LOCUS       sequence-71734-e        6861 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial nuclease-null TD Cas9 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 4849 to 4883)
  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 6861)
  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-TDcasN-
FEATURES             Location/Qualifiers
     source          1..6861
                     /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"
     promoter        4849..4883
                     /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   4900..4935
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     rep_origin      5117..5855
                     /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        5903..5994
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5995..6786
                     /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 gcaaaaatga agaaggagat caaggactac ttcctgggcc tggccgtggg
      421 caccggcagc gtgggctggg ccgtgaccga caccgactac aagctgctga aggccaaccg
      481 caaggacctg tggggcatgc gctgcttcga gactgctgag accgccgagg tgcgccggct
      541 gcaccgcggt gctcgccggc gcatcgagcg ccgcaagaag cgcatcaagc tgctgcagga
      601 gctgttcagc caggagatcg ccaagaccga cgagggcttc ttccagcgca tgaaggagag
      661 ccccttctac gccgaggaca agaccatcct gcaggagaac accctgttca acgacaagga
      721 cttcgccgac aagacctacc acaaggccta ccccaccatc aaccacctga tcaaggcctg
      781 gatcgagaac aaggtgaagc ccgaccctcg cctgctgtac ctggcctgcc acaacatcat
      841 caagaagcgc ggccacttcc tgttcgaggg cgacttcgac agcgagaacc agttcgacac
      901 cagcatccag gccctgttcg agtacctgcg cgaggacatg gaggtggaca tcgacgccga
      961 cagccagaag gtgaaggaga tcctgaagga cagcagcctg aagaacagcg agaagcagag
     1021 ccgcctgaac aagatcctgg gcctgaagcc cagcgacaag cagaagaagg ccatcaccaa
     1081 cctgatcagc ggcaacaaga tcaacttcgc cgacctgtac gacaaccccg acctgaagga
     1141 cgccgagaag aacagcatca gcttcagcaa ggacgacttc gacgccctga gcgacgacct
     1201 ggccagcatc ctgggcgaca gcttcgagct gctgctgaag gccaaggccg tgtacaactg
     1261 cagcgtgctg agcaaggtga tcggcgacga gcagtacctg agcttcgcca aggtgaagat
     1321 ctacgagaag cacaagaccg accttaccaa gctgaagaac gtgatcaaga agcacttccc
     1381 caaggactac aagaaggtgt tcggctacaa caagaacgag aagaacaaca acaactacag
     1441 cggctacgtg ggcgtgtgca agaccaagag caagaagctg atcatcaaca acagcgtgaa
     1501 ccaggaggac ttctacaagt tcctgaagac catcctgagc gccaagagcg agatcaagga
     1561 ggtgaacgac atcctgaccg agatcgagac cggcaccttc ctgcccaagc agatcagcaa
     1621 gagcaacgcc gagatcccct accagctgcg caagatggag ctggagaaga tcctgagcaa
     1681 cgccgagaag cacttcagct tcctgaagca gaaggacgag aagggcctga gccacagcga
     1741 gaagatcatc atgctgctga ccttcaagat cccctactac atcggcccca tcaacgacaa
     1801 ccacaagaag ttcttccccg accgctgctg ggtggtgaag aaggagaaga gccccagcgg
     1861 caagaccacc ccctggaact tcttcgacca catcgacaag gagaagaccg ccgaggcctt
     1921 catcaccagc cgcaccaact tctgcacata cctggtgggc gagagcgtgc tgcccaagag
     1981 cagcctgctg tacagcgagt acaccgtgct gaacgagatc aacaacctgc agatcatcat
     2041 cgacggcaag aacatctgcg acatcaagct gaagcagaag atctacgagg acctgttcaa
     2101 gaagtacaag aagatcaccc agaagcagat cagcaccttc atcaagcacg agggcatctg
     2161 caacaagacc gacgaggtga tcatcctggg catcgacaag gagtgcacca gcagcctgaa
     2221 gagctacatc gagctgaaga acatcttcgg caagcaggtg gacgagatca gcaccaagaa
     2281 catgctggag gagatcatcc gctgggccac catctacgac gagggcgagg gcaagaccat
     2341 cctgaagacc aagatcaagg ccgagtacgg caagtactgc agcgacgagc agatcaagaa
     2401 gatcctgaac ctgaagttca gcggctgggg ccgcctgagc cgcaagttcc tggagaccgt
     2461 gaccagcgag atgcccggct tcagcgagcc cgtgaacatc atcaccgcca tgcgcgagac
     2521 ccagaacaac ctgatggagc tgctgagcag cgagttcacc ttcaccgaga acatcaagaa
     2581 gatcaacagc ggcttcgagg acgccgagaa gcagttcagc tacgacggcc tggtgaagcc
     2641 cctgttcctg agccccagcg tgaagaagat gctgtggcag accctgaagc tggtgaagga
     2701 gatcagccac atcacccagg ctcctcctaa gaagatcttc atcgagatgg ccaagggcgc
     2761 cgagctggag cctgctcgca ccaagacccg cctgaagatc ctgcaggacc tgtacaacaa
     2821 ctgcaagaac gacgccgacg cattcagcag cgagatcaag gacctgagcg gcaagatcga
     2881 gaacgaggac aacctgcgcc tgcgcagcga caagctgtac ctgtactaca cccagctggg
     2941 caagtgcatg tactgcggca agcccatcga gatcggccac gtgttcgaca ccagcaacta
     3001 cgacatcgct gctatctacc cccagagcaa gatcaaggac gacagcatca gcaaccgcgt
     3061 gctggtgtgc agcagctgcg ccaagaacaa ggaggacaag taccctctga agagcgagat
     3121 ccagagcaag cagcgcggct tctggaactt cctgcagcgc aacaacttca tcagcctgga
     3181 gaagctgaac cgcctgaccc gcgccacccc catcagcgac gacgagaccg ccaagttcat
     3241 cgcccgccag ctggtggaga ctcgccaagc taccaaggtg gccgccaagg tgctggagaa
     3301 gatgttcccc gagaccaaga tcgtgtacag caaggccgag accgtgagca tgttccgcaa
     3361 caagttcgac atcgtgaagt gccgcgagat caacgacttc caccacgccc acgacgccta
     3421 cctgaacatc gtggtgggca acgtgtacaa caccaagttc accaacaacc cctggaattt
     3481 cattaaggag aagcgcgaca accccaagat cgccgacacc tacaactact acaaggtgtt
     3541 cgactacgac gtgaagcgca acaacatcac cgcctgggag aagggcaaga ccatcatcac
     3601 cgtgaaggac atgctgaagc gcaacacccc catctacacc cgccaggccg cctgcaagaa
     3661 gggcgagctg ttcaaccaga ccatcatgaa gaagggcctg ggccagcacc ccctgaagaa
     3721 ggagggcccc ttcagcaaca tcagcaagta cggcggctac aacaaggtga gcgccgccta
     3781 ctacaccctg atcgagtacg aggagaaggg caacaagatc cgcagcctgg agaccatccc
     3841 cctgtacctg gtgaaggaca tccagaagga ccaggacgtg ctgaagagct acctgaccga
     3901 cctgctgggc aagaaggagt tcaagatcct ggtgcccaag atcaagatca acagcctgct
     3961 gaagatcaac ggcttcccct gccacatcac cggcaagacc aacgacagct tcctgctgcg
     4021 ccccgccgtg cagttctgct gcagcaacaa cgaggtgctg tacttcaaga agatcatccg
     4081 cttcagcgag atccgcagcc agcgcgagaa gatcggcaag accatcagcc cctacgagga
     4141 cctgagcttc cgcagctaca tcaaggagaa cctgtggaag aagaccaaga acgacgagat
     4201 cggcgagaag gagttctacg acctgctgca gaagaagaac ctggagatct acgacatgct
     4261 gctgaccaag cacaaggaca ccatctacaa gaagcgcccc aacagcgcca ccatcgacat
     4321 cctggtgaag ggcaaggaga agttcaagag cctgatcatc gagaaccagt tcgaggtgat
     4381 cctggagatc ctgaagctgt tcagcgccac ccgcaacgtg agcgacctgc agcacatcgg
     4441 cggcagcaag tacagcggcg tggccaagat cggcaacaag atcagcagcc tggacaactg
     4501 catcctgatc taccagagca tcaccggcat cttcgagaag cgcatcgacc tgctgaaggt
     4561 gtaagctaag cttggtgagg gataacccga ccgtccgctg aataaaaaac gcccggcggc
     4621 aaccgagcgt tctgggacct tgaccattcc tttaaaaagt attataataa cagcatacac
     4681 aatttaagat ccatcttaaa ttacacaacg agttcaaata agaattcatc aaaatcgtcc
     4741 cttttgggac cgctcattgt ggagcatcaa ggcttaacat ggttaagcct tttttttcat
     4801 ccgcggcacc gcatcttggg acaatgaaaa cgttagtcat ggcgcgcctt gacggctagc
     4861 tcagtcctag gtacagtgct agcttaatta gtctacgagg ttttagagct atgctgtttt
     4921 gaatggtccc aaaacctgca gacaagcccg gccggcctaa ggcgatgccc cctcgacctc
     4981 gatcagggag gcgttcagga cgactcacaa agaaagccgg gcaatgcccg gctttttcca
     5041 cgcctcctgg gctgacttca ggtgctacat ttgaagagat aaattgcact gaaatctaga
     5101 gcggttcagt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5161 tttgccctgt aaacgaaaaa accacctggg gaggtggttt gatcgaaggt taagtcagtt
     5221 ggggaactgc ttaaccgtgg taactggctt tcgcagagca cagcaaccaa atctgtcctt
     5281 ccagtgtagc cggactttgg cgcacacttc aagagcaacc gcgtgtttag ctaaacaaat
     5341 cctctgcgaa ctcccagtta ccaatggctg ctgccagtgg cgttttaccg tgcttttccg
     5401 ggttggactc aagtgaacag ttaccggata aggcgcagca gtcgggctga acggggagtt
     5461 cttgcttaca gcccagcttg gagcgaacga cctacaccga gccgagatac cagtgtgtga
     5521 gctatgagaa agcgccacac ttcccgtaag ggagaaaggc ggaacaggta tccggtaaac
     5581 ggcagggtcg gaacaggaga gcgcaagagg gagcgacccg ccggaaacgg tggggatctt
     5641 taagtcctgt cgggtttcgc ccgtactgtc agattcatgg ttgagcctca cggctcccac
     5701 agatgcaccg gaaaagcgtc tgtttatgtg aactctggca ggagggcgga gcctatggaa
     5761 aaacgccacc ggcgcggccc tgctgttttg cctcacatgt tagtcccctg cttatccacg
     5821 gaatctgtgg gtaactttgt atgtgtccgc agcgcccgcc gcagtctcac gcccggagcg
     5881 tagcgaccga gtgagctagc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5941 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagg
     6001 gaagcggtga tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc
     6061 catctcgaac cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg
     6121 aagccacaca gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg
     6181 cggcgagctt tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt
     6241 ctccgcgctg tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca
     6301 gctaagcgcg aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc
     6361 gagccagcca cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc
     6421 gttgccttgg taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta
     6481 tttgaggcgc taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat
     6541 gagcgaaatg tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc
     6601 gcgccgaagg atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc
     6661 gtcatacttg aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc
     6721 gcagatcagt tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc
     6781 aaataatgtc taacaattcg ttcaagccga ggggccgcaa gatccggcca cgatgacccg
     6841 gtcgtcggtt cagggcaggg t
//
