LOCUS       sequence-146271-        7227 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses rTetR-EED 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 7227)
  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 7227)
  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-EED
FEATURES             Location/Qualifiers
     source          1..7227
                     /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(4404..4423)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4405..4423)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    4697..4818
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4734..4753)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4788..4807
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4825..5280)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4962..4983)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5174..5193
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5307..5411
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    5426..5459
                     /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(5501..6292,6293..6361))
                     /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     6124..6143
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6531..7119
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7020..7039
                     /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 atgctccgag
     3061 agggaagtgt cgactgcgcc ggcgggaaca gacatgcccg cggccaagaa gcagaagttg
     3121 agcagcgacg agaacagcaa cccggacctc tcgggagacg aaaatgacga tgctgtcagt
     3181 attgagagtg gcacaaacac agaacgcccg gacacgccca caaatacgcc aaatgcacca
     3241 ggaaggaaaa gctggggaaa gggaaaatgg aagtcaaaga aatgcaaata ttctttcaaa
     3301 tgtgtgaaca gcctcaagga agatcataac cagccattgt ttggagttca gtttaactgg
     3361 cacagtaaag aaggagaccc tctggtgttt gcaactgtgg gaagcaacag agtaacctta
     3421 tacgaatgcc attcacaggg ggagatacgg ttattgcagt cctatgtaga tgctgatgca
     3481 gatgaaaact tttacacttg tgcatggacc tatgatagca acaccagcca ccctctatta
     3541 gcagttgctg gatctagagg cattataaga ataattaatc ctataacaat gcagtgtata
     3601 aagcactatg ttggccatgg aaatgctatc aatgagctga aattccaccc acgagaccca
     3661 aaccttctcc tgtcagtaag taaagatcat gctttacggt tatggaatat ccaaacagac
     3721 actcttgtgg caatattcgg aggtgtggaa gggcacagag atgaagttct gagtgctgat
     3781 tatgatcttt tgggtgaaaa aataatgtcc tgtggtatgg atcactctct taaactgtgg
     3841 agaatcaact caaagaggat gatgaatgca attaaggagt cttatgatta taacccaaac
     3901 aaaactaaca ggccatttat ttcccagaaa atccactttc ctgacttttc taccagagac
     3961 atacatagga attatgttga ttgtgtgcga tggttaggcg atttgatact ttccaagtct
     4021 tgtgaaaatg ccattgtatg ctggaaacct ggcaaaatgg aggatgatat agataaaatt
     4081 aaacctagtg agtctaatgt gactattctt gggcgatttg attacagcca gtgtgacatt
     4141 tggtacatga ggttttctat ggatttctgg caaaagatgc ttgcattggg caatcaggtt
     4201 ggcaaactgt atgtttggga tttagaagta gaagatcctc ataaagccaa atgcacaaca
     4261 ctgacccatc ataaatgtgg cgcggctatt cgacaaacca gtttcagtag ggatagcagc
     4321 atcctcatag ctgtctgcga tgatgccagc atttggcgct gggatcgact tcgataaggt
     4381 accaggtaag tgtacccaat tcgccctata gtgagtcgta ttacaattca ctcgatcgcc
     4441 cttcccaaca gttgcgcagc ctgaatggcg aatggagatc caatttttaa gtgtataatg
     4501 tgttaaacta ctgattctaa ttgtttgtgt attttagatt cacagtccca aggctcattt
     4561 caggcccctc agtcctcaca gtctgttcat gatcataatc agccatacca catttgtaga
     4621 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     4681 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4741 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4801 actcatcaat gtatcttaac gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     4861 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     4921 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     4981 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     5041 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     5101 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     5161 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     5221 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     5281 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5341 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5401 aaaggaagaa tcctgaggcc gggccataac ttcgtataat gtatgctata cgaagttatc
     5461 catgggcccc ccctcgacat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5521 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5581 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5641 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5701 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5761 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5821 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5881 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5941 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6001 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6061 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6121 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6181 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6241 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6301 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6361 tactcttcct caggttactc atatatactt tagattgatt taaaacttca tttttaattt
     6421 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     6481 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     6541 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     6601 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     6661 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     6721 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     6781 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     6841 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     6901 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     6961 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     7021 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     7081 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     7141 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     7201 gattctgtgg ataaccgtat taccgcc
//
