LOCUS       sequence-364107-        6902 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  VPR activation domain coupled to reverse Tet repressor; labeled with
            GFP; binds tetO sites upon doxycycline addition.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6902)
  AUTHORS   Trojanowski J, Frank L, Rademacher A, Mucke N, Grigaitis P, Rippe K
  TITLE     Transcription activation is enhanced by multivalent interactions 
            independent of phase separation.
  JOURNAL   Mol Cell. 2022 May 4. pii: S1097-2765(22)00328-8. doi: 
            10.1016/j.molcel.2022.04.017.
  PUBMED    35537448
REFERENCE   2  (bases 1 to 6902)
  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: "Mol Cell. 
            2022 May 4. pii: S1097-2765(22)00328-8. doi: 
            10.1016/j.molcel.2022.04.017."
COMMENT     Sequence Label: rTetR-GFP-VPR
FEATURES             Location/Qualifiers
     source          1..6902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        146..449
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        450..653
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     604..624
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1352..1354,1355..1357,1358..2068)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1352..1354,1355..1357,1358..2068)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1397..1418)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1658..1677)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2005..2026
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2102..2122
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2189..2338
                     /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             2363..2383
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2813..3169
                     /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             3188..3757
                     /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"
     polyA_signal    3775..3896
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3812..3831)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3866..3885
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3903..4358)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4040..4061)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4252..4271
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4385..4489
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4491..4848
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4491..4682
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4514..4534)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4699..4834
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4883..5677
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4883..5677
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4937..4956)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5547..5566
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5865..5884)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5909..5956
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6285..6873
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6774..6793
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
       61 ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag
      121 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
      181 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
      241 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
      301 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
      361 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
      421 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
      481 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
      541 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
      601 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
      661 tgaaccgtca gatccgctag cgctaccgga ctcagatctc gaggccacca tgtctagact
      721 ggacaagagc aaagtcataa actctgctct ggaattactc aatggagtcg gtatcgaagg
      781 cctgacgaca aggaaactcg ctcaaaagct gggagttgag cagcctaccc tgtactggca
      841 cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc gagatgctgg acaggcatca
      901 tacccactcc tgccccctgg aaggcgagtc atggcaagac tttctgcgga acaacgccaa
      961 gtcataccgc tgtgctctcc tctcacatcg cgacggggct aaagtgcatc tcggcacccg
     1021 cccaacagag aaacagtacg aaaccctgga aaatcagctc gcgttcctgt gtcagcaagg
     1081 cttctccctg gagaacgcac tgtacgctct gtccgccgtg ggccacttta cactgggctg
     1141 cgtattggag gaacaggagc atcaagtagc aaaagaggaa agagagacac ctaccaccga
     1201 ttctatgccc ccacttctga aacaagcaat tgagctgttc gaccggcagg gagccgaacc
     1261 tgccttcctt ttcggcctgg aactaatcat atgtggcctg gagaaacagc taaagtgcga
     1321 aagcggcggg ccgacccgat cggcccgggt catggtgagc aagggcgagg agctgttcac
     1381 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     1441 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     1501 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     1561 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     1621 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     1681 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     1741 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
     1801 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
     1861 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
     1921 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa
     1981 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
     2041 cactctcggc atggacgagc tgtacaagag cggccgcagg gggcgtcgac tgggtacccc
     2101 tcctaagaag aagaggaagg ttgcccccac catggataaa aagaagagaa aggccccccc
     2161 gaccgatgtc agcctggggg acgagctcga cgcattggac gattttgatc tggatatgct
     2221 gggaagtgac gccctcgatg attttgacct tgacatgctt ggttcggatg cccttgatga
     2281 ctttgacctc gacatgctcg gcagtgacgc ccttgatgat ttcgacctgg acatgctgat
     2341 taactctaga agttccggat ctccgaaaaa gaaacgcaaa gttggtagcc agtacctgcc
     2401 cgacaccgac gaccggcacc ggatcgagga aaagcggaag cggacctacg agacattcaa
     2461 gagcatcatg aagaagtccc ccttcagcgg ccccaccgac cctagacctc cacctagaag
     2521 aatcgccgtg cccagcagat ccagcgccag cgtgccaaaa cctgcccccc agccttaccc
     2581 cttcaccagc agcctgagca ccatcaacta cgacgagttc cctaccatgg tgttccccag
     2641 cggccagatc tctcaggcct ctgctctggc tccagcccct cctcaggtgc tgcctcaggc
     2701 tcctgctcct gcaccagctc cagccatggt gtctgcactg gctcaggcac cagcacccgt
     2761 gcctgtgctg gctcctggac ctccacaggc tgtggctcca ccagccccta aacctacaca
     2821 ggccggcgag ggcacactgt ctgaagctct gctgcagctg cagttcgacg acgaggatct
     2881 gggagccctg ctgggaaaca gcaccgatcc tgccgtgttc accgacctgg ccagcgtgga
     2941 caacagcgag ttccagcagc tgctgaacca gggcatccct gtggcccctc acaccaccga
     3001 gcccatgctg atggaatacc ccgaggccat cacccggctc gtgacaggcg ctcagaggcc
     3061 tcctgatcca gctcctgccc ctctgggagc accgggcctg cctaatggac tgctgtctgg
     3121 cgacgaggac ttcagctcta tcgccgatat ggatttctca gccttgctgg gctctggcag
     3181 cggcagccgg gattccaggg aagggatgtt tttgccgaag cctgaggccg gctccgctat
     3241 tagtgacgtg tttgagggcc gcgaggtgtg ccagccaaaa cgaatccggc catttcatcc
     3301 tccaggaagt ccatgggcca accgcccact ccccgccagc ctcgcaccaa caccaaccgg
     3361 tccagtacat gagccagtcg ggtcactgac cccggcacca gtccctcagc cactggatcc
     3421 agcgcccgca gtgactcccg aggccagtca cctgttggag gatcccgatg aagagacgag
     3481 ccaggctgtc aaagcccttc gggagatggc cgatactgtg attccccaga aggaagaggc
     3541 tgcaatctgt ggccaaatgg acctttccca tccgccccca aggggccatc tggatgagct
     3601 gacaaccaca cttgagtcca tgaccgagga tctgaacctg gactcacccc tgaccccgga
     3661 attgaacgag attctggata ccttcctgaa cgacgagtgc ctcttgcatg ccatgcatat
     3721 cagcacagga ctgtccatct tcgacacatc tctgttttaa caattgttgt tgttaacttg
     3781 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3841 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     3901 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3961 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     4021 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     4081 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     4141 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     4201 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     4261 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     4321 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     4381 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4441 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     4501 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4561 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4621 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4681 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4741 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4801 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4861 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4921 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4981 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     5041 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     5101 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     5161 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     5221 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     5281 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5341 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5401 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5461 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5521 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5581 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5641 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5701 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5761 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5821 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     5881 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     5941 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     6001 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     6061 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     6121 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6181 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6241 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6301 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6361 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6421 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6481 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6541 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6601 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6661 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6721 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6781 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6841 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6901 tt
//
