LOCUS       sequence-146274-        9155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses rTetR-HDAC4 for gene silencing and the nuclear H2B-mCherry
            marker in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9155)
  AUTHORS   Bintu L, Yong J, Antebi YE, McCue K, Kazuki Y, Uno N, Oshimura M, 
            Elowitz MB
  TITLE     Dynamics of epigenetic regulation at the single-cell level.
  JOURNAL   Science. 2016 Feb 12;351(6274):720-4. doi: 10.1126/science.aab2956.
  PUBMED    26912859
REFERENCE   2  (bases 1 to 9155)
  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: "10.1126/science"; 
            journalName: "Science"; date: "2016-02-12- 12"; volume: "351"; 
            issue: "6274"; pages: "720-4"
COMMENT     Sequence Label: pEx1-pEF-H2B-mCherry-T2A-rTetR-HDAC4
FEATURES             Location/Qualifiers
     source          1..9155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          214..1152
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1109..1129
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        1185..1203
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             1264..1641
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             1648..2355
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1793..1811)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2061..2080
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2359..2412
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(6332..6351)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6333..6351)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    6625..6746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6662..6681)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6716..6735
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(6753..7208)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6890..6911)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7102..7121
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        7235..7339
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    7354..7387
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(7429..8220,8221..8289))
                     /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     8052..8071
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      8459..9047
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8948..8967
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgcatgagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
       61 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
      121 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
      181 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg
      241 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac ctggctgcag
      301 tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc gaggccttgc
      361 gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc
      421 cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc
      481 atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag tcttgtaaat
      541 gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc
      601 cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc accgagaatc
      661 ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc gccgccgtgt
      721 atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg agcggaaaga
      781 tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag
      841 cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca
      901 tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc gagcttttgg
      961 agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc ccacactgag
     1021 tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt ggaatttgcc
     1081 ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca aagttttttt
     1141 cttccatttc aggtgtcgtg agctagcgat tacgccaagc tcgaaattaa ccctcactaa
     1201 agggaacaaa agctggagct ccaccgcggt ggcggccgct ctagcccggg cggatccgcc
     1261 accatgccag agccagcgaa gtctgctccc gccccgaaaa agggctccaa gaaggcggtg
     1321 actaaggcgc agaagaaagg cggcaagaag cgcaagcgca gccgcaagga gagctattcc
     1381 atctatgtgt acaaggttct gaagcaggtc caccctgaca ccggcatttc gtccaaggcc
     1441 atgggcatca tgaattcgtt tgtgaacgac attttcgagc gcatcgcagg tgaggcttcc
     1501 cgcctggcgc attacaacaa gcgctcgacc atcacctcca gggagatcca gacggccgtg
     1561 cgcctgctgc tgcctgggga gttggccaag cacgccgtgt ccgagggtac taaggccatc
     1621 accaagtaca ccagcgctaa gcctaggatg gtgagcaagg gcgaggagga taacatggcc
     1681 atcatcaagg agttcatgcg cttcaaggtg cacatggagg gctccgtgaa cggccacgag
     1741 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac cgccaagctg
     1801 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcctgtcccc tcagttcatg
     1861 tacggctcca aggcctacgt gaagcacccc gccgacattc ccgactactt gaagctgtcc
     1921 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     1981 gtgacccagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     2041 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc
     2101 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg agatcaagca gaggctgaag
     2161 ctgaaggacg gcggccacta cgacgctgag gtcaagacca cctacaaggc caagaagccc
     2221 gtgcagctgc ccggcgccta caacgtcaac atcaagttgg acatcacctc ccacaacgag
     2281 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     2341 gacgagctgt acaagtctga gggcaggggg agcctcctaa cttgcggaga cgtcgaggag
     2401 aatcctggcc catctagact ggacaagagc aaagtcataa acggagctct ggaattactc
     2461 aatggtgtcg gtatcgaagg cctgacgaca aggaaactcg ctcaaaagct gggagttgag
     2521 cagcctaccc tgtactggca cgtgaagaac aagcgggccc tgctcgatgc cctgccaatc
     2581 gagatgctgg acaggcatca tacccacttc tgccccctgg aaggcgagtc atggcaagac
     2641 tttctgcgga acaacgccaa gtcataccgc tgtgctctcc tctcacatcg cgacggggct
     2701 aaagtgcatc tcggcacccg cccaacagag aaacagtacg aaaccctgga aaatcagctc
     2761 gcgttcctgt gtcagcaagg cttctccctg gagaacgcac tgtacgctct gtccgccgtg
     2821 ggccacttta cactgggctg cgtattggag gaacaggagc atcaagtagc aaaagaggaa
     2881 agagagacac ctaccaccga ttctatgccc ccacttctga gacaagcaat tgagctgttc
     2941 gaccggcagg gagccgaacc tgccttcctt ttcggcctgg aactaatcat atgtggcctg
     3001 gagaaacagc taaagtgcga aagcggcggg ggtagcggca gcggtagcgc atgcagctcc
     3061 caaagccatc cagatggact ttctggccga gaccagccag tggagctgct gaatcctgcc
     3121 cgcgtgaacc acatgcccag cacggtggat gtggccacgg cgctgcctct gcaagtggcc
     3181 ccctcggcag tgcccatgga cctgcgcctg gaccaccagt tctcactgcc tgtggcagag
     3241 ccggccctgc gggagcagca gctgcagcag gagctcctgg cgctcaagca gaagcagcag
     3301 atccagaggc agatcctcat cgctgagttc cagaggcagc acgagcagct ctcccggcag
     3361 cacgaggcgc agctccacga gcacatcaag caacaacagg agatgctggc catgaagcac
     3421 cagcaggagc tgctggaaca ccagcggaag ctggagaggc accgccagga gcaggagctg
     3481 gagaagcagc accgggagca gaagctgcag cagctcaaga acaaggagaa gggcaaagag
     3541 agtgccgtgg ccagcacaga agtgaagatg aagttacaag aatttgtcct caataaaaag
     3601 aaggcgctgg cccaccggaa tctgaaccac tgcatttcca gcgaccctcg ctactggtac
     3661 gggaaaacgc agcacagttc ccttgaccag agttctccac cccagagcgg agtgtcgacc
     3721 tcctataacc acccggtcct gggaatgtac gacgccaaag atgacttccc tcttaggaaa
     3781 acagcttctg aaccgaatct gaaattacgg tccaggctaa agcagaaagt ggccgaaaga
     3841 cggagcagcc ccctgttacg caggaaagac gggccagtgg tcactgctct aaaaaagcgt
     3901 ccgttggatg tcacagactc cgcgtgcagc agcgccccag gctccggacc cagctcaccc
     3961 aacaacagct ccgggagcgt cagcgcggag aacggtatcg cgcccgccgt ccccagcatc
     4021 ccggcggaga cgagtttggc gcacagactt gtggcacgag aaggctcggc cgctccactt
     4081 cccctctaca catcgccatc cttgcccaac atcacgctgg gcctgcctgc caccggcccc
     4141 tctgcgggca cggcgggcca gcaggacgcc gagagactca cccttcccgc cctccagcag
     4201 aggctctccc ttttccccgg cacccacctc actccctacc tgagcacctc gcccttggag
     4261 cgggacggag gggcagcgca cagccctctt ctgcagcaca tggtcttact ggagcagccg
     4321 ccggcacaag cacccctcgt cacaggcctg ggagcactgc ccctccacgc acagtccttg
     4381 gttggtgcag accgggtgtc cccctccatc cacaagctgc ggcagcaccg cccactgggg
     4441 cggacccagt cggccccgct gccccagaac gcccaggctc tgcagcacct ggtcatccag
     4501 cagcagcatc agcagtttct ggagaaacac aagcagcagt tccagcagca gcaactgcag
     4561 atgaacaaga tcatccccaa gccaagcgag ccagcccggc agccggagag ccacccggag
     4621 gagacggagg aggagctccg tgagcaccag gctctgctgg acgagcccta cctggaccgg
     4681 ctgccggggc agaaggaggc gcacgcacag gccggcgtgc aggtgaagca ggagcccatt
     4741 gagagcgatg aggaagaggc agagccccca cgggaggtgg agccgggcca gcgccagccc
     4801 agtgagcagg agctgctctt cagacagcaa gccctcctgc tggagcagca gcggatccac
     4861 cagctgagga actaccaggc gtccatggag gccgccggca tccccgtgtc cttcggcggc
     4921 cacaggcctc tgtcccgggc gcagtcctca cccgcgtctg ccaccttccc cgtgtctgtg
     4981 caggagcccc ccaccaagcc gaggttcacg acaggcctcg tgtatgacac gctgatgctg
     5041 aagcaccagt gcacctgcgg gagtagcagc agccaccccg agcacgccgg gaggatccag
     5101 agcatctggt cccgcctgca ggagacgggc ctccggggca aatgcgagtg catccgcgga
     5161 cgcaaggcca ccctggagga gctacagacg gtgcactcgg aagcccacac cctcctgtat
     5221 ggcacgaacc ccctcaaccg gcagaaactg gacagtaaga aacttctagg ctcgctcgcc
     5281 tccgtgttcg tccggctccc ttgcggtggt gttggggtgg acagtgacac catatggaac
     5341 gaggtgcact cggcgggggc agcccgcctg gctgtgggct gcgtggtaga gctggtcttc
     5401 aaggtggcca caggggagct gaagaatggc tttgctgtgg tccgcccccc tggacaccat
     5461 gcggaggaga gcacgcccat gggcttttgc tacttcaact ccgtggccgt ggcagccaag
     5521 cttctgcagc agaggttgag cgtgagcaag atcctcatcg tggactggga cgtgcaccat
     5581 ggaaacggga cccagcaggc tttctacagc gaccctagcg tcctgtacat gtccctccac
     5641 cgctacgacg atgggaactt cttcccaggc agcggggctc ctgatgaggt gggcacaggg
     5701 cccggcgtgg gtttcaacgt caacatggct ttcaccggcg gcctggaccc ccccatggga
     5761 gacgctgagt acttggcggc cttcagaacg gtggtcatgc cgatcgccag cgagtttgcc
     5821 ccggatgtgg tgctggtgtc atcaggcttc gatgccgtgg agggccaccc cacccctctt
     5881 gggggctaca acctctccgc cagatgcttc gggtacctga cgaagcagct gatgggcctg
     5941 gctggcggcc ggattgtcct ggccctcgag ggaggccacg acctgaccgc catttgcgac
     6001 gcctcggaag catgtgtttc tgccttgctg ggaaacgagc ttgatcctct cccagaaaag
     6061 gttttacagc aaagacccaa tgcaaacgct gtccgttcca tggagaaagt catggagatc
     6121 cacagcaagt actggcgctg cctgcagcgc acaacctcca cagcggggcg ttctctgatc
     6181 gaggctcaga cttgcgagaa cgaagaagcc gagacggtca ccgccatggc ctcgctgtcc
     6241 gtgggcgtga agcccgccga aaagagacca gatgaggagc ccatggaaga ggagccgccc
     6301 ctgtaaggtc caggtaagtg tacccaattc gccctatagt gagtcgtatt acaattcact
     6361 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggagatcca atttttaagt
     6421 gtataatgtg ttaaactact gattctaatt gtttgtgtat tttagattca cagtcccaag
     6481 gctcatttca ggcccctcag tcctcacagt ctgttcatga tcataatcag ccataccaca
     6541 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     6601 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     6661 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     6721 ttgtccaaac tcatcaatgt atcttaacgc gtaaattgta agcgttaata ttttgttaaa
     6781 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     6841 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     6901 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     6961 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     7021 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     7081 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     7141 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     7201 gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     7261 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     7321 atattgaaaa aggaagaatc ctgaggccgg gccataactt cgtataatgt atgctatacg
     7381 aagttatcca tgggcccccc ctcgacatga gtaaacttgg tctgacagtt accaatgctt
     7441 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     7501 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     7561 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     7621 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     7681 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     7741 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     7801 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7861 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7921 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7981 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     8041 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     8101 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     8161 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     8221 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     8281 aatactcata ctcttcctca ggttactcat atatacttta gattgattta aaacttcatt
     8341 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     8401 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     8461 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     8521 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     8581 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     8641 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     8701 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     8761 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     8821 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     8881 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     8941 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     9001 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     9061 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     9121 tatcccctga ttctgtggat aaccgtatta ccgcc
//
