LOCUS       sequence-364151-        7061 bp    DNA     circular SYN 15-APR-2025
DEFINITION  PCP tandem dimer binding to PP7 RNA stem loops coupled to VP16 
            activation domain; labeled with GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7061)
  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 7061)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: NLS-tdPCP-GFP-VPR
FEATURES             Location/Qualifiers
     source          1..7061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        68..371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        372..575
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     526..546
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             644..664
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1433..1435,1436..1438,1439..2149)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1433..1435,1436..1438,1439..2149)
                     /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(1478..1499)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1739..1758)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2086..2107
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2183..2203
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2270..2419
                     /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             2444..2464
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2894..3250
                     /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             3269..3838
                     /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    3856..3977
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3893..3912)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3947..3966
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3984..4439)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4121..4142)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4333..4352
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4466..4570
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4572..4929
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4572..4763
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4595..4615)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4780..4915
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4964..5758
                     /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             4964..5758
                     /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(5018..5037)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5628..5647
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5946..5965)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5990..6037
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6366..6954
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6855..6874
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
       61 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      121 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      181 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      241 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      301 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      361 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgaca
      421 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      481 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      541 ggcgtgtacg gtgggaggtc ggggggggca gagctggttt agtgaaccgt cagatccgct
      601 agcgctaccg gactcagatc tcgagctcaa gcttctcatg ggcccaaaaa agaaaagaaa
      661 agttgggtca catatgctag cctccaaaac catcgttctt tcggtcggcg aggctactcg
      721 cactctgact gagatccagt ccaccgcaga ccgtcagatc ttcgaagaga aggtcgggcc
      781 tctggtgggt cggctgcgcc tcacggcttc gctccgtcaa aacggagcca agaccgcgta
      841 tcgcgtcaac ctaaaactgg atcaggcgga cgtcgttgat tccggacttc cgaaagtgcg
      901 ctacactcag gtatggtcgc acgacgtgac aatcgttgcg aatagcaccg aggcctcgcg
      961 caaatcgttg tacgatttga ccaagtccct cgtcgcgacc tcgcaggtcg aagatcttgt
     1021 cgtcaacctt gtgccgctgg gccgtgcgga tccgctagcc tccaaaacca tcgttctttc
     1081 ggtcggcgag gctactcgca ctctgactga gatccagtcc accgcagacc gtcagatctt
     1141 cgaagagaag gtcgggcctc tggtgggtcg gctgcgcctc acggcttcgc tccgtcaaaa
     1201 cggagccaag accgcgtatc gcgtcaacct aaaactggat caggcggacg tcgttgattc
     1261 cggacttccg aaagtgcgct acactcaggt atggtcgcac gacgtgacaa tcgttgcgaa
     1321 tagcaccgag gcctcgcgca aatcgttgta cgatttgacc aagtccctcg tcgcgacctc
     1381 gcaggtcgaa gatcttgtcg tcaaccttgt gccgctgggc cgtccaccgg tcatggtgag
     1441 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     1501 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     1561 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     1621 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     1681 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     1741 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     1801 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     1861 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     1921 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     1981 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     2041 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     2101 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaaga gcggccgcag
     2161 ggggcgtcga ctgggtaccc ctcctaagaa gaagaggaag gttgccccca ccatggataa
     2221 aaagaagaga aaggcccccc cgaccgatgt cagcctgggg gacgagctcg acgcattgga
     2281 cgattttgat ctggatatgc tgggaagtga cgccctcgat gattttgacc ttgacatgct
     2341 tggttcggat gcccttgatg actttgacct cgacatgctc ggcagtgacg cccttgatga
     2401 tttcgacctg gacatgctga ttaactctag aagttccgga tctccgaaaa agaaacgcaa
     2461 agttggtagc cagtacctgc ccgacaccga cgaccggcac cggatcgagg aaaagcggaa
     2521 gcggacctac gagacattca agagcatcat gaagaagtcc cccttcagcg gccccaccga
     2581 ccctagacct ccacctagaa gaatcgccgt gcccagcaga tccagcgcca gcgtgccaaa
     2641 acctgccccc cagccttacc ccttcaccag cagcctgagc accatcaact acgacgagtt
     2701 ccctaccatg gtgttcccca gcggccagat ctctcaggcc tctgctctgg ctccagcccc
     2761 tcctcaggtg ctgcctcagg ctcctgctcc tgcaccagct ccagccatgg tgtctgcact
     2821 ggctcaggca ccagcacccg tgcctgtgct ggctcctgga cctccacagg ctgtggctcc
     2881 accagcccct aaacctacac aggccggcga gggcacactg tctgaagctc tgctgcagct
     2941 gcagttcgac gacgaggatc tgggagccct gctgggaaac agcaccgatc ctgccgtgtt
     3001 caccgacctg gccagcgtgg acaacagcga gttccagcag ctgctgaacc agggcatccc
     3061 tgtggcccct cacaccaccg agcccatgct gatggaatac cccgaggcca tcacccggct
     3121 cgtgacaggc gctcagaggc ctcctgatcc agctcctgcc cctctgggag caccaggcct
     3181 gcctaatgga ctgctgtctg gcgacgagga cttcagctct atcgccgata tggatttctc
     3241 agccttgctg ggctctggca gcggcagccg ggattccagg gaagggatgt ttttgccgaa
     3301 gcctgaggcc ggctccgcta ttagtgacgt gtttgagggc cgcgaggtgt gccagccaaa
     3361 acgaatccgg ccatttcatc ctccaggaag tccatgggcc aaccgcccac tccccgccag
     3421 cctcgcacca acaccaaccg gtccagtaca tgagccagtc gggtcactga ccccggcacc
     3481 agtccctcag ccactggatc cagcgcccgc agtgactccc gaggccagtc acctgttgga
     3541 ggatcccgat gaagagacga gccaggctgt caaagccctt cgggagatgg ccgatactgt
     3601 gattccccag aaggaagagg ctgcaatctg tggccaaatg gacctttccc atccgccccc
     3661 aaggggccat ctggatgagc tgacaaccac acttgagtcc atgaccgagg atctgaacct
     3721 ggactcaccc ctgaccccgg aattgaacga gattctggat accttcctga acgacgagtg
     3781 cctcttgcat gccatgcata tcagcacagg actgtccatc ttcgacacat ctctgtttta
     3841 acaattgttg ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3901 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3961 ctcatcaatg tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt
     4021 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     4081 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     4141 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     4201 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     4261 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     4321 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     4381 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     4441 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4501 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4561 aaggaagagt cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     4621 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4681 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4741 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4801 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4861 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     4921 ttttgcaaag atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg
     4981 attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca
     5041 acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt
     5101 tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg
     5161 gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga
     5221 agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca
     5281 ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct
     5341 tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac
     5401 tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc
     5461 gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt
     5521 gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt
     5581 catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg
     5641 tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat
     5701 cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc
     5761 gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc
     5821 gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc
     5881 tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg
     5941 ctaactgaaa cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa
     6001 agacagaata aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     6061 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     6121 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     6181 ggggcggcag gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa
     6241 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6301 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6361 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6421 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6481 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6541 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6601 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6661 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6721 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     6781 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     6841 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     6901 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6961 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7021 cctgcgttat cccctgattc tgtggataac cgtattaccg c
//
