LOCUS       sequence-331761-        7177 bp    DNA     circular SYN 15-APR-2025
DEFINITION  regulated dCas9 generator.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7177)
  AUTHORS   Huang HH, Bellato M, Qian Y, Cardenas P, Pasotti L, Magni P, Del 
            Vecchio D
  TITLE     dCas9 regulator to neutralize competition in CRISPRi circuits.
  JOURNAL   Nat Commun. 2021 Mar 16;12(1):1692. doi: 10.1038/s41467-021-21772-6.
  PUBMED    33727557
REFERENCE   2  (bases 1 to 7177)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-03-16"; volume: "12(1):1692. doi"; pages: " 
            10.1038/s41467-021-21772-6"
COMMENT     Sequence Label: pdCas9_CL
FEATURES             Location/Qualifiers
     source          1..7177
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     65..83
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_RNA        159..234
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    289..310
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        join(329..334,335..351,352..357)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     RBS             395..403
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             join(409..435,436..438,439..2925,2926..2928,2929..4515)
                     /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"
     terminator      4539..4610
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4626..4653
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    4660..4680
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     CDS             complement(4820..5635)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4820..5635)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5543..5562
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5627..5646
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      complement(6230..6774)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(6309..6328)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agtctattcc atggtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
       61 taaaaatagg cgtatcacga ggcagggcgc gccaaaaaga gtattgactt cgcatctttt
      121 tgtacctata atagattcag tattgactat taatcatcgt tttagagcta gaaatagcaa
      181 gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt
      241 tccaattatt gaacacccta acgggtgttt tttttttttt ggtctcccga attcgcggcc
      301 gcttctagag ggcgcgccaa aaagagtatt gactattaat catccggctc gtataataga
      361 ttcactctag acgagaggaa acgcggtttt aatataagga ggttattaat ggataagaaa
      421 tactcaatag gcttagctat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
      481 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
      541 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
      601 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
      661 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
      721 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
      781 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
      841 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
      901 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
      961 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt
     1021 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
     1081 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
     1141 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
     1201 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
     1261 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
     1321 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
     1381 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
     1441 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
     1501 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
     1561 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
     1621 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
     1681 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
     1741 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
     1801 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
     1861 ggtgcttcag ctcaatcatt tattgaacgc atgacaaact ttgataaaaa tcttccaaat
     1921 gaaaaagtac taccaaaaca tagtttgctt tatgagtatt ttacggttta taacgaattg
     1981 acaaaggtca aatatgttac tgaaggaatg cgaaaaccag catttctttc aggtgaacag
     2041 aagaaagcca ttgttgattt actcttcaaa acaaatcgaa aagtaaccgt taagcaatta
     2101 aaagaagatt atttcaaaaa aatagaatgt tttgatagtg ttgaaatttc aggagttgaa
     2161 gatagattta atgcttcatt aggtacctac catgatttgc taaaaattat taaagataaa
     2221 gattttttgg ataatgaaga aaatgaagat atcttagagg atattgtttt aacattgacc
     2281 ttatttgaag atagggagat gattgaggaa agacttaaaa catatgctca cctctttgat
     2341 gataaggtga tgaaacagct taaacgtcgc cgttatactg gttggggacg tttgtctcga
     2401 aaattgatta atggtattag ggataagcaa tctggcaaaa caatattaga ttttttgaaa
     2461 tcagatggtt ttgccaatcg caattttatg cagctgatcc atgatgatag tttgacattt
     2521 aaagaagaca ttcaaaaagc acaagtgtct ggacaaggcg atagtttaca tgaacatatt
     2581 gcaaatttag ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt
     2641 gatgaattgg tcaaagtaat ggggcggcat aagccagaaa atatcgttat tgaaatggca
     2701 cgtgaaaatc agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc
     2761 gaagaaggta tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact
     2821 caattgcaaa atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg
     2881 gaccaagaat tagatattaa tcgtttaagt gattatgatg tcgatgccat tgttccacaa
     2941 agtttcctta aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt
     3001 ggtaaatcgg ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga
     3061 caacttctaa acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa
     3121 cgtggaggtt tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact
     3181 cgccaaatca ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat
     3241 gaaaatgata aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct
     3301 gacttccgaa aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc
     3361 catgatgcgt atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt
     3421 gaatcggagt ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag
     3481 tctgagcaag aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac
     3541 ttcttcaaaa cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa
     3601 actaatgggg aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc
     3661 aaagtattgt ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga
     3721 ttctccaagg agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa
     3781 gactgggatc caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta
     3841 gtggttgcta aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta
     3901 gggatcacaa ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct
     3961 aaaggatata aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt
     4021 gagttagaaa acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat
     4081 gagctggctc tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag
     4141 ttgaagggta gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat
     4201 tatttagatg agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat
     4261 gccaatttag ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa
     4321 caagcagaaa atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt
     4381 aaatattttg atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat
     4441 gccactctta tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag
     4501 ctaggaggtg actaactcga gtaaggatct ccaggcatca aataaaacga aaggctcagt
     4561 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc
     4621 acactggctc accttcgggt gggcctttct gcgtttatat actagtagcg gccgctgcag
     4681 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4741 cactcaaagg cggtaatctc gagtcccgtc aagtcagcgt aatgctctgc cagtgttaca
     4801 accaattaac caattctgat tagaaaaact catcgagcat caaatgaaac tgcaatttat
     4861 tcatatcagg attatcgata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
     4921 actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc
     4981 gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
     5041 aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc
     5101 agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac
     5161 cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
     5221 aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
     5281 tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag
     5341 tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca
     5401 taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac
     5461 ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg
     5521 tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca
     5581 tgttggaatt taatcgcggc ctggagcaag acgtttcccg ttgaatatgg ctcataacac
     5641 cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat
     5701 cttgtgcaat gtaacatcag agattttgag acacaacgtg gctttgttga ataaatcgaa
     5761 cttttgctga gttgaaggat cagatcacgc atcttcccga caacgcagac cgttccgtgg
     5821 caaagcaaaa gttcaaaatc accaactggt ccacctacaa caaagctctc atcaaccgtg
     5881 gctccctcac tttctggctg gatgatgggg cgattcaggc ctggtatgag tcagcaacac
     5941 cttcttcacg aggcagacct cagcgctagc ggagtgtata ctggcttact atgttggcac
     6001 tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc accggtgcgt
     6061 cagcagaata tgtgatacag gatatattcc gcttcctcgc tcactgactc gctacgctcg
     6121 gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg gcggagattt cctggaagat
     6181 gccaggaaga tacttaacag ggaagtgaga gggccgcggc aaagccgttt ttccataggc
     6241 tccgccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc gaaacccgac
     6301 aggactataa agataccagg cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc
     6361 tgcctttcgg tttaccggtg tcattccgct gttatggccg cgtttgtctc attccacgcc
     6421 tgacactcag ttccgggtag gcagttcgct ccaagctgga ctgtatgcac gaaccccccg
     6481 ttcagtccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggaaagac
     6541 atgcaaaagc accactggca gcagccactg gtaattgatt tagaggagtt agtcttgaag
     6601 tcatgcgccg gttaaggcta aactgaaagg acaagttttg gtgactgcgc tcctccaagc
     6661 cagttacctc ggttcaaaga gttggtagct cagagaacct tcaaaaaacc gccctgcaag
     6721 gcggtttttt cgttttcaga gcaagagatt acgcgcagac caaaacgatc tcaagaagat
     6781 catcttatta aggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6841 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6901 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6961 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     7021 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     7081 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7141 cgcagaagtg gtcctgcaac tttatccgcc tccatcc
//
