LOCUS       Exported                7113 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  pMAGIC R4-R3 entry plasmid, contains 2x NLS x-dCas9(3.7) fused to 
            the KRAB transcriptional repressor for 3 or 4-component MultiSite 
            Gateway Pro assembly..
ACCESSION   .
VERSION     .
KEYWORDS    KN901: pMAGIC (R4-R3) NLS-x dCas9(3.7)-NLS-KRAB
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7113)
  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 7113)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7113
                     /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"
     CDS             4968..5153
                     /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
                     QLTKPDVILRLEKGEEP"
     protein_bind    5176..5299
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5354..5373)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5355..5373)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5378..5394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5378..5394)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5496..5515)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5507..6316
                     /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(5574..5593)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6463..7051
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6952..6971
                     /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 gggcggaagc gatgctaagt cactaactgc ctggtcccgg acactggtga
     4981 ccttcaagga tgtatttgtg gacttcacca gggaggagtg gaagctgctg gacactgctc
     5041 agcagatcgt gtacagaaat gtgatgctgg agaactataa gaacctggtt tccttgggtt
     5101 atcagcttac taagccagat gtgatcctcc ggttggagaa gggagaagag cccggcggtt
     5161 ccggcggagg gagcgcaact ttgtataata aagttgaacg agaaacgtaa aatgatataa
     5221 atatcaatat attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac
     5281 aacatatcca gtcactatga atcaactact tagatggtat tagtgacctg tactgcaggg
     5341 cggccgcgat atcccctata gtgagtcgta ttacatggtc atagctgttt cctggcagct
     5401 ctggcccgtg tctcaaaatc tctgatgtta cattgcacaa gataaaaata tatcatcatg
     5461 aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga gccatattca
     5521 acgggaaacg tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa
     5581 atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc
     5641 cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga
     5701 tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt
     5761 tatccgtact cctgatgatg catggttact caccactgcg atccccggaa aaacagcatt
     5821 ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt
     5881 cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt
     5941 tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga
     6001 tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata aacttttgcc
     6061 attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga
     6121 cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca
     6181 ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct
     6241 ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct
     6301 cgatgagttt ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct
     6361 gacttgacgg gacggcgcaa gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt
     6421 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6481 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6541 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6601 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6661 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6721 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6781 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6841 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6901 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6961 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     7021 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     7081 cctggccttt tgctggcctt ttgctcacat gtt
//
