LOCUS       Exported                4401 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  pMAGIC R4-R3 entry plasmid, contains 2x NLS/VPR scaffold enabling 
            addition of dCas9 mutants into pMAGIC. dCas9 can be inserted via 
            unique SrfI/PmeI restriction sites.
ACCESSION   .
VERSION     .
KEYWORDS    KL001: pMAGIC (R4-R3) NLS-(SrfI/PmeI)-NLS-VPR
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4401)
  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 4401)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..4401
                     /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             813..860
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             891..1040
                     /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             1065..1085
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1515..1871
                     /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             1890..2459
                     /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    2464..2587
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2642..2661)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2643..2661)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2666..2682)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2666..2682)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2784..2803)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2795..3604
                     /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(2862..2881)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      3751..4339
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4240..4259
                     /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 ggatccgagg ccagcggttc cggacgggct gacgcattgg
      901 acgattttga tctggatatg ctgggaagtg acgccctcga tgattttgac cttgacatgc
      961 ttggttcgga tgcccttgat gactttgacc tcgacatgct cggcagtgac gcccttgatg
     1021 atttcgacct ggacatgctg attaactcta gaagttccgg atctccgaaa aagaaacgca
     1081 aagttggtag ccagtacctg cccgacaccg acgaccggca ccggatcgag gaaaagcgga
     1141 agcggaccta cgagacattc aagagcatca tgaagaagtc ccccttcagc ggccccaccg
     1201 accctagacc tccacctaga agaatcgccg tgcccagcag atccagcgcc agcgtgccaa
     1261 aacctgcccc ccagccttac cccttcacca gcagcctgag caccatcaac tacgacgagt
     1321 tccctaccat ggtgttcccc agcggccaga tctctcaggc ctctgctctg gctccagccc
     1381 ctcctcaggt gctgcctcag gctcctgctc ctgcaccagc tccagccatg gtgtctgcac
     1441 tggctcaggc accagcaccc gtgcctgtgc tggctcctgg acctccacag gctgtggctc
     1501 caccagcccc taaacctaca caggccggcg agggcacact gtctgaagct ctgctgcagc
     1561 tgcagttcga cgacgaggat ctgggagccc tgctgggaaa cagcaccgat cctgccgtgt
     1621 tcaccgacct ggccagcgtg gacaacagcg agttccagca gctgctgaac cagggcatcc
     1681 ctgtggcccc tcacaccacc gagcccatgc tgatggaata ccccgaggcc atcacccggc
     1741 tcgtgacagg cgctcagagg cctcctgatc cagctcctgc ccctctggga gcaccaggcc
     1801 tgcctaatgg actgctgtct ggcgacgagg acttcagctc tatcgccgat atggatttct
     1861 cagccttgct gggctctggc agcggcagcc gggattccag ggaagggatg tttttgccga
     1921 agcctgaggc cggctccgct attagtgacg tgtttgaggg ccgcgaggtg tgccagccaa
     1981 aacgaatccg gccatttcat cctccaggaa gtccatgggc caaccgccca ctccccgcca
     2041 gcctcgcacc aacaccaacc ggtccagtac atgagccagt cgggtcactg accccggcac
     2101 cagtccctca gccactggat ccagcgcccg cagtgactcc cgaggccagt cacctgttgg
     2161 aggatcccga tgaagagacg agccaggctg tcaaagccct tcgggagatg gccgatactg
     2221 tgattcccca gaaggaagag gctgcaatct gtggccaaat ggacctttcc catccgcccc
     2281 caaggggcca tctggatgag ctgacaacca cacttgagtc catgaccgag gatctgaacc
     2341 tggactcacc cctgaccccg gaattgaacg agattctgga taccttcctg aacgacgagt
     2401 gcctcttgca tgccatgcat atcagcacag gactgtccat cttcgacaca tctctgtttg
     2461 gagcaacttt gtataataaa gttgaacgag aaacgtaaaa tgatataaat atcaatatat
     2521 taaattagat tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt
     2581 cactatgaat caactactta gatggtatta gtgacctgta ctgcagggcg gccgcgatat
     2641 cccctatagt gagtcgtatt acatggtcat agctgtttcc tggcagctct ggcccgtgtc
     2701 tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa caataaaact
     2761 gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc
     2821 gaggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga
     2881 taatgtcggg caatcaggtg cgacaatcta tcgcttgtat gggaagcccg atgcgccaga
     2941 gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag
     3001 actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc
     3061 tgatgatgca tggttactca ccactgcgat ccccggaaaa acagcattcc aggtattaga
     3121 agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt
     3181 gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca
     3241 ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa
     3301 tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga
     3361 ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt
     3421 aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat
     3481 cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata
     3541 tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt
     3601 ctaatcagaa ttggttaatt ggttgtaaca ctggcagagc attacgctga cttgacggga
     3661 cggcgcaagc tcatgaccaa aatcccttaa cgtgagttac gcgtcgttcc actgagcgtc
     3721 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     3781 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     3841 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct
     3901 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     3961 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4021 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4081 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4141 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4201 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
     4261 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4321 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4381 ctggcctttt gctcacatgt t
//
