LOCUS       Exported                8514 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pMAGIC R4-R3 entry plasmid, contains 2x NLS x-dCas9(3.7) fused to 
            the Dnmt3a-3L DNA methyltransferase for 3 or 4-component MultiSite 
            Gateway Pro assembly..
ACCESSION   .
VERSION     .
KEYWORDS    KO101: pMAGIC (R4-R3) NLS-x dCas9(3.7)-NLS-Dnmt3a3L
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8514)
  AUTHORS   Haldeman JM, Conway AE, Arlotto ME, Slentz DH, Muoio DM, Becker TC, 
            Newgard CB
  TITLE     Creation of versatile cloning platforms for transgene expression and
            dCas9-based epigenome editing.
  JOURNAL   Nucleic Acids Res. 2018 Dec 27. pii: 5264291. doi: 
            10.1093/nar/gky1286.
  PUBMED    30590691
REFERENCE   2  (bases 1 to 8514)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8514
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      268..295
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      387..473
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(603..727)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      726..735
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             738..758
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             783..4883
                     /codon_start=1
                     /product="catalytically dead Streptococcus pyogenes Cas9 
                     mutant with broad PAM compatibility (Hu et al., 2018)"
                     /label=dxCas9(3.7)
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFIQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             4884..4931
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     protein_bind    6577..6700
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(6755..6774)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6756..6774)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6779..6795)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6779..6795)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6897..6916)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6908..7717
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(6975..6994)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      7864..8452
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8353..8372
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcgggcccct acaggtcact aataccatct aagtagttga
      601 ttcatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt ttatgcaaaa
      661 tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcaact tttctataca
      721 aagttgccac catggcccca aagaagaagc ggaaggtcgg tatccacgga gtcccagcag
      781 ccgacaagaa gtactccatt gggctcgcta tcggcacaaa cagcgtcggc tgggccgtca
      841 ttacggacga gtacaaggtg ccgagcaaaa aattcaaagt tctgggcaat accgatcgcc
      901 acagcataaa gaagaacctc attggcgccc tcctgttcga ctccggggag acggccgaag
      961 ccacgcggct caaaagaaca gcacggcgca gatatacccg cagaaagaat cggatctgct
     1021 acctgcagga gatctttagt aatgagatgg ctaaggtgga tgactctttc ttccataggc
     1081 tggaggagtc ctttttggtg gaggaggata aaaagcacga gcgccaccca atctttggca
     1141 atatcgtgga cgaggtggcg taccatgaaa agtacccaac catatatcat ctgaggaaga
     1201 agcttgtaga cagtactgat aaggctgact tgcggttgat ctatctcgcg ctggcgcata
     1261 tgatcaaatt tcggggacac ttcctcatcg agggggacct gaacccagac aacagcgatg
     1321 tcgacaaact ctttatccaa ctggttcaga cttacaatca gcttttcgaa gagaacccga
     1381 tcaacgcatc cggagttgac gccaaagcaa tcctgagcgc taggctgtcc aaatcccggc
     1441 ggctcgaaaa cctcatcgca cagctccctg gggagaagaa gaacggcctg tttggtaatc
     1501 ttatcgccct gtccctcggg ctgaccccca actttaaatc taacttcgac ctggccgaag
     1561 ataccaagct tcaactgagc aaagacacct acgatgatga tctcgacaat ctgctggccc
     1621 agatcggcga ccagtacgca gacctttttt tggcggcaaa gaacctgtca gacgccattc
     1681 tgctgagtga tattctgcga gtgaacacgg agatcaccaa agctccgctg agcgctagta
     1741 tgatcaagct ctatgatgag caccaccaag acttgacttt gctgaaggcc cttgtcagac
     1801 agcaactgcc tgagaagtac aaggaaattt tcttcgatca gtctaaaaat ggctacgccg
     1861 gatacattga cggcggagca agccaggagg aattttacaa atttattaag cccatcttgg
     1921 aaaaaatgga cggcaccgag gagctgctgg taaagcttaa cagagaagat ctgttgcgca
     1981 aacagcgcac tttcgacaat ggaatcatcc cccaccagat tcacctgggc gaactgcacg
     2041 ctatcctcag gcggcaagag gatttctacc cctttttgaa agataacagg gaaaagattg
     2101 agaaaatcct cacatttcgg ataccctact atgtaggccc cctcgcccgg ggaaattcca
     2161 gattcgcgtg gatgactcgc aaatcagaag agaccatcac tccctggaac ttcgagaaag
     2221 tcgtggataa gggggcctct gcccagtcct tcatcgaaag gatgactaac tttgataaaa
     2281 atctgcctaa cgaaaaggtg cttcctaaac actctctgct gtacgagtac ttcacagttt
     2341 ataacgagct caccaaggtc aaatacgtca cagaagggat gagaaagcca gcattcctgt
     2401 ctggagatca gaagaaagct attgtggacc tcctcttcaa gacgaaccgg aaagttaccg
     2461 tgaaacagct caaagaagac tatttcaaaa agattgaatg tttcgactct gttgaaatca
     2521 gcggagtgga ggatcgcttc aacgcatccc tgggaacgta tcacgatctc ctgaaaatca
     2581 ttaaagacaa ggacttcctg gacaatgagg agaacgagga cattcttgag gacattgtcc
     2641 tcacccttac gttgtttgaa gatagggaga tgattgaaga acgcttgaaa acttacgctc
     2701 atctcttcga cgacaaagtc atgaagcagc tcaagaggcg ccgatataca ggatgggggc
     2761 ggctgtcaag aaaactgatc aatggcatcc gagacaagca gagtggaaag acaatcctgg
     2821 attttcttaa gtccgatgga tttgccaacc ggaacttcat tcagttgatc catgatgact
     2881 ctctcacctt taaggaggac atccagaaag cacaagtttc tggccagggg gacagtcttc
     2941 acgagcacat cgctaatctt gcaggtagcc cagctatcaa aaagggaata ctgcagaccg
     3001 ttaaggtcgt ggatgaactc gtcaaagtaa tgggaaggca taagcccgag aatatcgtta
     3061 tcgagatggc ccgagagaac caaaccaccc agaagggaca gaagaacagt agggaaagga
     3121 tgaagaggat tgaagagggt ataaaagaac tggggtccca aatccttaag gaacacccag
     3181 ttgaaaacac ccagcttcag aatgagaagc tctacctgta ctacctgcag aacggcaggg
     3241 acatgtacgt ggatcaggaa ctggacatca atcggctctc cgactacgac gtggacgcta
     3301 tcgtgcccca gtcttttctc aaagatgatt ctattgataa taaagtgttg acaagatccg
     3361 ataaaaacag agggaagagt gataacgtcc cctcagaaga agttgtcaag aaaatgaaaa
     3421 attattggcg gcagctgctg aacgccaaac tgatcacaca acggaagttc gataatctga
     3481 ctaaggctga acgaggtggc ctgtctgagt tggataaagc cggtttcatc aaaaggcagc
     3541 ttgttgagac acgccagatc accaagcacg tggcccaaat tctcgattca cgcatgaaca
     3601 ccaagtacga tgaaaatgac aaactgattc gagaggtgaa agttattact ctgaagtcta
     3661 agctggtctc agatttcaga aaggactttc agttttataa ggtgagagag atcaacaatt
     3721 accaccatgc gcatgatgcc tacctgaatg cagtggtagg cactgcactt atcaaaaaat
     3781 atcccaagct tgaatctgaa tttgtttacg gagactataa agtgtacgat gttaggaaaa
     3841 tgatcgcaaa gtctgagcag gaaataggca aggccaccgc taagtacttc ttttacagca
     3901 atattatgaa ttttttcaag accgagatta cactggccaa tggagagatt cggaagcgac
     3961 cacttatcga aacaaacgga gaaacaggag aaatcgtgtg ggacaagggt agggatttcg
     4021 cgacagtccg gaaggtcctg tccatgccgc aggtgaacat cgttaaaaag accgaagtac
     4081 agaccggagg tttctccaag gaaagtatcc tcccgaaaag gaacagcgac aagctgatcg
     4141 cacgcaaaaa agattgggac cccaagaaat acggcggatt cgattctcct acagtcgctt
     4201 acagtgtact ggttgtggcc aaagtggaga aagggaagtc taaaaaactc aaaagcgtca
     4261 aggaactgct gggcatcaca atcatggagc gatcaagctt cgaaaaaaac cccatcgact
     4321 ttctcgaggc gaaaggatat aaagaggtca aaaaagacct catcattaag cttcccaagt
     4381 actctctctt tgagcttgaa aacggccgga aacgaatgct cgctagtgcg ggcgtgctgc
     4441 agaaaggtaa cgagctggca ctgccctcta aatacgttaa tttcttgtat ctggccagcc
     4501 actatgaaaa gctcaaaggg tctcccgaag ataatgagca gaagcagctg ttcgtggaac
     4561 aacacaaaca ctaccttgat gagatcatcg agcaaataag cgaattctcc aaaagagtga
     4621 tcctcgccga cgctaacctc gataaggtgc tttctgctta caataagcac agggataagc
     4681 ccatcaggga gcaggcagaa aacattatcc acttgtttac tctgaccaac ttgggcgcgc
     4741 ctgcagcctt caagtacttc gacactacca tagacagaaa gcggtacacc tctacaaagg
     4801 aggtcctgga cgccacactg attcatcagt caattacggg gctctatgaa acaagaatcg
     4861 acctctctca gctcggtgga gacaaacggc cggcagccac gaagaaggcc ggccaggcaa
     4921 aaaagaaaaa gggaggggga ggcagcaacc atgaccagga atttgaccct ccaaaggttt
     4981 acccacctgt gccagctgag aagaggaagc ccatccgcgt gctgtctctc tttgatggga
     5041 ttgctacagg gctcctggtg ctgaaggacc tgggcatcca agtggaccgc tacattgcct
     5101 ccgaggtgtg tgaggactcc atcacagtgg gcatggtgcg gcaccaggga aagatcatgt
     5161 acgtcgggga cgtccgcagc gtcacacaga agcatatcca ggagtggggc ccattcgacc
     5221 tggtgattgg aggcagtccc tgcaatgacc tctccattgt caaccctgcc cgcaagggac
     5281 tttatgaggg tactggccgc ctcttctttg agttctaccg cctcctgcat gatgctcggc
     5341 ccaaggaggg agatgatcgc cccttcttct ggctctttga gaatgtggtg gccatgggcg
     5401 ttagtgacaa gagggacatc tcccgatttc ttgagtctaa ccccgtgatg attgacgcca
     5461 aagaagtgtc tgctgcacac agggccagat acttctgggg taaccttcct ggcatgaaca
     5521 ggcctttggc atccactgtg aatgataagc tggagctgca agagtgtctg gagcacggca
     5581 gaatagccaa gttcagcaaa gtgaggacca ttaccaccag gtcaaactct ataaagcagg
     5641 gcaaagacca gcatttcccc gtcttcatga acgagaagga ggacatcctg tggtgcactg
     5701 aaatggaaag ggtgtttggc ttccccgtcc actacacaga cgtctccaac atgagccgct
     5761 tggccaggca gagactgctg ggccgatcct ggagcgtgcc ggtcatccgc cacctcttcg
     5821 ctccgctgaa ggaatatttt gcttgtgtga gcagcggcaa cagcaacgcc aacagccgcg
     5881 gccccagctt cagcagcggc ctggtgcccc tgagcctgcg cggcagccac atgggcccta
     5941 tggagatata caagacagtg tctgcatgga agagacagcc agtgcgggtg ctgagcctgt
     6001 tcagaaacat cgataaagtg ctgaagagtt tgggcttctt ggaaagcggt tctggttctg
     6061 ggggaggaac cctgaagtac gtggaagatg tcacaaatgt cgtgaggaga gacgtggaga
     6121 aatggggccc ctttgacctg gtgtacggct ccactcagcc cctgggcagc tcttgtgatc
     6181 gctgtcccgg ctggtacatg ttccagttcc accgaatcct gcagtatgcc ctgcctcgcc
     6241 aggagagtca gcggcccttc ttctggatat tcatggacaa tctgctgctg actgaggatg
     6301 accaagagac aactacccgc ttccttcaga cagaggctgt gaccctccag gatgtcagag
     6361 gcagagacta ccagaatgct atgcgggtgt ggagcaacat tccagggctg aagagcaagc
     6421 atgctcccct gaccccaaag gaagaagagt atctgcaagc ccaagtcaga agcaggagca
     6481 agctggacgc cccgaaagtt gacctcctgg tgaagaactg ccttctcccg ctgagagagt
     6541 acttcaagta tttttctcaa aactcacttc ctctcgcaac tttgtataat aaagttgaac
     6601 gagaaacgta aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga
     6661 ctacataata ctgtaaaaca caacatatcc agtcactatg aatcaactac ttagatggta
     6721 ttagtgacct gtactgcagg gcggccgcga tatcccctat agtgagtcgt attacatggt
     6781 catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca
     6841 agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag
     6901 gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga
     6961 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     7021 ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     7081 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     7141 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     7201 gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     7261 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     7321 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     7381 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     7441 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     7501 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     7561 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     7621 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     7681 attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta
     7741 acactggcag agcattacgc tgacttgacg ggacggcgca agctcatgac caaaatccct
     7801 taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     7861 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     7921 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     7981 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     8041 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     8101 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     8161 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     8221 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     8281 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     8341 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     8401 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     8461 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtt
//
