LOCUS       sequence-237391-        3042 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pMAGIC R4-R3 entry plasmid, contains 2x NLS/KRAB scaffold enabling 
            addition of dCas9 mutants into pMAGIC. dCas9 can be inserted via 
            unique SrfI/PmeI restriction sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3042)
  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 3042)
  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: KL101: pMAGIC (R4-R3) NLS-(SrfI/PmeI)-NLS-KRAB
FEATURES             Location/Qualifiers
     source          1..3042
                     /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             813..860
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             897..1082
                     /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    1105..1228
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1283..1302)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1284..1302)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1307..1323)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1307..1323)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1425..1444)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1436..2245
                     /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(1503..1522)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2392..2980
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2881..2900
                     /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 cccgggcgag accatgctcg cggtctcagt ttaaacggcc ggcagccacg aagaaggccg
      841 gccaggcaaa aaagaaaaag ggcggaagcg atgctaagtc actaactgcc tggtcccgga
      901 cactggtgac cttcaaggat gtatttgtgg acttcaccag ggaggagtgg aagctgctgg
      961 acactgctca gcagatcgtg tacagaaatg tgatgctgga gaactataag aacctggttt
     1021 ccttgggtta tcagcttact aagccagatg tgatcctccg gttggagaag ggagaagagc
     1081 ccggcggttc cggcggaggg agcgcaactt tgtataataa agttgaacga gaaacgtaaa
     1141 atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact
     1201 gtaaaacaca acatatccag tcactatgaa tcaactactt agatggtatt agtgacctgt
     1261 actgcagggc ggccgcgata tcccctatag tgagtcgtat tacatggtca tagctgtttc
     1321 ctggcagctc tggcccgtgt ctcaaaatct ctgatgttac attgcacaag ataaaaatat
     1381 atcatcatga acaataaaac tgtctgctta cataaacagt aatacaaggg gtgttatgag
     1441 ccatattcaa cgggaaacgt cgaggccgcg attaaattcc aacatggatg ctgatttata
     1501 tgggtataaa tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgcttgta
     1561 tgggaagccc gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga
     1621 tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat
     1681 caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga tccccggaaa
     1741 aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct
     1801 ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga
     1861 tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag
     1921 tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa
     1981 acttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct
     2041 tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga
     2101 ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca
     2161 gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca
     2221 tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac actggcagag
     2281 cattacgctg acttgacggg acggcgcaag ctcatgacca aaatccctta acgtgagtta
     2341 cgcgtcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2401 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2461 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2521 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2581 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2641 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2701 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2761 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2821 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2881 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2941 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3001 tttacggttc ctggcctttt gctggccttt tgctcacatg tt
//
