LOCUS       sequence-271589-        5422 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DmrC with VP64 fused to it's C-terminus; Mammalian expression 
            vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5422)
  AUTHORS   Tak YE, Horng JE, Perry NT, Schultz HT, Iyer S, Yao Q, Zou LS, Aryee
            MJ, Pinello L, Joung JK
  TITLE     Augmenting and directing long-range CRISPR-mediated activation in 
            human cells.
  JOURNAL   Nat Methods. 2021 Sep;18(9):1075-1081. doi: 
            10.1038/s41592-021-01224-1. Epub 2021 Aug 5.
  PUBMED    34354266
REFERENCE   2  (bases 1 to 5422)
  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"; doi: 
            "10.1038/s41592-021-01224-1"; journalName: "Nat Methods"; date: 
            "2021-09"; volume: "18"; issue: "9"; pages: "1075-1081"
COMMENT     Sequence Label: BPK880-pCAG-DmrC-NLS-3xFLAG-VP64
FEATURES             Location/Qualifiers
     source          1..5422
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        382..659
                     /label=chicken beta-actin promoter
     intron          660..1676
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1600..1622
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1684..1703
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1769..1778
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1787..2065
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     CDS             2078..2098
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2105..2170
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2201..2350
                     /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"
     primer_bind     complement(2440..2459)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2505..2560
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2559..2578)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2921..2937)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2921..2937)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2934..2956)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2945..2961
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2969..2975,2976..2993,2994..2999))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3014..3035
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3094..3289
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3140..3275
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3295..3429
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3332..3351)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3386..3405
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3497..3514)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3668..4256)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3748..3767)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4427..5218,5219..5287))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5050..5069
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5288..5392)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct
      421 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga
      481 tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg
      541 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
      601 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
      661 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
      721 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
      781 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
      841 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct
      961 gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg
     1021 gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     1081 gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc
     1141 cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg
     1201 gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg
     1261 ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg
     1321 gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc
     1381 agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc
     1441 cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg
     1501 gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc
     1561 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
     1621 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
     1681 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc tgcagtcgac
     1741 ggtaccgcgg gcccgggatc caccggtcgc caccatggga tccagaatcc tctggcatga
     1801 gatgtggcat gaaggcctgg aagaggcatc tcgtttgtac tttggggaaa ggaacgtgaa
     1861 aggcatgttt gaggtgctgg agcccttgca tgctatgatg gaacggggac cccagactct
     1921 gaaggaaaca tcctttaatc aggcctatgg tcgagattta atggaggccc aagagtggtg
     1981 caggaagtac atgaaatcag ggaatgtcaa ggacctcctc caagcctggg acctctatta
     2041 tcatgtgttc cgacgaatct caaagggcgg cggatccccc aagaagaaga ggaaagtctc
     2101 gagcgactac aaagaccatg acggtgatta taaagatcat gacatcgatt acaaggatga
     2161 cgatgacaag gctgcaggag gcggtggaag cgggcgcgcc gacgcgctgg acgatttcga
     2221 tctcgacatg ctgggttctg atgccctcga tgactttgac ctggatatgt tgggaagcga
     2281 cgcattggat gactttgatc tggacatgct cggctccgat gctctggacg atttcgatct
     2341 cgatatgtta taaagcggcc gcactcctca ggtgcaggct gcctatcaga aggtggtggc
     2401 tggtgtggcc aatgccctgg ctcacaaata ccactgagat ctttttccct ctgccaaaaa
     2461 ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa ggaaatttat
     2521 tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga catatgggag
     2581 ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa catatgccca
     2641 tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata tgaaacagcc
     2701 ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga ttttttttat
     2761 attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac atgttttact
     2821 agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc ttctcttatg
     2881 gagatccctc gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     2941 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     3001 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     3061 tccagtcggg aaacctgtcg tgccagcgga tccgcatctc aattagtcag caaccatagt
     3121 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     3181 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     3241 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctaacttg
     3301 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3361 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     3421 gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     3481 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     3541 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     3601 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     3661 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3721 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3781 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3841 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3901 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3961 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4021 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4081 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     4141 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4201 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4261 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4321 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4381 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4441 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     4501 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     4561 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     4621 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     4681 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     4741 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     4801 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     4861 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     4921 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4981 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5041 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5101 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5161 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5221 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5281 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5341 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5401 cccgaaaagt gccacctggg tc
//
