LOCUS       sequence-71690-e        5974 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial N. meningitidis Cas9 (NM) + tracrRNA expression, 
            cloDF13/spectinomycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 3962 to 3996)
  AUTHORS   Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM
  TITLE     Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.
  JOURNAL   Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681.
  PUBMED    24076762
REFERENCE   2  (bases 1 to 5974)
  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. 2013 Sep 29. doi: 10.1038/nmeth.2681."
COMMENT     Sequence Label: DS-NMcas
FEATURES             Location/Qualifiers
     source          1..5974
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      153..180
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     CDS             377..3622
                     /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="MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRV
                     FERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIK
                     SLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVA
                     DNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYSHTFSRKDLQAELILLFEKQKEF
                     GNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLT
                     KLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKD
                     NAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRL
                     KDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT
                     EEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRK
                     EIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
                     RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEF
                     KARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKR
                     VFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAF
                     DGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAE
                     KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKD
                     LEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQK
                     TGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQ
                     LIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILE
                     GIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR"
     regulatory      3209..3218
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        3813..3905
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        3962..3996
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     repeat_region   4013..4048
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     rep_origin      4230..4968
                     /direction=RIGHT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     promoter        5016..5107
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5108..5899
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
ORIGIN
        1 accaggcacg cctaaccgtc agtgagattg gatgagtgaa cgatattgat cgagaagagc
       61 cctgcgcagc cgctgccgtg cctgcaggaa gcaacggccc ggagggtggc gggcaggacg
      121 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
      181 tgcgtttcta caaactctgc tagcttctag agcacagcta acaccacgtc gtccctatct
      241 gctgccctag gtctatgagt ggttgctgga taactttacg ggcatgcata aggctcgtat
      301 gatatattca gggagaccac aacggtttcc ctctacaaat aattttgttt aacttttact
      361 agaggaggag gcaaaaatgg ccgccttcaa gcccaacccc atcaactaca tcctgggcct
      421 ggacatcggc atcgccagcg tgggctgggc catggtggag atcgacgagg acgagaaccc
      481 catctgcctg atcgacctgg gtgtgcgcgt gttcgagcgc gctgaggtgc ccaagactgg
      541 tgacagtctg gctatggctc gccggcttgc tcgctctgtt cggcgcctta ctcgccggcg
      601 cgctcaccgc cttctgcgcg ctcgccgcct gctgaagcgc gagggtgtgc tgcaggctgc
      661 cgacttcgac gagaacggcc tgatcaagag cctgcccaac actccttggc agctgcgcgc
      721 tgccgctctg gaccgcaagc tgactcctct ggagtggagc gccgtgctgc tgcacctgat
      781 caagcaccgc ggctacctga gccagcgcaa gaacgagggc gagaccgccg acaaggagct
      841 gggtgctctg ctgaagggcg tggccgacaa cgcccacgcc ctgcagactg gtgacttccg
      901 cactcctgct gagctggccc tgaacaagtt cgagaaggag agcggccaca tccgcaacca
      961 gcgcggcgac tacagccaca ccttcagccg caaggacctg caggccgagc tgatcctgct
     1021 gttcgagaag cagaaggagt tcggcaaccc ccacgtgagc ggcggcctga aggagggcat
     1081 cgagaccctg ctgatgaccc agcgccccgc cctgagcggc gacgccgtgc agaagatgct
     1141 gggccactgc accttcgagc cagccgagcc caaggccgcc aagaacacct acaccgccga
     1201 gcgcttcatc tggctgacca agctgaacaa cctgcgcatc ctggagcagg gcagcgagcg
     1261 ccccctgacc gacaccgagc gcgccaccct gatggacgag ccctaccgca agagcaagct
     1321 gacctacgcc caggcccgca agctgctggg tctggaggac accgccttct tcaagggcct
     1381 gcgctacggc aaggacaacg ccgaggccag caccctgatg gagatgaagg cctaccacgc
     1441 catcagccgc gccctggaga aggagggcct gaaggacaag aagagtcctc tgaacctgag
     1501 ccccgagctg caggacgaga tcggcaccgc cttcagcctg ttcaagaccg acgaggacat
     1561 caccggccgc ctgaaggacc gcatccagcc cgagatcctg gaggccctgc tgaagcacat
     1621 cagcttcgac aagttcgtgc agatcagcct gaaggccctg cgccgcatcg tgcccctgat
     1681 ggagcagggc aagcgctacg acgaggcctg cgccgagatc tacggcgacc actacggcaa
     1741 gaagaacacc gaggagaaga tctacctgcc tcctatcccc gccgacgaga tccgcaaccc
     1801 cgtggtgctg cgcgccctga gccaggcccg caaggtgatc aacggcgtgg tgcgccgcta
     1861 cggcagcccc gcccgcatcc acatcgagac cgcccgcgag gtgggcaaga gcttcaagga
     1921 ccgcaaggag atcgagaagc gccaggagga gaaccgcaag gaccgcgaga aggccgccgc
     1981 caagttccgc gagtacttcc ccaacttcgt gggcgagccc aagagcaagg acatcctgaa
     2041 gctgcgcctg tacgagcagc agcacggcaa gtgcctgtac agcggcaagg agatcaacct
     2101 gggccgcctg aacgagaagg gctacgtgga gatcgaccac gccctgccct tcagccgcac
     2161 ctgggacgac agcttcaaca acaaggtgct ggtgctgggc agcgagaacc agaacaaggg
     2221 caaccagacc ccctacgagt acttcaacgg caaggacaac agccgcgagt ggcaggagtt
     2281 caaggcccgc gtggagacca gccgcttccc ccgcagcaag aagcagcgca tcctgctgca
     2341 gaagttcgac gaggacggct tcaaggagcg caacctgaac gacacccgct acgtgaaccg
     2401 cttcctgtgc cagttcgtgg ccgaccgcat gcgcctgacc ggcaagggca agaagcgcgt
     2461 gttcgccagc aacggccaga tcaccaacct gctgcgcggc ttctggggcc tgcgcaaggt
     2521 gcgcgccgag aacgaccgcc accacgccct ggacgccgtg gtggtggcct gcagcaccgt
     2581 ggccatgcag cagaagatca cccgcttcgt gcgctacaag gagatgaacg ccttcgacgg
     2641 taaaaccatc gacaaggaga ccggcgaggt gctgcaccag aagacccact tcccccagcc
     2701 ctgggagttc ttcgcccagg aggtgatgat ccgcgtgttc ggcaagcccg acggcaagcc
     2761 cgagttcgag gaggccgaca cccccgagaa gctgcgcacc ctgctggccg agaagctgag
     2821 cagccgccct gaggccgtgc acgagtacgt gactcctctg ttcgtgagcc gcgcccccaa
     2881 ccgcaagatg agcggtcagg gtcacatgga gaccgtgaag agcgccaagc gcctggacga
     2941 gggcgtgagc gtgctgcgcg tgcccctgac ccagctgaag ctgaaggacc tggagaagat
     3001 ggtgaaccgc gagcgcgagc ccaagctgta cgaggccctg aaggcccgcc tggaggccca
     3061 caaggacgac cccgccaagg ccttcgccga gcccttctac aagtacgaca aggccggcaa
     3121 ccgcacccag caggtgaagg ccgtgcgcgt ggagcaggtg cagaagaccg gcgtgtgggt
     3181 gcgcaaccac aacggcatcg ccgacaacgc caccatggtg cgcgtggacg tgttcgagaa
     3241 gggcgacaag tactacctgg tgcccatcta cagctggcag gtggccaagg gcatcctgcc
     3301 cgaccgcgcc gtggtgcagg gcaaggacga ggaggactgg cagctgatcg acgacagctt
     3361 caacttcaag ttcagcctgc accccaacga cctggtggag gtgatcacca agaaggcccg
     3421 catgttcggc tacttcgcca gctgccaccg cggcaccggc aacatcaaca tccgcatcca
     3481 cgacctggac cacaagatcg gcaagaacgg catcctggag ggcatcggcg tgaagaccgc
     3541 cctgagcttc cagaagtacc agatcgacga gctgggcaag gagatccgcc cctgccgcct
     3601 gaagaagcgc cctcctgtgc gctaagacaa gcccaagctg gacttctaac atccgcggca
     3661 ccgcccactc cagaaaaaat tagcgcaaga agacaaaaat caccttgcgc taatgctctg
     3721 ttacaggtca ccttattgtc ttatttatat acaaatagat tattgactta tcatctcaca
     3781 acggctagaa tcccaaacat attgtcgcac tgcgaaatga gaaccgttgc tacaataagg
     3841 ccgtctgaaa agatgtgccg caacgctctg ccccttaaag cttctgcttt aaggggcatc
     3901 gtttatttgt tgcctgcagg ctcccatctt gggacaatga aaacgttagt catggcgcgc
     3961 cttgacggct agctcagtcc taggtacagt gctagcttaa ttagtctacg aggttttaga
     4021 gctatgctgt tttgaatggt cccaaaacct gcagacaagc ccggccggcc taaggcgatg
     4081 ccccctcgac ctcgatcagg gaggcgttca ggacgactca caaagaaagc cgggcaatgc
     4141 ccggcttttt ccacgcctcc tgggctgact tcaggtgcta catttgaaga gataaattgc
     4201 actgaaatct agagcggttc agtagaaaag atcaaaggat cttcttgaga tccttttttt
     4261 ctgcgcgtaa tcttttgccc tgtaaacgaa aaaaccacct ggggaggtgg tttgatcgaa
     4321 ggttaagtca gttggggaac tgcttaaccg tggtaactgg ctttcgcaga gcacagcaac
     4381 caaatctgtc cttccagtgt agccggactt tggcgcacac ttcaagagca accgcgtgtt
     4441 tagctaaaca aatcctctgc gaactcccag ttaccaatgg ctgctgccag tggcgtttta
     4501 ccgtgctttt ccgggttgga ctcaagtgaa cagttaccgg ataaggcgca gcagtcgggc
     4561 tgaacgggga gttcttgctt acagcccagc ttggagcgaa cgacctacac cgagccgaga
     4621 taccagtgtg tgagctatga gaaagcgcca cacttcccgt aagggagaaa ggcggaacag
     4681 gtatccggta aacggcaggg tcggaacagg agagcgcaag agggagcgac ccgccggaaa
     4741 cggtggggat ctttaagtcc tgtcgggttt cgcccgtact gtcagattca tggttgagcc
     4801 tcacggctcc cacagatgca ccggaaaagc gtctgtttat gtgaactctg gcaggagggc
     4861 ggagcctatg gaaaaacgcc accggcgcgg ccctgctgtt ttgcctcaca tgttagtccc
     4921 ctgcttatcc acggaatctg tgggtaactt tgtatgtgtc cgcagcgccc gccgcagtct
     4981 cacgcccgga gcgtagcgac cgagtgagct agctatttgt ttatttttct aaatacattc
     5041 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     5101 gaagagtatg agggaagcgg tgatcgccga agtatcgact caactatcag aggtagttgg
     5161 cgtcatcgag cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt
     5221 ggatggcggc ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct
     5281 tgatgaaaca acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg
     5341 agagagcgag attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc
     5401 gtggcgttat ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct
     5461 tgcaggtatc ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc
     5521 aagagaacat agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc
     5581 tgaacaggat ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga
     5641 ctgggctggc gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt
     5701 aaccggcaaa atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc
     5761 ccagtatcag cccgtcatac ttgaagctag acaggcttat cttggacaag aagaagatcg
     5821 cttggcctcg cgcgcagatc agttggaaga atttgtccac tacgtgaaag gcgagatcac
     5881 caaggtagtc ggcaaataat gtctaacaat tcgttcaagc cgaggggccg caagatccgg
     5941 ccacgatgac ccggtcgtcg gttcagggca gggt
//
