgtcggaga
     3061 acccaaatcc aaagatattc tgaaactgcg cctgtacgag caacaacacg gcaaatgcct
     3121 gtattcgggc aaagaaatca acttaggccg tctgaacgaa aaaggctatg tcgaaatcga
     3181 ccatgccctg ccgttctcgc gcacatggga cgacagtttc aacaataaag tactggtatt
     3241 gggcagcgaa aaccaaaaca aaggcaatca aaccccttac gaatacttca acggcaaaga
     3301 caacagccgc gaatggcagg aatttaaagc gcgtgtcgaa accagccgtt tcccgcgcag
     3361 taaaaaacaa cggattctgc tgcaaaaatt cgatgaagac ggctttaaag aacgcaatct
     3421 gaacgacacg cgctacgtca accgtttcct gtgtcaattt gttgccgacc gtatgcggct
     3481 gacaggtaaa ggcaagaaac gtgtctttgc atccaacgga caaattacca atctgttgcg
     3541 cggcttttgg ggattgcgca aagtgcgtgc ggaaaacgac cgccatcacg ccttggacgc
     3601 cgtcgtcgtt gcctgctcga ccgttgccat gcagcagaaa attacccgtt ttgtacgcta
     3661 taaagagatg aacgcgtttg acggtaaaac catagacaaa gaaacaggag aagtgctgca
     3721 tcaaaaaaca cacttcccac aaccttggga atttttcgca caagaagtca tgattcgcgt
     3781 cttcggcaaa ccggacggca aacccgaatt cgaagaagcc gataccctag aaaaactgcg
     3841 cacgttgctt gccgaaaaat tatcatctcg ccccgaagcc gtacacgaat acgttacgcc
     3901 actgtttgtt tcacgcgcgc ccaatcggaa gatgagcggg caagggcata tggagaccgt
     3961 caaatccgcc aaacgactgg acgaaggcgt cagcgtgttg cgcgtaccgc tgacacagtt
     4021 aaaactgaaa gacttggaaa aaatggtcaa tcgggagcgc gaacctaagc tatacgaagc
     4081 actgaaagca cggctggaag cacataaaga cgatcctgcc aaagcctttg ccgagccgtt
     4141 ttacaaatac gataaagcag gcaaccgcac ccaacaggta aaagccgtac gcgtagagca
     4201 agtacagaaa accggcgtat gggtgcgcaa ccataacggt attgccgaca acgcaaccat
     4261 ggtgcgcgta gatgtgtttg agaaaggcga caagtattat ctggtaccga tttacagttg
     4321 gcaggtagcg aaagggattt tgccggatag ggctgttgta caaggaaaag atgaagaaga
     4381 ttggcaactt attgatgata gtttcaactt taaattctca ttacacccta atgatttagt
     4441 cgaggttata acaaaaaaag ctagaatgtt tggttacttt gccagctgcc atcgaggcac
     4501 aggtaatatc aatatacgca ttcatgatct tgatcataaa attggcaaaa atggaatact
     4561 ggaaggtatc ggcgtcaaaa ccgccctttc attccaaaaa taccaaattg acgaactggg
     4621 caaagaaatc agaccatgcc gtctgaaaaa acgcccgcct gtccgtgatt ataaggatga
     4681 tgatgataag taactcgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     4741 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     4801 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     4861 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     4921 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctcgac cgatgccctt
     4981 gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc
     5041 acttatgact gtcttcttta tcatgcaact cgtaggacag gtgcctggcc ggggtccccc
     5101 ggaaactcgg ccgtggtgac catgcaggaa aaggacaagc agcgaaaatt cacgccccct
     5161 tgggaggtgg cggcatatgc aaaggatagc actcccactc tactactggg tatcatatgc
     5221 tgactgtata tgcatgagga tagcatatgc tacccggata cagattagga tagcatatac
     5281 tacccagata tagattagga tagcatatgc tacccagata tagattagga tagcctatgc
     5341 tacccagata taaattagga tagcatatac tacccagata tagattagga tagcatatgc
     5401 tacccagata tagattagga tagcctatgc tacccagata tagattagga tagcatatgc
     5461 tacccagata tagattagga tagcatatgc tatccagata tttgggtagt atatgctacc
     5521 cagatataaa ttaggatagc atatactacc ctaatctcta ttaggatagc atatgctacc
     5581 cggatacaga ttaggatagc atatactacc cagatataga ttaggatagc atatgctacc
     5641 cagatataga ttaggatagc ctatgctacc cagatataaa ttaggatagc atatactacc
     5701 cagatataga ttaggatagc atatgctacc cagatataga ttaggatagc ctatgctacc
     5761 cagatataga ttaggatagc atatgctatc cagatatttg ggtagtatat gctacccatg
     5821 gcaacattag cccaccgtgc tctcagcgac ctcgtgaata tgaggaccaa caaccctgtg
     5881 cttggcgctc aggcgcaagt gtgtgtaatt tgtcctccag atcgcagcaa tcgcgcccct
     5941 atcttggccc gcccacctac ttatgcaggt attccccggg gtgccattag tggttttgtg
     6001 ggcaagtggt ttgaccgcag tggttagcgg ggttacaatc agccaagtta ttacaccctt
     6061 attttacagt ccaaaaccgc agggcggcgt gtgggggctg acgcgatcgc tgttccttag
     6121 gaccctttta ctaaccctaa ttcgatagca tatgcttccc gttgggtaac atatgctatt
     6181 gaattagggt tagtctggat agtatatact actacccggg aagcatatgc tacccgttta
     6241 gggttatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg
     6301 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     6361 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     6421 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     6481 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     6541 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     6601 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     6661 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6721 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6781 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6841 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc
     6901 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6961 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     7021 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     7081 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
     7141 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     7201 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     7261 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     7321 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     7381 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     7441 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     7501 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     7561 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     7621 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     7681 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7741 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7801 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7861 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7921 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7981 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     8041 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     8101 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     8161 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     8221 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     8281 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     8341 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     8401 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c
//
