LOCUS       sequence-237380-        7527 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMAGIC R4-R3 entry plasmid, contains 2x NLS Sa-dCas9 fused to the 
            VPR transcriptional activator for 3 or 4-component MultiSite Gateway
            Pro assembly..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7527)
  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 7527)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2018 Dec 27. pii: 5264291. doi: 10.1093/nar/gky1286."
COMMENT     Sequence Label: IA304: pMAGIC (R4-R3) NLS-Sa dCas9-NLS-VPR
FEATURES             Location/Qualifiers
     source          1..7527
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(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..726)
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      726..735
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             738..758
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3939..3986
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             4017..4166
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             4191..4211
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4641..4997
                     /codon_start=1
                     /product="transcriptional activation domain of human RelA, 
                     also known as p65 (O'Shea and Perkins, 2008)"
                     /label=RelA (p65) AD
                     /translation="PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNS
                     EFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGD
                     EDFSSIADMDFSALL"
     CDS             5016..5585
                     /codon_start=1
                     /product="transcriptional activation domain from the human 
                     herpesvirus 4 (Epstein-Barr virus) replication and 
                     transcription activator Rta/BRLF1 (Hardwick et al., 1992; 
                     Chavez et al., 2015)"
                     /label=Rta AD
                     /translation="RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWAN
                     RPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKAL
                     REMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEIL
                     DTFLNDECLLHAMHISTGLSIFDTSLF"
     protein_bind    5590..5713
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5768..5787)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5769..5787)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5792..5808)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5792..5808)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5910..5929)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5921..6730
                     /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(5988..6007)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      6877..7465
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7366..7385
                     /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 ccaagcggaa ctacatcctg ggcctggcca tcggcatcac cagcgtgggc tacggcatca
      841 tcgactacga gacacgggac gtgatcgatg ccggcgtgcg gctgttcaaa gaggccaacg
      901 tggaaaacaa cgagggcagg cggagcaaga gaggcgccag aaggctgaag cggcggaggc
      961 ggcatagaat ccagagagtg aagaagctgc tgttcgacta caacctgctg accgaccaca
     1021 gcgagctgag cggcatcaac ccctacgagg ccagagtgaa gggcctgagc cagaagctga
     1081 gcgaggaaga gttctctgcc gccctgctgc acctggccaa gagaagaggc gtgcacaacg
     1141 tgaacgaggt ggaagaggac accggcaacg agctgtccac caaagagcag atcagccgga
     1201 acagcaaggc cctggaagag aaatacgtgg ccgaactgca gctggaacgg ctgaagaaag
     1261 acggcgaagt gcggggcagc atcaacagat tcaagaccag cgactacgtg aaagaagcca
     1321 aacagctgct gaaggtgcag aaggcctacc accagctgga ccagagcttc atcgacacct
     1381 acatcgacct gctggaaacc cggcggacct actatgaggg acctggcgag ggcagcccct
     1441 tcggctggaa ggacatcaaa gaatggtacg agatgctgat gggccactgc acctacttcc
     1501 ccgaggaact gcggagcgtg aagtacgcct acaacgccga cctgtacaac gccctgaacg
     1561 acctgaacaa tctcgtgatc accagggacg agaacgagaa gctggaatat tacgagaagt
     1621 tccagatcat cgagaacgtg ttcaagcaga agaagaagcc caccctgaag cagatcgcca
     1681 aagaaatcct cgtgaacgaa gaggatatta agggctacag agtgaccagc accggcaagc
     1741 ccgagttcac caacctgaag gtgtaccacg acatcaagga cattaccgcc cggaaagaga
     1801 ttattgagaa cgccgagctg ctggatcaga ttgccaagat cctgaccatc taccagagca
     1861 gcgaggacat ccaggaagaa ctgaccaatc tgaactccga gctgacccag gaagagatcg
     1921 agcagatctc taatctgaag ggctataccg gcacccacaa cctgagcctg aaggccatca
     1981 acctgatcct ggacgagctg tggcacacca acgacaacca gatcgctatc ttcaaccggc
     2041 tgaagctggt gcccaagaag gtggacctgt cccagcagaa agagatcccc accaccctgg
     2101 tggacgactt catcctgagc cccgtcgtga agagaagctt catccagagc atcaaagtga
     2161 tcaacgccat catcaagaag tacggcctgc ccaacgacat cattatcgag ctggcccgcg
     2221 agaagaactc caaggacgcc cagaaaatga tcaacgagat gcagaagcgg aaccggcaga
     2281 ccaacgagcg gatcgaggaa atcatccgga ccaccggcaa agagaacgcc aagtacctga
     2341 tcgagaagat caagctgcac gacatgcagg aaggcaagtg cctgtacagc ctggaagcca
     2401 tccctctgga agatctgctg aacaacccct tcaactatga ggtggaccac atcatcccca
     2461 gaagcgtgtc cttcgacaac agcttcaaca acaaggtgct cgtgaagcag gaagaagcca
     2521 gcaagaaggg caaccggacc ccattccagt acctgagcag cagcgacagc aagatcagct
     2581 acgaaacctt caagaagcac atcctgaatc tggccaaggg caagggcaga atcagcaaga
     2641 ccaagaaaga gtatctgctg gaagaacggg acatcaacag gttctccgtg cagaaagact
     2701 tcatcaaccg gaacctggtg gataccagat acgccaccag aggcctgatg aacctgctgc
     2761 ggagctactt cagagtgaac aacctggacg tgaaagtgaa gtccatcaat ggcggcttca
     2821 ccagctttct gcggcggaag tggaagttta agaaagagcg gaacaagggg tacaagcacc
     2881 acgccgagga cgccctgatc attgccaacg ccgatttcat cttcaaagag tggaagaaac
     2941 tggacaaggc caaaaaagtg atggaaaacc agatgttcga ggaaaagcag gccgagagca
     3001 tgcccgagat cgaaaccgag caggagtaca aagagatctt catcaccccc caccagatca
     3061 agcacattaa ggacttcaag gactacaagt acagccaccg ggtggacaag aagcctaata
     3121 gagagctgat taacgacacc ctgtactcca cccggaagga cgacaagggc aacaccctga
     3181 tcgtgaacaa tctgaacggc ctgtacgaca aggacaatga caagctgaaa aagctgatca
     3241 acaagagccc cgaaaagctg ctgatgtacc accacgaccc ccagacctac cagaaactga
     3301 agctgattat ggaacagtac ggcgacgaga agaatcccct gtacaagtac tacgaggaaa
     3361 ccgggaacta cctgaccaag tactccaaaa aggacaacgg ccccgtgatc aagaagatta
     3421 agtattacgg caacaaactg aacgcccatc tggacatcac cgacgactac cccaacagca
     3481 gaaacaaggt cgtgaagctg tccctgaagc cctacagatt cgacgtgtac ctggacaatg
     3541 gcgtgtacaa gttcgtgacc gtgaagaatc tggatgtgat caaaaaagaa aactactacg
     3601 aagtgaatag caagtgctat gaggaagcta agaagctgaa gaagatcagc aaccaggccg
     3661 agtttatcgc ctccttctac aacaacgatc tgatcaagat caacggcgag ctgtatagag
     3721 tgatcggcgt gaacaacgac ctgctgaacc ggatcgaagt gaacatgatc gacatcacct
     3781 accgcgagta cctggaaaac atgaacgaca agaggccccc caggatcatt aagacaatcg
     3841 cctccaagac ccagagcatt aagaagtaca gcacagacat tctgggcaac ctgtatgaag
     3901 tgaaatctaa gaagcaccct cagatcatca aaaagggcaa aaggccggcg gccacgaaaa
     3961 aggccggcca ggcaaaaaag aaaaagggat ccgaggccag cggttccgga cgggctgacg
     4021 cattggacga ttttgatctg gatatgctgg gaagtgacgc cctcgatgat tttgaccttg
     4081 acatgcttgg ttcggatgcc cttgatgact ttgacctcga catgctcggc agtgacgccc
     4141 ttgatgattt cgacctggac atgctgatta actctagaag ttccggatct ccgaaaaaga
     4201 aacgcaaagt tggtagccag tacctgcccg acaccgacga ccggcaccgg atcgaggaaa
     4261 agcggaagcg gacctacgag acattcaaga gcatcatgaa gaagtccccc ttcagcggcc
     4321 ccaccgaccc tagacctcca cctagaagaa tcgccgtgcc cagcagatcc agcgccagcg
     4381 tgccaaaacc tgccccccag ccttacccct tcaccagcag cctgagcacc atcaactacg
     4441 acgagttccc taccatggtg ttccccagcg gccagatctc tcaggcctct gctctggctc
     4501 cagcccctcc tcaggtgctg cctcaggctc ctgctcctgc accagctcca gccatggtgt
     4561 ctgcactggc tcaggcacca gcacccgtgc ctgtgctggc tcctggacct ccacaggctg
     4621 tggctccacc agcccctaaa cctacacagg ccggcgaggg cacactgtct gaagctctgc
     4681 tgcagctgca gttcgacgac gaggatctgg gagccctgct gggaaacagc accgatcctg
     4741 ccgtgttcac cgacctggcc agcgtggaca acagcgagtt ccagcagctg ctgaaccagg
     4801 gcatccctgt ggcccctcac accaccgagc ccatgctgat ggaatacccc gaggccatca
     4861 cccggctcgt gacaggcgct cagaggcctc ctgatccagc tcctgcccct ctgggagcac
     4921 caggcctgcc taatggactg ctgtctggcg acgaggactt cagctctatc gccgatatgg
     4981 atttctcagc cttgctgggc tctggcagcg gcagccggga ttccagggaa gggatgtttt
     5041 tgccgaagcc tgaggccggc tccgctatta gtgacgtgtt tgagggccgc gaggtgtgcc
     5101 agccaaaacg aatccggcca tttcatcctc caggaagtcc atgggccaac cgcccactcc
     5161 ccgccagcct cgcaccaaca ccaaccggtc cagtacatga gccagtcggg tcactgaccc
     5221 cggcaccagt ccctcagcca ctggatccag cgcccgcagt gactcccgag gccagtcacc
     5281 tgttggagga tcccgatgaa gagacgagcc aggctgtcaa agcccttcgg gagatggccg
     5341 atactgtgat tccccagaag gaagaggctg caatctgtgg ccaaatggac ctttcccatc
     5401 cgcccccaag gggccatctg gatgagctga caaccacact tgagtccatg accgaggatc
     5461 tgaacctgga ctcacccctg accccggaat tgaacgagat tctggatacc ttcctgaacg
     5521 acgagtgcct cttgcatgcc atgcatatca gcacaggact gtccatcttc gacacatctc
     5581 tgtttggagc aactttgtat aataaagttg aacgagaaac gtaaaatgat ataaatatca
     5641 atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa acacaacata
     5701 tccagtcact atgaatcaac tacttagatg gtattagtga cctgtactgc agggcggccg
     5761 cgatatcccc tatagtgagt cgtattacat ggtcatagct gtttcctggc agctctggcc
     5821 cgtgtctcaa aatctctgat gttacattgc acaagataaa aatatatcat catgaacaat
     5881 aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata ttcaacggga
     5941 aacgtcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc
     6001 tcgcgataat gtcgggcaat caggtgcgac aatctatcgc ttgtatggga agcccgatgc
     6061 gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat
     6121 ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc attttatccg
     6181 tactcctgat gatgcatggt tactcaccac tgcgatcccc ggaaaaacag cattccaggt
     6241 attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg
     6301 ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct
     6361 cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga
     6421 gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc
     6481 accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg
     6541 gaaattaata ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct
     6601 tgccatccta tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca
     6661 aaaatatggt attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga
     6721 gtttttctaa tcagaattgg ttaattggtt gtaacactgg cagagcatta cgctgacttg
     6781 acgggacggc gcaagctcat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg
     6841 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     6901 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     6961 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     7021 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     7081 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7141 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7201 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7261 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7321 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7381 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7441 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7501 cttttgctgg ccttttgctc acatgtt
//
