LOCUS       40924_585        8461 bp DNA     circular SYN 11-JUN-2021
DEFINITION  Improved dCas9 repressor-dCas9-KRAB-MeCP2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8461)
  AUTHORS   Yeo NC, Chavez A, Lance-Byrne A, Chan Y, Menn D, Milanova D, Kuo CC,
            Guo X, Sharma S, Tung A, Cecchi RJ, Tuttle M, Pradhan S, Lim ET, 
            Davidsohn N, Ebrahimkhani MR, Collins JJ, Lewis NE, Kiani S, Church 
            GM
  TITLE     An enhanced CRISPR repressor for targeted mammalian gene regulation.
  JOURNAL   Nat Methods. 2018 Jul 16. pii: 10.1038/s41592-018-0048-5. doi: 
            10.1038/s41592-018-0048-5.
  PUBMED    30013045
REFERENCE   2  (bases 1 to 8461)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8461)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Methods. 2018 Jul 16. pii: 10.1038/s41592-018-0048-5. doi: 
            10.1038/s41592-018-0048-5."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             748..4851
                     /codon_start=1
                     /label=Cas9m4
                     /note="catalytically dead mutant of the Cas9 endonuclease
                     from the Streptococcus pyogenes Type II CRISPR/Cas system"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVAAIVPQSFLKDDSIDNKVLTRSDKARGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             4864..4884
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4945..5139
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     CDS             5173..5193
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5203..6060
                     /codon_start=1
                     /product="transcriptional repression domain from rat 
                     methyl-CpG-binding protein 2 (Nan et al., 1997)"
                     /label=MeCP2
                     /translation="VQVKRVLEKSPGKLLVKMPFQASPGGKGEGGGATTSAQVMVIKRP
                     GRKRKAEADPQAIPKKRGRKPGSVVAAAAAEAKKKAVKESSIRSVQETVLPIKKRKTRE
                     TVSIEVKEVVKPLLVSTLGEKSGKGLKTCKSPGRKSKESSPKGRSSSASSPPKKEHHHH
                     HHHAESPKAPMPLLPPPPPPEPQSSEDPISPPEPQDLSSSICKEEKMPRAGSLESDGCP
                     KEPAKTQPMVAAAATTTTTTTTTVAEKYKHRGEGERKDIVSSSMPRPNREEPVDSRTPV
                     TERVS"
     primer_bind     complement(6115..6134)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6159..6207
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      complement(6521..7109)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(7283..8140)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(8141..8245)
                     /label=AmpR promoter
     primer_bind     complement(8320..8339)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagagg atcgaaccct tgccaccatg gacaagaagt actccattgg gctcgctatc
      781 ggcacaaaca gcgtcggctg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa
      841 ttcaaagttc tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc
      901 ctgttcgact ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga
      961 tatacccgca gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct
     1021 aaggtggatg actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa
     1081 aagcacgagc gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag
     1141 tacccaacca tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg
     1201 cggttgatct atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag
     1261 ggggacctga acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact
     1321 tacaatcagc ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc
     1381 ctgagcgcta ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg
     1441 gagaagaaga acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac
     1501 tttaaatcta acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac
     1561 gatgatgatc tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg
     1621 gcggcaaaga acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag
     1681 atcaccaaag ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac
     1741 ttgactttgc tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc
     1801 ttcgatcagt ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa
     1861 ttttacaaat ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta
     1921 aagcttaaca gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc
     1981 caccagattc acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc
     2041 tttttgaaag ataacaggga aaagattgag aaaatcctca catttcggat accctactat
     2101 gtaggccccc tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag
     2161 accatcactc cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc
     2221 atcgaaagga tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac
     2281 tctctgctgt acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca
     2341 gaagggatga gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc
     2401 ctcttcaaga cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag
     2461 attgaatgtt tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg
     2521 ggaacgtatc acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag
     2581 aacgaggaca ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg
     2641 attgaagaac gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc
     2701 aagaggcgcc gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga
     2761 gacaagcaga gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg
     2821 aacttcatgc agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca
     2881 caagtttctg gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca
     2941 gctatcaaaa agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg
     3001 ggaaggcata agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag
     3061 aagggacaga agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg
     3121 gggtcccaaa tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc
     3181 tacctgtact acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat
     3241 cggctctccg actacgacgt ggctgctatc gtgccccagt cttttctcaa agatgattct
     3301 attgataata aagtgttgac aagatccgat aaagctagag ggaagagtga taacgtcccc
     3361 tcagaagaag ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg
     3421 atcacacaac ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg
     3481 gataaagccg gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg
     3541 gcccaaattc tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga
     3601 gaggtgaaag ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag
     3661 ttttataagg tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca
     3721 gtggtaggca ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga
     3781 gactataaag tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag
     3841 gccaccgcta agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca
     3901 ctggccaatg gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa
     3961 atcgtgtggg acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag
     4021 gtgaacatcg ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc
     4081 ccgaaaagga acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac
     4141 ggcggattcg attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa
     4201 gggaagtcta aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga
     4261 tcaagcttcg aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa
     4321 aaagacctca tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa
     4381 cgaatgctcg ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa
     4441 tacgttaatt tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat
     4501 aatgagcaga agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag
     4561 caaataagcg aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt
     4621 tctgcttaca ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac
     4681 ttgtttactc tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata
     4741 gacagaaagc ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca
     4801 attacggggc tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct
     4861 gaccccaaga agaagaggaa ggtgagtggt ggaggaagtg gcgggtcagg gtcgatggac
     4921 gcgaaatcac ttacggcatg gtcgagaaca ctggttacgt tcaaggacgt gtttgtggac
     4981 tttacacgtg aggagtggaa attgctggat actgcgcaac aaattgtgta tcgaaatgtc
     5041 atgcttgaga attacaagaa cctcgtcagt ctcggatacc agttgacgaa accggatgtg
     5101 atccttaggc tcgaaaaggg ggaagaacct tggctggtat cgggaggtgg ttcgggtggc
     5161 tctggatcaa gcccaaagaa gaaacggaag gtggaagcct cagtgcaggt gaaaagggtg
     5221 ctggaaaaat cccccggcaa actcctcgtg aagatgccct tccaggcttc ccctggcgga
     5281 aaaggtgaag ggggtggcgc aaccacatct gcccaggtca tggtcatcaa gcgacctgga
     5341 aggaaaagaa aggccgaggc tgaccctcag gccattccaa agaaacgggg acgcaagcca
     5401 gggtccgtgg tcgcagctgc agcagctgag gctaagaaaa aggcagtgaa ggaaagctcc
     5461 atccgcagtg tgcaggagac tgtcctgccc atcaagaaga ggaagactag ggagaccgtg
     5521 tccatcgagg tcaaagaagt ggtcaagccc ctgctcgtgt ccaccctggg cgaaaaatct
     5581 ggaaaggggc tcaaaacatg caagtcacct ggacggaaaa gcaaggagtc tagtccaaag
     5641 gggcgctcaa gctccgcttc tagtccccct aaaaaggaac accatcacca tcaccatcac
     5701 gccgagtctc ctaaggctcc tatgccactg ctcccaccac ctccaccacc tgagccacag
     5761 tcaagcgaag accccatcag cccacccgag cctcaggatc tgtcctctag tatttgcaaa
     5821 gaggaaaaga tgcccagagc aggcagcctg gagagtgatg gctgtccaaa agaacccgcc
     5881 aagacccagc ctatggtggc agccgctgca actaccacca caaccacaac taccacagtg
     5941 gccgaaaaat acaagcatcg cggcgagggc gaacgaaagg acattgtgtc aagctccatg
     6001 cccagaccta accgggagga accagtcgat agtaggacac ccgtgactga gagagtctca
     6061 tagaagaatt cgatccctac cggttagtaa tgagtttaaa cgggggaggc taactgaaac
     6121 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     6181 aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     6241 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     6301 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     6361 gccctccata gtcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     6421 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     6481 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6541 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6601 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6661 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6721 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6781 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6841 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6901 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6961 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     7021 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     7081 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     7141 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     7201 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     7261 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     7321 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     7381 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     7441 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     7501 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7561 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7621 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     7681 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7741 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7801 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7861 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7921 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7981 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     8041 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     8101 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     8161 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     8221 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg
     8281 gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc
     8341 atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag
     8401 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
     8461 g
//
