LOCUS       sequence-463626-        6228 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  p65 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 6228)
  TITLE     HP1 binding creates a local barrier against transcription activation
            and persists during chromatin decondensation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6228)
  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: 
            "Unpublished"
COMMENT     Sequence Label: rTetR-GFP-p65
FEATURES             Location/Qualifiers
     source          1..6228
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        147..450
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        451..654
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     605..625
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(1353..1355,1356..1358,1359..2069)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1353..1355,1356..1358,1359..2069)
                     /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(1398..1419)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1659..1678)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2006..2027
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2103..2123
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2613..2969
                     /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"
     polyA_signal    3102..3223
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3139..3158)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3193..3212
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3230..3685)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3367..3388)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3579..3598
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3712..3816
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3818..4175
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3818..4009
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3841..3861)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4026..4161
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4210..5004
                     /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             4210..5004
                     /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(4264..4283)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4874..4893
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5192..5211)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5236..5283
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5612..6200
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6101..6120
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
       61 tggataaccg tattaccgcc atgcattagt tattaatagt aatcaattac ggggtcatta
      121 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
      181 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
      241 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
      301 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
      361 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
      421 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
      481 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
      541 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
      601 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta
      661 gtgaaccgtc agatccgcta gcgctaccgg actcagatct cgaggccacc atgtctagac
      721 tggacaagag caaagtcata aactctgctc tggaattact caatggagtc ggtatcgaag
      781 gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc ctgtactggc
      841 acgtgaagaa caagcgggcc ctgctcgatg ccctgccaat cgagatgctg gacaggcatc
      901 atacccactc ctgccccctg gaaggcgagt catggcaaga ctttctgcgg aacaacgcca
      961 agtcataccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat ctcggcaccc
     1021 gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag
     1081 gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt acactgggct
     1141 gcgtattgga ggaacaggag catcaagtag caaaagagga aagagagaca cctaccaccg
     1201 attctatgcc cccacttctg aaacaagcaa ttgagctgtt cgaccggcag ggagccgaac
     1261 ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag ctaaagtgcg
     1321 aaagcggcgg gccgacccga tcggcccggg tcatggtgag caagggcgag gagctgttca
     1381 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1441 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1501 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1561 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1621 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1681 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1741 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     1801 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     1861 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1921 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     1981 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2041 tcactctcgg catggacgag ctgtacaaga gcggccgcag ggggcgtcga ctgggtaccc
     2101 ctcctaagaa gaagaggaag gttgccccca ccatggataa aaagaagaga aaggcccccc
     2161 cgaccgatgt cagcctgggg gacgagctcc agtacctgcc cgacaccgac gaccggcacc
     2221 ggatcgagga aaagcggaag cggacctacg agacattcaa gagcatcatg aagaagtccc
     2281 ccttcagcgg ccccaccgac cctagacctc cacctagaag aatcgccgtg cccagcagat
     2341 ccagcgccag cgtgccaaaa cctgcccccc agccttaccc cttcaccagc agcctgagca
     2401 ccatcaacta cgacgagttc cctaccatgg tgttccccag cggccagatc tctcaggcct
     2461 ctgctctggc tccagcccct cctcaggtgc tgcctcaggc tcctgctcct gcaccagctc
     2521 cagccatggt gtctgcactg gctcaggcac cagcacccgt gcctgtgctg gctcctggac
     2581 ctccacaggc tgtggctcca ccagccccta aacctacaca ggccggcgag ggcacactgt
     2641 ctgaagctct gctgcagctg cagttcgacg acgaggatct gggagccctg ctgggaaaca
     2701 gcaccgatcc tgccgtgttc accgacctgg ccagcgtgga caacagcgag ttccagcagc
     2761 tgctgaacca gggcatccct gtggcccctc acaccaccga gcccatgctg atggaatacc
     2821 ccgaggccat cacccggctc gtgacaggcg ctcagaggcc tcctgatcca gctcctgccc
     2881 ctctgggagc accaggcctg cctaatggac tgctgtctgg cgacgaggac ttcagctcta
     2941 tcgccgatat ggatttctca gccttgctgt aaggatccac cggatctagc ggctagatca
     3001 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     3061 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     3121 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     3181 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc
     3241 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     3301 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     3361 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     3421 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     3481 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3541 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3601 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3661 cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3721 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3781 tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt
     3841 ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     3901 aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag
     3961 gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc
     4021 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4081 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4141 gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga
     4201 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4261 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4321 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4381 aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4441 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4501 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4561 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     4621 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     4681 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc
     4741 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     4801 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4861 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4921 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     4981 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     5041 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     5101 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5161 agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag
     5221 gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg
     5281 ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc
     5341 attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg
     5401 aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact
     5461 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     5521 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     5581 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     5641 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     5701 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     5761 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     5821 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     5881 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     5941 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6001 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     6061 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     6121 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     6181 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggt
//
