LOCUS       sequence-248640-        6862 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial T. denticola Cas9 (TD) + tracrRNA expression, 
            cloDF13/spectinomycin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 6595 to 6629)
  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 6862)
  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-TDcas
FEATURES             Location/Qualifiers
     source          1..6862
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..739
                     /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        787..878
                     /gene="bla"
                     /label=AmpR promoter
     CDS             879..1670
                     /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"
     terminator      1898..1925
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        6595..6629
                     /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   6646..6681
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
ORIGIN
        1 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atcttttgcc ctgtaaacga
       61 aaaaaccacc tggggaggtg gtttgatcga aggttaagtc agttggggaa ctgcttaacc
      121 gtggtaactg gctttcgcag agcacagcaa ccaaatctgt ccttccagtg tagccggact
      181 ttggcgcaca cttcaagagc aaccgcgtgt ttagctaaac aaatcctctg cgaactccca
      241 gttaccaatg gctgctgcca gtggcgtttt accgtgcttt tccgggttgg actcaagtga
      301 acagttaccg gataaggcgc agcagtcggg ctgaacgggg agttcttgct tacagcccag
      361 cttggagcga acgacctaca ccgagccgag ataccagtgt gtgagctatg agaaagcgcc
      421 acacttcccg taagggagaa aggcggaaca ggtatccggt aaacggcagg gtcggaacag
      481 gagagcgcaa gagggagcga cccgccggaa acggtgggga tctttaagtc ctgtcgggtt
      541 tcgcccgtac tgtcagattc atggttgagc ctcacggctc ccacagatgc accggaaaag
      601 cgtctgttta tgtgaactct ggcaggaggg cggagcctat ggaaaaacgc caccggcgcg
      661 gccctgctgt tttgcctcac atgttagtcc cctgcttatc cacggaatct gtgggtaact
      721 ttgtatgtgt ccgcagcgcc cgccgcagtc tcacgcccgg agcgtagcga ccgagtgagc
      781 tagctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
      841 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagggaagcg gtgatcgccg
      901 aagtatcgac tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt
      961 tgctggccgt acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata
     1021 ttgatttgct ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca
     1081 acgacctttt ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag
     1141 tcaccattgt tgtgcacgac gacatcattc cttggcgtta tccagctaag cgcgaactgc
     1201 aatttggaga atggcagcgc aatgacattc ttgcgggtat cttcgagcca gccacgatcg
     1261 acattgatct ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc
     1321 cagcggcgga ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg
     1381 aaaccttaac gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc
     1441 ttacgttgtc ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg
     1501 ctgccgactg ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta
     1561 gacaggctta tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag
     1621 aatttgtcca ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa tgtctaacaa
     1681 ttcgttcaag ccgaggggcc gcaagatccg gccacgatga cccggtcgtc ggttcagggc
     1741 agggtaccag gcacgcctaa ccgtcagtga gattggatga gtgaacgata ttgatcgaga
     1801 agagccctgc gcagccgctg ccgtgcctgc aggaagcaac ggcccggagg gtggcgggca
     1861 ggacgcccgc cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc
     1921 ctttttgcgt ttctacaaac tctgctagct tctagagcac agctaacacc acgtcgtccc
     1981 tatctgctgc cctaggtcta tgagtggttg ctggataact ttacgggcat gcataaggct
     2041 cgtatgatat attcagggag accacaacgg tttccctcta caaataattt tgtttaactt
     2101 ttactagagg aggaggcaaa aatgaagaag gagatcaagg actacttcct gggcctggac
     2161 gtgggcaccg gcagcgtggg ctgggccgtg accgacaccg actacaagct gctgaaggcc
     2221 aaccgcaagg acctgtgggg catgcgctgc ttcgagactg ctgagaccgc cgaggtgcgc
     2281 cggctgcacc gcggtgctcg ccggcgcatc gagcgccgca agaagcgcat caagctgctg
     2341 caggagctgt tcagccagga gatcgccaag accgacgagg gcttcttcca gcgcatgaag
     2401 gagagcccct tctacgccga ggacaagacc atcctgcagg agaacaccct gttcaacgac
     2461 aaggacttcg ccgacaagac ctaccacaag gcctacccca ccatcaacca cctgatcaag
     2521 gcctggatcg agaacaaggt gaagcccgac cctcgcctgc tgtacctggc ctgccacaac
     2581 atcatcaaga agcgcggcca cttcctgttc gagggcgact tcgacagcga gaaccagttc
     2641 gacaccagca tccaggccct gttcgagtac ctgcgcgagg acatggaggt ggacatcgac
     2701 gccgacagcc agaaggtgaa ggagatcctg aaggacagca gcctgaagaa cagcgagaag
     2761 cagagccgcc tgaacaagat cctgggcctg aagcccagcg acaagcagaa gaaggccatc
     2821 accaacctga tcagcggcaa caagatcaac ttcgccgacc tgtacgacaa ccccgacctg
     2881 aaggacgccg agaagaacag catcagcttc agcaaggacg acttcgacgc cctgagcgac
     2941 gacctggcca gcatcctggg cgacagcttc gagctgctgc tgaaggccaa ggccgtgtac
     3001 aactgcagcg tgctgagcaa ggtgatcggc gacgagcagt acctgagctt cgccaaggtg
     3061 aagatctacg agaagcacaa gaccgacctt accaagctga agaacgtgat caagaagcac
     3121 ttccccaagg actacaagaa ggtgttcggc tacaacaaga acgagaagaa caacaacaac
     3181 tacagcggct acgtgggcgt gtgcaagacc aagagcaaga agctgatcat caacaacagc
     3241 gtgaaccagg aggacttcta caagttcctg aagaccatcc tgagcgccaa gagcgagatc
     3301 aaggaggtga acgacatcct gaccgagatc gagaccggca ccttcctgcc caagcagatc
     3361 agcaagagca acgccgagat cccctaccag ctgcgcaaga tggagctgga gaagatcctg
     3421 agcaacgccg agaagcactt cagcttcctg aagcagaagg acgagaaggg cctgagccac
     3481 agcgagaaga tcatcatgct gctgaccttc aagatcccct actacatcgg ccccatcaac
     3541 gacaaccaca agaagttctt ccccgaccgc tgctgggtgg tgaagaagga gaagagcccc
     3601 agcggcaaga ccaccccctg gaacttcttc gaccacatcg acaaggagaa gaccgccgag
     3661 gccttcatca ccagccgcac caacttctgc acatacctgg tgggcgagag cgtgctgccc
     3721 aagagcagcc tgctgtacag cgagtacacc gtgctgaacg agatcaacaa cctgcagatc
     3781 atcatcgacg gcaagaacat ctgcgacatc aagctgaagc agaagatcta cgaggacctg
     3841 ttcaagaagt acaagaagat cacccagaag cagatcagca ccttcatcaa gcacgatggg
     3901 catctgcaac aagaccgacg aggtgatcat cctgggcatc gacaaggagt gcaccagcag
     3961 cctgaagagc tacatcgagc tgaagaacat cttcggcaag caggtggacg agatcagcac
     4021 caagaacatg ctggaggaga tcatccgctg ggccaccatc tacgacgagg gcgagggcaa
     4081 gaccatcctg aagaccaaga tcaaggccga gtacggcaag tactgcagcg acgagcagat
     4141 caagaagatc ctgaacctga agttcagcgg ctggggccgc ctgagccgca agttcctgga
     4201 gaccgtgacc agcgagatgc ccggcttcag cgagcccgtg aacatcatca ccgccatgcg
     4261 cgagacccag aacaacctga tggagctgct gagcagcgag ttcaccttca ccgagaacat
     4321 caagaagatc aacagcggct tcgaggacgc cgagaagcag ttcagctacg acggcctggt
     4381 gaagcccctg ttcctgagcc ccagcgtgaa gaagatgctg tggcagaccc tgaagctggt
     4441 gaaggagatc agccacatca cccaggctcc tcctaagaag atcttcatcg agatggccaa
     4501 gggcgccgag ctggagcctg ctcgcaccaa gacccgcctg aagatcctgc aggacctgta
     4561 caacaactgc aagaacgacg ccgacgcatt cagcagcgag atcaaggacc tgagcggcaa
     4621 gatcgagaac gaggacaacc tgcgcctgcg cagcgacaag ctgtacctgt actacaccca
     4681 gctgggcaag tgcatgtact gcggcaagcc catcgagatc ggccacgtgt tcgacaccag
     4741 caactacgac atcgaccaca tctaccccca gagcaagatc aaggacgaca gcatcagcaa
     4801 ccgcgtgctg gtgtgcagca gctgcaacaa gaacaaggag gacaagtacc ctctgaagag
     4861 cgagatccag agcaagcagc gcggcttctg gaacttcctg cagcgcaaca acttcatcag
     4921 cctggagaag ctgaaccgcc tgacccgcgc cacccccatc agcgacgacg agaccgccaa
     4981 gttcatcgcc cgccagctgg tggagactcg ccaagctacc aaggtggccg ccaaggtgct
     5041 ggagaagatg ttccccgaga ccaagatcgt gtacagcaag gccgagaccg tgagcatgtt
     5101 ccgcaacaag ttcgacatcg tgaagtgccg cgagatcaac gacttccacc acgcccacga
     5161 cgcctacctg aacatcgtgg tgggcaacgt gtacaacacc aagttcacca acaacccctg
     5221 gaatttcatt aaggagaagc gcgacaaccc caagatcgcc gacacctaca actactacaa
     5281 ggtgttcgac tacgacgtga agcgcaacaa catcaccgcc tgggagaagg gcaagaccat
     5341 catcaccgtg aaggacatgc tgaagcgcaa cacccccatc tacacccgcc aggccgcctg
     5401 caagaagggc gagctgttca accagaccat catgaagaag ggcctgggcc agcaccccct
     5461 gaagaaggag ggccccttca gcaacatcag caagtacggc ggctacaaca aggtgagcgc
     5521 cgcctactac accctgatcg agtacgagga gaagggcaac aagatccgca gcctggagac
     5581 catccccctg tacctggtga aggacatcca gaaggaccag gacgtgctga agagctacct
     5641 gaccgacctg ctgggcaaga aggagttcaa gatcctggtg cccaagatca agatcaacag
     5701 cctgctgaag atcaacggct tcccctgcca catcaccggc aagaccaacg acagcttcct
     5761 gctgcgcccc gccgtgcagt tctgctgcag caacaacgag gtgctgtact tcaagaagat
     5821 catccgcttc agcgagatcc gcagccagcg cgagaagatc ggcaagacca tcagccccta
     5881 cgaggacctg agcttccgca gctacatcaa ggagaacctg tggaagaaga ccaagaacga
     5941 cgagatcggc gagaaggagt tctacgacct gctgcagaag aagaacctgg agatctacga
     6001 catgctgctg accaagcaca aggacaccat ctacaagaag cgccccaaca gcgccaccat
     6061 cgacatcctg gtgaagggca aggagaagtt caagagcctg atcatcgaga accagttcga
     6121 ggtgatcctg gagatcctga agctgttcag cgccacccgc aacgtgagcg acctgcagca
     6181 catcggcggc agcaagtaca gcggcgtggc caagatcggc aacaagatca gcagcctgga
     6241 caactgcatc ctgatctacc agagcatcac cggcatcttc gagaagcgca tcgacctgct
     6301 gaaggtgtaa gctaagcttg gtgagggata acccgaccgt ccgctgaata aaaaacgccc
     6361 ggcggcaacc gagcgttctg ggaccttgac cattccttta aaaagtatta taataacagc
     6421 atacacaatt taagatccat cttaaattac acaacgagtt caaataagaa ttcatcaaaa
     6481 tcgtcccttt tgggaccgct cattgtggag catcaaggct taacatggtt aagccttttt
     6541 tttcatccgc ggcaccgcat cttgggacaa tgaaaacgtt agtcatggcg cgccttgacg
     6601 gctagctcag tcctaggtac agtgctagct taattagtct acgaggtttt agagctatgc
     6661 tgttttgaat ggtcccaaaa cctgcagaca agcccggccg gcctaaggcg atgccccctc
     6721 gacctcgatc agggaggcgt tcaggacgac tcacaaagaa agccgggcaa tgcccggctt
     6781 tttccacgcc tcctgggctg acttcaggtg ctacatttga agagataaat tgcactgaaa
     6841 tctagagcgg ttcagtagaa aa
//
