LOCUS       sequence-237375-        6168 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMAGIC R4-R3 entry plasmid, contains 2x NLS Sa-dCas9 fused to the 
            KRAB transcriptional repressor 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 6168)
  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 6168)
  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: IB801: pMAGIC (R4-R3) NLS-Sa dCas9-NLS-KRAB
FEATURES             Location/Qualifiers
     source          1..6168
                     /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             4023..4208
                     /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    4231..4354
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4409..4428)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4410..4428)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4433..4449)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4433..4449)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4551..4570)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4562..5371
                     /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(4629..4648)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      5518..6106
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6007..6026
                     /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 aaaaagggcg gaagcgatgc taagtcacta actgcctggt
     4021 cccggacact ggtgaccttc aaggatgtat ttgtggactt caccagggag gagtggaagc
     4081 tgctggacac tgctcagcag atcgtgtaca gaaatgtgat gctggagaac tataagaacc
     4141 tggtttcctt gggttatcag cttactaagc cagatgtgat cctccggttg gagaagggag
     4201 aagagcccgg cggttccggc ggagggagcg caactttgta taataaagtt gaacgagaaa
     4261 cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa cagactacat
     4321 aatactgtaa aacacaacat atccagtcac tatgaatcaa ctacttagat ggtattagtg
     4381 acctgtactg cagggcggcc gcgatatccc ctatagtgag tcgtattaca tggtcatagc
     4441 tgtttcctgg cagctctggc ccgtgtctca aaatctctga tgttacattg cacaagataa
     4501 aaatatatca tcatgaacaa taaaactgtc tgcttacata aacagtaata caaggggtgt
     4561 tatgagccat attcaacggg aaacgtcgag gccgcgatta aattccaaca tggatgctga
     4621 tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg
     4681 cttgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc
     4741 caatgatgtt acagatgaga tggtcagact aaactggctg acggaattta tgcctcttcc
     4801 gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc
     4861 cggaaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga
     4921 tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa
     4981 cagcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttga
     5041 tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat
     5101 gcataaactt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga
     5161 taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat
     5221 cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc
     5281 attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca
     5341 gtttcatttg atgctcgatg agtttttcta atcagaattg gttaattggt tgtaacactg
     5401 gcagagcatt acgctgactt gacgggacgg cgcaagctca tgaccaaaat cccttaacgt
     5461 gagttacgcg tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5521 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5581 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5641 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5701 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5761 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5821 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5881 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5941 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6001 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6061 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6121 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtt
//
