LOCUS       sequence-71732-e        6028 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial nuclease-null ST1 Cas9 expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 4016 to 4050)
  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 6028)
  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-ST1casN-
FEATURES             Location/Qualifiers
     source          1..6028
                     /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             join(377..400,401..403,404..2167,2168..2173,2174..2239,
                     2240..2242,2243..3739)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus thermophilus Type II 
                     CRISPR/Cas system"
                     /label=St1dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D9A, D598A, 
                     H599A, and N622A"
                     /translation="MSDLVLGLAIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLV
                     RRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEE
                     LFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQTYG
                     QLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINRYLEILTGK
                     RKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNLL
                     NDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRID
                     KSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADG
                     SFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKT
                     TSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNED
                     DEKKAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERC
                     LYTGKTISIHDLINNSNQFEVAAILPLSITFDDSLANKVLVYATAAQEKGQRTPYQALD
                     SMDDAWSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVV
                     LNALQEHFRAHKIDTKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNL
                     WKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSY
                     QVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSK
                     FLMYRHDPQTFEKVIEPILENYPNKQINDKGKEVPCNPFLKYKEEHGYIRKYSKKGNGP
                     EIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQ
                     FDKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRT
                     MPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQ
                     HIIKNEGDKPKLDF"
     promoter        4016..4050
                     /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   4067..4102
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     rep_origin      4284..5022
                     /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        5070..5161
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5162..5953
                     /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 gcgacctggt gctgggcctg gccatcggca tcggcagcgt
      421 gggcgtgggc atcctgaaca aggtgaccgg cgagatcatc cacaagaaca gtcgcatctt
      481 ccctgctgct caggctgaga acaacctggt gcgccgcacc aaccgccagg gtcgccggct
      541 tgctcgccgc aagaagcacc ggcgcgtgcg cctgaaccgc ctgttcgagg agagcggcct
      601 gatcaccgac ttcaccaaga tcagcatcaa cctgaacccc taccagctgc gcgtgaaggg
      661 cctgaccgac gagctgagca acgaggagct gttcatcgcc ctgaagaaca tggtgaagca
      721 ccgcggcatc agctacctgg acgacgccag cgacgacggc aacagcagcg tgggcgacta
      781 cgcccagatc gtgaaggaga acagcaagca gctggagacc aagacccccg gccagatcca
      841 gctggagcgc taccagacct acggccagct gcgcggcgac ttcaccgtgg agaaggacgg
      901 caagaagcac cgcctgatca acgtgttccc caccagcgcc taccgcagcg aggccctgcg
      961 catcctgcag acccagcagg agttcaaccc ccagatcacc gacgagttca tcaaccgcta
     1021 cctggagatc ctgaccggca agcgcaagta ctaccacggc cccggcaacg agaagagccg
     1081 caccgactac ggccgctacc gcaccagcgg cgagaccctg gacaacatct tcggcatcct
     1141 gatcggcaag tgcaccttct accccgacga gttccgcgcc gccaaggcca gctacaccgc
     1201 ccaggagttc aacctgctga acgacctgaa caacctgacc gtgcccaccg agaccaagaa
     1261 gctgagcaag gagcagaaga accagatcat caactacgtg aagaacgaga aggccatggg
     1321 ccccgccaag ctgttcaagt acatcgccaa gctgctgagc tgcgacgtgg ccgacatcaa
     1381 gggctaccgc atcgacaaga gcggcaaggc cgagatccac accttcgagg cctaccgcaa
     1441 gatgaagacc ctggagaccc tggacatcga gcagatggac cgcgagaccc tggacaagct
     1501 ggcctacgtg ctgaccctga acaccgagcg cgagggcatc caggaggccc tggagcacga
     1561 gttcgccgac ggcagcttca gccagaagca ggtggacgag ctggtgcagt tccgcaaggc
     1621 caacagcagc atcttcggca agggctggca caacttcagc gtgaagctga tgatggagct
     1681 gatccccgag ctgtacgaga ccagcgagga gcagatgacc atcctgaccc gcctgggcaa
     1741 gcagaagacc accagcagca gcaacaagac caagtacatc gacgagaagc tgctgaccga
     1801 ggagatctac aaccccgtgg tggccaagag cgtgcgccag gccatcaaga tcgtgaacgc
     1861 cgccatcaag gagtacggcg acttcgacaa catcgtgatc gagatggccc gcgagaccaa
     1921 cgaggacgac gagaagaagg ccatccagaa gatccagaag gccaacaagg acgagaagga
     1981 cgccgccatg ctgaaggccg ccaaccagta caacggcaag gccgagctgc cccacagcgt
     2041 gttccacggc cacaagcagc tggccaccaa gatccgcctg tggcaccagc agggcgagcg
     2101 ctgcctgtac accggcaaga ccatcagcat ccacgacctg atcaacaaca gcaaccagtt
     2161 cgaggtggct gccatcctgc ccctgagcat caccttcgac gacagcctgg ccaacaaggt
     2221 gctggtgtac gccaccgccg ctcaggagaa gggccagcgc accccctacc aggccctgga
     2281 cagcatggac gacgcctgga gcttccgcga gctgaaggcc ttcgtgcgcg agagcaagac
     2341 cctgagcaac aagaagaagg agtacctgct gaccgaggag gacatcagca agttcgacgt
     2401 gcgcaagaag ttcatcgagc gcaacctggt ggacacccgc tacgccagcc gcgtggtgct
     2461 gaacgccctg caggagcact tccgcgccca caagatcgac accaaggtga gcgtggtgcg
     2521 cggccagttc accagccagc tgcgccgcca ctggggcatc gagaagaccc gcgacaccta
     2581 ccaccaccac gccgtggacg ccctgatcat tgcggcttct agccagctga acctgtggaa
     2641 gaagcagaag aacaccctgg tgagctacag cgaggaccag ctgctggaca tcgagaccgg
     2701 cgagctgatc agcgacgacg agtacaagga gagcgtgttc aaggccccct accagcactt
     2761 cgtggacacc ctgaagagca aggagttcga ggacagcatc ctgttcagct accaggtgga
     2821 cagcaagttc aaccgcaaga tcagcgacgc caccatctac gccacccgcc aggccaaggt
     2881 gggcaaggac aaggccgacg agacctacgt gctgggcaag atcaaggaca tctacaccca
     2941 ggacggctac gacgccttca tgaagatcta caagaaggac aagagcaagt tcctgatgta
     3001 ccgccacgac ccccagacct tcgagaaggt gatcgagccc atcctggaga actaccccaa
     3061 caagcagatc aacgataaag gcaaggaggt gccctgcaac cccttcctga agtacaagga
     3121 ggagcacggc tacatccgca agtacagcaa gaagggcaac ggccccgaga tcaagagcct
     3181 gaagtactac gacagcaagc tgggcaacca catcgacatc acccccaagg acagcaacaa
     3241 caaggtggtg ctgcagagcg tgagcccctg gcgcgccgac gtgtacttca acaagaccac
     3301 cggcaagtac gagatcctgg gcctgaagta cgccgacctg cagtttgata agggcaccgg
     3361 cacctacaag atcagccagg agaagtacaa cgacatcaag aagaaggagg gcgtggacag
     3421 cgacagcgag ttcaagttca ccctgtacaa gaacgacctt ctgctggtga aggacaccga
     3481 gaccaaggag caacagctgt tccgcttcct gagccgcacc atgcccaagc agaagcacta
     3541 cgtggagctg aagccctacg acaagcagaa gttcgagggc ggcgaggccc tgatcaaggt
     3601 gctgggcaac gtggccaaca gcggccagtg caagaagggc ctgggcaaga gcaacatcag
     3661 catctacaag gtgcgcaccg acgtgctggg caaccagcac atcatcaaga acgagggcga
     3721 caagcccaag ctggacttct aacatccgcg gcaccgccta agctagattt ttaatattaa
     3781 ttgttagaaa gtgttgcaat tatagttatc atatactata ataatagtgt aagggacgcc
     3841 ttacacagtt acttaaatct tgcagaagct acaaagataa ggcttcatgc cgaaatcaac
     3901 accctgtcat tttatggcag ggtgttttcg ttatttaaag aggagaagaa atgacttgga
     3961 gagttgcctg caggctccca tcttgggaca atgaaaacgt tagtcatggc gcgccttgac
     4021 ggctagctca gtcctaggta cagtgctagc ttaattagtc tacgaggttt tagagctatg
     4081 ctgttttgaa tggtcccaaa acctgcagac aagcccggcc ggcctaaggc gatgccccct
     4141 cgacctcgat cagggaggcg ttcaggacga ctcacaaaga aagccgggca atgcccggct
     4201 ttttccacgc ctcctgggct gacttcaggt gctacatttg aagagataaa ttgcactgaa
     4261 atctagagcg gttcagtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     4321 gtaatctttt gccctgtaaa cgaaaaaacc acctggggag gtggtttgat cgaaggttaa
     4381 gtcagttggg gaactgctta accgtggtaa ctggctttcg cagagcacag caaccaaatc
     4441 tgtccttcca gtgtagccgg actttggcgc acacttcaag agcaaccgcg tgtttagcta
     4501 aacaaatcct ctgcgaactc ccagttacca atggctgctg ccagtggcgt tttaccgtgc
     4561 ttttccgggt tggactcaag tgaacagtta ccggataagg cgcagcagtc gggctgaacg
     4621 gggagttctt gcttacagcc cagcttggag cgaacgacct acaccgagcc gagataccag
     4681 tgtgtgagct atgagaaagc gccacacttc ccgtaaggga gaaaggcgga acaggtatcc
     4741 ggtaaacggc agggtcggaa caggagagcg caagagggag cgacccgccg gaaacggtgg
     4801 ggatctttaa gtcctgtcgg gtttcgcccg tactgtcaga ttcatggttg agcctcacgg
     4861 ctcccacaga tgcaccggaa aagcgtctgt ttatgtgaac tctggcagga gggcggagcc
     4921 tatggaaaaa cgccaccggc gcggccctgc tgttttgcct cacatgttag tcccctgctt
     4981 atccacggaa tctgtgggta actttgtatg tgtccgcagc gcccgccgca gtctcacgcc
     5041 cggagcgtag cgaccgagtg agctagctat ttgtttattt ttctaaatac attcaaatat
     5101 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     5161 tatgagggaa gcggtgatcg ccgaagtatc gactcaacta tcagaggtag ttggcgtcat
     5221 cgagcgccat ctcgaaccga cgttgctggc cgtacatttg tacggctccg cagtggatgg
     5281 cggcctgaag ccacacagtg atattgattt gctggttacg gtgaccgtaa ggcttgatga
     5341 aacaacgcgg cgagctttga tcaacgacct tttggaaact tcggcttccc ctggagagag
     5401 cgagattctc cgcgctgtag aagtcaccat tgttgtgcac gacgacatca ttccgtggcg
     5461 ttatccagct aagcgcgaac tgcaatttgg agaatggcag cgcaatgaca ttcttgcagg
     5521 tatcttcgag ccagccacga tcgacattga tctggctatc ttgctgacaa aagcaagaga
     5581 acatagcgtt gccttggtag gtccagcggc ggaggaactc tttgatccgg ttcctgaaca
     5641 ggatctattt gaggcgctaa atgaaacctt aacgctatgg aactcgccgc ccgactgggc
     5701 tggcgatgag cgaaatgtag tgcttacgtt gtcccgcatt tggtacagcg cagtaaccgg
     5761 caaaatcgcg ccgaaggatg tcgctgccga ctgggcaatg gagcgcctgc cggcccagta
     5821 tcagcccgtc atacttgaag ctagacaggc ttatcttgga caagaagaag atcgcttggc
     5881 ctcgcgcgca gatcagttgg aagaatttgt ccactacgtg aaaggcgaga tcaccaaggt
     5941 agtcggcaaa taatgtctaa caattcgttc aagccgaggg gccgcaagat ccggccacga
     6001 tgacccggtc gtcggttcag ggcagggt
//
