LOCUS       Exported                6876 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  pMAGIC R4-R3 entry plasmid, contains 2x NLS xCas9(3.7) (nuclease) 
            fusion for 3 or 4-component MultiSite Gateway Pro assembly..
ACCESSION   .
VERSION     .
KEYWORDS    KO401: pMAGIC (R4-R3) NLS-x Cas9(3.7)-NLS
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6876)
  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 6876)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6876
                     /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="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFIQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     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    4939..5062
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5117..5136)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5118..5136)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5141..5157)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5141..5157)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5259..5278)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5270..6079
                     /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(5337..5356)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6226..6814
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6715..6734
                     /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 gggctcgata 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 gtggaccata
     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 gggatccgca actttgtata ataaagttga acgagaaacg taaaatgata
     4981 taaatatcaa tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa
     5041 cacaacatat ccagtcacta tgaatcaact acttagatgg tattagtgac ctgtactgca
     5101 gggcggccgc gatatcccct atagtgagtc gtattacatg gtcatagctg tttcctggca
     5161 gctctggccc gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc
     5221 atgaacaata aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat
     5281 tcaacgggaa acgtcgaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     5341 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgct tgtatgggaa
     5401 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     5461 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     5521 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg gaaaaacagc
     5581 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     5641 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     5701 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     5761 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt
     5821 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     5881 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     5941 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     6001 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     6061 gctcgatgag tttttctaat cagaattggt taattggttg taacactggc agagcattac
     6121 gctgacttga cgggacggcg caagctcatg accaaaatcc cttaacgtga gttacgcgtc
     6181 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6241 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6301 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6361 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6421 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6481 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6541 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6601 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6661 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6721 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6781 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6841 gttcctggcc ttttgctggc cttttgctca catgtt
//
