LOCUS       sequence-169645-        8117 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pOP576 with gRNA key.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8117)
  AUTHORS   Purcell O, Wang J, Siuti P, Lu TK
  TITLE     Encryption and steganography of synthetic gene circuits.
  JOURNAL   Nat Commun. 2018 Nov 22;9(1):4942. doi: 10.1038/s41467-018-07144-7.
  PUBMED    30467337
REFERENCE   2  (bases 1 to 8117)
  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 
            Commun."; date: "2018-11-22"; volume: "9(1):4942. doi"; pages: " 
            10.1038/s41467-018-07144-7"
COMMENT     Sequence Label: pOP592
FEATURES             Location/Qualifiers
     source          1..8117
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(207..233,234..236,237..2723,2724..2726,2727..4313)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     promoter        4326..4360
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        4389..4464
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4476..4562
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        4597..4631
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        4660..4735
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      4747..4833
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        4868..4902
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     misc_RNA        4931..5006
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      5018..5104
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(5284..6234)
                     /codon_start=1
                     /gene="rep101 encoding an A56V mutation"
                     /product="temperature-sensitive version of the RepA protein
                     needed for replication with the pSC101 origin (Armstrong et
                     al., 1984)"
                     /label=Rep101(Ts)
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYVQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      6282..6504
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     CDS             complement(join(7126..7917,7918..7986))
                     /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     7749..7768
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7987..8091)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catcgattta gcacagctaa caccacgtcg tccctatctg ctgccctagg tctatgagtg
       61 gttgctggat aactttacgg gcatgcataa ggctcgtata atatattcag ggagaccaca
      121 acggtttccc tctacaaata attttgttta actttgcatg ccagttgtag atcagcatag
      181 catagacaac caaggaggta gtgcacatgg ataagaaata ctctattggt ttggctatcg
      241 gtacaaactc tgttggttgg gctgttatta ctgatgaata caaggttcca tccaagaagt
      301 tcaaggtttt gggtaacact gatagacact ccatcaaaaa gaacttgatt ggtgccttgt
      361 tgttcgattc tggtgaaact gctgaagcta ctagattgaa aagaaccgct agaagaagat
      421 acaccagaag aaagaacaga atctgctact tgcaagaaat cttctccaac gaaatggcca
      481 aggttgatga ttcattcttc cacagattgg aagaatcctt cttggtcgaa gaagataaga
      541 agcacgaaag acatccaatc ttcggtaaca tcgttgatga agttgcttac cacgaaaagt
      601 acccaactat ctaccatttg agaaagaagt tggttgactc taccgataag gctgatttga
      661 gattgatcta tttggctttg gcccacatga ttaagttcag aggtcatttc ttgatcgaag
      721 gtgatttgaa cccagataac tccgatgttg ataagttgtt catccaatta gtccaaacct
      781 acaatcaatt attcgaagaa aacccaatca acgcctctgg tgttgatgct aaagctattt
      841 tgtctgccag attgtccaag tccagaagat tagaaaattt gatcgcccaa ttaccaggtg
      901 aaaagaagaa tggtttgttc ggtaatttga ttgccttgtc tttgggtttg actccaaact
      961 tcaagtccaa tttcgatttg gctgaagatg ccaagttgca attatctaag gatacctacg
     1021 atgacgattt ggataacttg ttggctcaaa tcggtgatca atacgctgat ttgtttttgg
     1081 ctgctaagaa cttgtccgat gccattttgt tgtccgatat tttgagagtc aacaccgaaa
     1141 ttactaaggc tccattgtct gcctctatga tcaaaagata cgatgaacac caccaagact
     1201 tgactttgtt gaaggctttg gtcagacaac aattacctga aaagtacaaa gaaattttct
     1261 tcgatcaatc caagaacggt tacgccggtt atattgatgg tggtgcttct caagaagaat
     1321 tttacaagtt catcaagcca atcttggaaa agatggacgg tactgaagaa ttattggtca
     1381 agttgaacag agaagatttg ttgagaaagc aaagaacctt cgacaacggt tctattccac
     1441 atcaaattca cttgggtgaa ttgcacgcaa ttttgagaag acaagaagat ttttatccat
     1501 tcttgaagga caacagagaa aagatcgaaa agattctgac cttcagaatc ccttactacg
     1561 ttggtccatt ggctagaggt aattcaagat ttgcctggat gactagaaag tccgaagaaa
     1621 ctattactcc ttggaacttc gaagaagttg tagataaggg tgcttctgcc caatccttta
     1681 ttgaaagaat gaccaacttc gacaagaact tgccaaacga aaaggttttg ccaaagcact
     1741 ctttgttgta cgaatacttc accgtctaca acgaattgac taaggttaag tacgtcaccg
     1801 aaggtatgag aaaaccagct tttttatccg gtgaacaaaa gaaggctatc gtcgatttgt
     1861 tgttcaagac caacagaaag gttactgtca agcaattaaa agaagattac ttcaagaaaa
     1921 tcgaatgctt cgactccgtt gaaatttctg gtgtcgaaga tagattcaat gcctctttag
     1981 gtacttacca tgacttgttg aaaatcatca aggacaagga tttcttggac aacgaagaaa
     2041 acgaagatat tttggaagat attgtcttga cattgacctt gtttgaagat agagaaatga
     2101 ttgaagaaag attgaaaacc tacgcccact tgttcgatga taaggttatg aagcaattaa
     2161 agagaagaag atacactggt tggggtagat tgtccagaaa attgattaac ggtatcagag
     2221 acaagcaatc cggtaagacc attttggact ttttgaagtc tgatggtttc gctaacagaa
     2281 acttcatgca attaatccac gacgattcct tgactttcaa agaagatata caaaaggccc
     2341 aagtctctgg tcaaggtgat tctttacatg aacatatcgc taacttggct ggttctccag
     2401 ctattaagaa gggtatttta caaaccgtta aggtcgttga cgaattggtc aaagttatgg
     2461 gtagacataa gccagaaaac atcgttatcg aaatggctag agaaaatcaa accacccaaa
     2521 agggtcaaaa gaactccaga gaaagaatga agagaatcga agaaggtatc aaagaattgg
     2581 gttcccaaat tttgaaagaa cacccagttg aaaacaccca attacaaaac gaaaagttgt
     2641 acttgtacta cttgcaaaac ggtagagata tgtacgttga ccaagaattg gacatcaaca
     2701 gattgtctga ttacgatgtt gacgctatcg ttccacaatc ttttttgaag gatgactcca
     2761 ttgacaacaa ggtcttgact agatccgata agaatagagg taagtccgat aacgttccat
     2821 ctgaagaagt cgttaagaaa atgaagaact attggagaca attattgaac gccaagttga
     2881 tcacccaaag aaagtttgac aatttgacca aggctgaaag aggtggtttg tctgaattgg
     2941 ataaggcagg tttcatcaaa agacaattag tagaaaccag acaaatcacc aagcacgttg
     3001 ctcaaatttt ggatagtaga atgaacacta agtacgacga aaacgacaaa ttgatcagag
     3061 aagttaaggt cattaccttg aagtccaagt tggtttccga tttcagaaag gacttccaat
     3121 tctacaaggt cagagaaatc aacaactacc atcatgcaca tgatgcttac ttgaatgctg
     3181 ttgttggtac tgccttgatt aagaagtatc caaagttgga atccgaattt gtctacggtg
     3241 attacaaggt ttacgacgtt agaaagatga tcgccaagtc cgaacaagaa attggtaaag
     3301 ctactgccaa atacttcttc tactccaata ttatgaattt ctttaagacc gaaatcactt
     3361 tggccaacgg tgaaattaga aaaagaccat tgattgaaac taatggtgaa acaggtgaaa
     3421 tcgtttggga taagggtaga gattttgcca ctgttagaaa ggtattgtcc atgccacaag
     3481 taaacatcgt caaaaagacc gaagttcaaa ctggtggttt ctccaaagaa tccattttgc
     3541 ctaagagaaa ctccgataag ttgatcgcta gaaaaaaaga ctgggaccca aaaaagtacg
     3601 gtggttttga ttctccaact gttgcttact ctgttttggt tgttgctaag gtcgaaaagg
     3661 gtaagagtaa gaagttgaag tccgtcaaag aattattagg tatcactatc atggaaagat
     3721 cctcattcga aaagaatcct atcgactttt tggaagccaa gggttacaaa gaagtcaaga
     3781 aggacttgat cattaagttg ccaaagtaca gtttgttcga attggaaaat ggtagaaaga
     3841 gaatgttggc ttctgccggt gaattacaaa agggtaatga attggctttg ccatccaagt
     3901 acgttaattt cttatacttg gcctcccact acgaaaaatt gaaaggttct cctgaagata
     3961 acgaacaaaa gcaattattt gtcgaacaac acaagcacta cttggacgaa atcattgaac
     4021 aaatttccga attttccaaa agagtcattt tggctgacgc caatttggac aaagttttgt
     4081 cagcttacaa caagcacaga gataagccaa ttagagaaca agctgaaaac atcattcact
     4141 tgttcacttt gactaacttg ggtgctccag ctgcttttaa gtatttcgat accactatcg
     4201 acagaaagag atacacctct accaaagaag ttttggacgc tactttgatc caccaatcta
     4261 ttactggttt gtacgaaact agaatcgact tgtctcaatt aggtggtgat taacaagctt
     4321 gagctttgac agctagctca gtcctaggta taatgctagc ccctgataga gatactgagc
     4381 acatcagcgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt
     4441 gaaaaagtgg caccgagtcg gtgctttttt ggcatcaaat aaaacgaaag gctcagtcga
     4501 aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa
     4561 atccgccgcc ctagaatccc tagtcgatta gcacctttga cagctagctc agtcctaggt
     4621 ataatgctag cccccaatta tgatagattc aattgtgagg ttttagagct agaaatagca
     4681 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
     4741 tggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     4801 ttgtcggtga acgctctcct gagtaggaca aatccgccgc cctagacctc gataccctag
     4861 ctaatacttg acagctagct cagtcctagg tataatgcta gccccaaacc attaacacaa
     4921 gagcagcttg gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac
     4981 ttgaaaaagt ggcaccgagt cggtgctttt ttggcatcaa ataaaacgaa aggctcagtc
     5041 gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac
     5101 aaatccgccg ccctagaagc tcgaattctc tagagaatca ggtttgtgcc aataccagta
     5161 gaaacagacg aagaatccat gggtatggac agttttccct ttgatatgta acggtgaaca
     5221 gttgttctac ttttgtttgt tagtcttgat gcttcactga tagatacaag agccataaga
     5281 acctcagatc cttccgtatt tagccagtat gttctctagt gtggttcgtt gtttttgcgt
     5341 gagccatgag aacgaaccat tgagatcata cttactttgc atgtcactca aaaattttgc
     5401 ctcaaaactg gtgagctgaa tttttgcagt taaagcatcg tgtagtgttt ttcttagtcc
     5461 gttatgtagg taggaatctg atgtaatggt tgttggtatt ttgtcaccat tcatttttat
     5521 ctggttgttc tcaagttcgg ttacgagatc catttgtcta tctagttcaa cttggaaaat
     5581 caacgtatca gtcgggcggc ctcgcttatc aaccaccaat ttcatattgc tgtaagtgtt
     5641 taaatcttta cttattggtt tcaaaaccca ttggttaagc cttttaaact catggtagtt
     5701 attttcaagc attaacatga acttaaattc atcaaggcta atctctatat ttgccttgtg
     5761 agttttcttt tgtgttagtt cttttaataa ccactcataa atcctcatag agtatttgtt
     5821 ttcaaaagac ttaacatgtt ccagattata ttttatgaat ttttttaact ggaaaagata
     5881 aggcaatatc tcttcactaa aaactaattc taatttttcg cttgagaact tggcatagtt
     5941 tgtccactgg aaaatctcaa agcctttaac caaaggattc ctgatttcca cagttctcgt
     6001 catcagctct ctggttgctt tagctaatac accataagca ttttccctac tgatgttcat
     6061 catctgaacg tattggttat aagtgaacga taccgtccgt tctttccttg tagggttttc
     6121 aatcgtgggg ttgagtagtg ccacacagca taaaattagc ttggtttcat gctccgttaa
     6181 gtcatagcga ctaatcgcta gttcatttgc tttgaaaaca actaattcag acatacatct
     6241 caattggtct aggtgatttt aatcactata ccaattgaga tgggctagtc aatgataatt
     6301 actagtcctt ttcctttgag ttgtgggtat ctgtaaattc tgctagacct ttgctggaaa
     6361 acttgtaaat tctgctagac cctctgtaaa ttccgctaga cctttgtgtg ttttttttgt
     6421 ttatattcaa gtggttataa tttatagaat aaagaaagaa taaaaaaaga taaaaagaat
     6481 agatcccagc cctgtgtata actcactact ttagtcagtt ccgcagtatt acaaaaggat
     6541 gtcgcaaacg ctgtttgctc ctctacaaaa cagaccttaa aaccctaaag gcttaagtag
     6601 caccctcgca agctcggttg cggccgcaat cgggcaaatc gctgaatatt ccttttgtct
     6661 ccgaccatca ggcacctgag tcgctgtctt tttcgtgaca ttcagttcgc tgcgctcacg
     6721 gctctggcag tgaatggggg taaatggcac tacaggcgcc ttttatggat tcatgcaagg
     6781 aaactaccca taatacaaga aaagcccgtc acgggcttct cagggcgttt tatggcgggt
     6841 ctgctatgtg gtgctatctg actttttgct gttcagcagt tcctgccctc tgattttcca
     6901 gtctgaccac ttcggattat cccgtgacag gtcattcaga ctggctaatg cacccagtaa
     6961 ggcagcggta tcatcaacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     7021 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     7081 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     7141 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     7201 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     7261 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     7321 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     7381 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     7441 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     7501 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     7561 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     7621 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     7681 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     7741 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     7801 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     7861 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     7921 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     7981 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     8041 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     8101 ccgaaaagtg ccacctg
//
