LOCUS       Exported                7177 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  regulated dCas9 generator.
ACCESSION   .
VERSION     .
KEYWORDS    pdCas9_CL
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 May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7177
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     67..85
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_RNA        161..236
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    291..312
                     /label=BioBrick prefix
                     /note="BioBrick prefix for parts that do not start with 
                     ""ATG"""
     promoter        331..359
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     RBS             397..405
                     /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             411..4517
                     /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      4541..4612
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4628..4655
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    4662..4682
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     CDS             complement(4822..5637)
                     /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     5545..5564
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5629..5648
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      complement(6232..6776)
                     /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(6311..6330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ccagtctatt ccatggtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
       61 tataaaaata ggcgtatcac gaggcagggc gcgccaaaaa gagtattgac ttcgcatctt
      121 tttgtaccta taatagattc agtattgact attaatcatc gttttagagc tagaaatagc
      181 aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt
      241 tttccaatta ttgaacaccc taacgggtgt tttttttttt ttggtctccc gaattcgcgg
      301 ccgcttctag agggcgcgcc aaaaagagta ttgactatta atcatccggc tcgtataata
      361 gattcactct agacgagagg aaacgcggtt ttaatataag gaggttatta atggataaga
      421 aatactcaat aggcttagct atcggcacaa atagcgtcgg atgggcggtg atcactgatg
      481 aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc cacagtatca
      541 aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa gcgactcgtc
      601 tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt tatctacagg
      661 agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga cttgaagagt
      721 cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga aatatagtag
      781 atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa aaattggtag
      841 attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat atgattaagt
      901 ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat gtggacaaac
      961 tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct attaacgcaa
     1021 gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga cgattagaaa
     1081 atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat ctcattgctt
     1141 tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa gatgctaaat
     1201 tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg caaattggag
     1261 atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt ttactttcag
     1321 atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca atgattaaac
     1381 gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga caacaacttc
     1441 cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca ggttatattg
     1501 atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta gaaaaaatgg
     1561 atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc aagcaacgga
     1621 cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat gctattttga
     1681 gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt gaaaaaatct
     1741 tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt cgttttgcat
     1801 ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa gttgtcgata
     1861 aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa aatcttccaa
     1921 atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt tataacgaat
     1981 tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt tcaggtgaac
     2041 agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc gttaagcaat
     2101 taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt tcaggagttg
     2161 aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt attaaagata
     2221 aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt ttaacattga
     2281 ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct cacctctttg
     2341 atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga cgtttgtctc
     2401 gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta gattttttga
     2461 aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat agtttgacat
     2521 ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta catgaacata
     2581 ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact gtaaaagttg
     2641 ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt attgaaatgg
     2701 cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt atgaaacgaa
     2761 tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct gttgaaaata
     2821 ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga gacatgtatg
     2881 tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatgcc attgttccac
     2941 aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct gataaaaatc
     3001 gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa aactattgga
     3061 gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta acgaaagctg
     3121 aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa ttggttgaaa
     3181 ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat actaaatacg
     3241 atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct aaattagttt
     3301 ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat taccatcatg
     3361 cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa tatccaaaac
     3421 ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa atgattgcta
     3481 agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct aatatcatga
     3541 acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc cctctaatcg
     3601 aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt gccacagtgc
     3661 gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta cagacaggcg
     3721 gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt gctcgtaaaa
     3781 aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct tattcagtcc
     3841 tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt aaagagttac
     3901 tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac tttttagaag
     3961 ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa tatagtcttt
     4021 ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta caaaaaggaa
     4081 atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt cattatgaaa
     4141 agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag cagcataagc
     4201 attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt attttagcag
     4261 atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa ccaatacgtg
     4321 aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct cccgctgctt
     4381 ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa gaagttttag
     4441 atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt gatttgagtc
     4501 agctaggagg tgactaactc gagtaaggat ctccaggcat caaataaaac gaaaggctca
     4561 gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag
     4621 tcacactggc tcaccttcgg gtgggccttt ctgcgtttat atactagtag cggccgctgc
     4681 aggcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4741 ctcactcaaa ggcggtaatc tcgagtcccg tcaagtcagc gtaatgctct gccagtgtta
     4801 caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     4861 attcatatca ggattatcga taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     4921 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     4981 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     5041 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt
     5101 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     5161 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     5221 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     5281 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     5341 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     5401 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     5461 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     5521 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     5581 catgttggaa tttaatcgcg gcctggagca agacgtttcc cgttgaatat ggctcataac
     5641 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt
     5701 atcttgtgca atgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg
     5761 aacttttgct gagttgaagg atcagatcac gcatcttccc gacaacgcag accgttccgt
     5821 ggcaaagcaa aagttcaaaa tcaccaactg gtccacctac aacaaagctc tcatcaaccg
     5881 tggctccctc actttctggc tggatgatgg ggcgattcag gcctggtatg agtcagcaac
     5941 accttcttca cgaggcagac ctcagcgcta gcggagtgta tactggctta ctatgttggc
     6001 actgatgagg gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct gcaccggtgc
     6061 gtcagcagaa tatgtgatac aggatatatt ccgcttcctc gctcactgac tcgctacgct
     6121 cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat ttcctggaag
     6181 atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt ttttccatag
     6241 gctccgcccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg gcgaaacccg
     6301 acaggactat aaagatacca ggcgtttccc cctggcggct ccctcgtgcg ctctcctgtt
     6361 cctgcctttc ggtttaccgg tgtcattccg ctgttatggc cgcgtttgtc tcattccacg
     6421 cctgacactc agttccgggt aggcagttcg ctccaagctg gactgtatgc acgaaccccc
     6481 cgttcagtcc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggaaag
     6541 acatgcaaaa gcaccactgg cagcagccac tggtaattga tttagaggag ttagtcttga
     6601 agtcatgcgc cggttaaggc taaactgaaa ggacaagttt tggtgactgc gctcctccaa
     6661 gccagttacc tcggttcaaa gagttggtag ctcagagaac cttcaaaaaa ccgccctgca
     6721 aggcggtttt ttcgttttca gagcaagaga ttacgcgcag accaaaacga tctcaagaag
     6781 atcatcttat taaggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     6841 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     6901 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     6961 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     7021 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     7081 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     7141 agcgcagaag tggtcctgca actttatccg cctccat
//
