LOCUS       sequence-146273-        6102 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses rTetR-KRAB 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 6102)
  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 6102)
  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-KRAB
FEATURES             Location/Qualifiers
     source          1..6102
                     /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"
     CDS             3061..3186
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the rat zinc finger protein Kid-1 
                     (Witzgall et al., 1994)"
                     /label=KRAB-A
                     /translation="VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASM"
     primer_bind     complement(3279..3298)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3280..3298)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3572..3693
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3609..3628)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3663..3682
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3700..4155)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3837..3858)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4049..4068
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4182..4286
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    4301..4334
                     /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(4376..5167,5168..5236))
                     /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     4999..5018
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5406..5994
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5895..5914
                     /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 atgcctagca
     3061 gtgtcagtga catttgaaga tgtggctgtg ctctttactc gggacgagtg gaagaagctg
     3121 gatctgtctc agagaagcct gtaccgtgag gtgatgctgg agaattacag caacctggcc
     3181 tccatggcag gattcctgtt taccaaacca aaggtgatct ccctgttgca gcaaggagag
     3241 gatccctggt aaggtaccag gtaagtgtac ccaattcgcc ctatagtgag tcgtattaca
     3301 attcactcga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg agatccaatt
     3361 tttaagtgta taatgtgtta aactactgat tctaattgtt tgtgtatttt agattcacag
     3421 tcccaaggct catttcaggc ccctcagtcc tcacagtctg ttcatgatca taatcagcca
     3481 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     3541 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     3601 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3661 ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt
     3721 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     3781 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     3841 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     3901 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     3961 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4021 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     4081 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     4141 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4201 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4261 ttcaataata ttgaaaaagg aagaatcctg aggccgggcc ataacttcgt ataatgtatg
     4321 ctatacgaag ttatccatgg gccccccctc gacatgagta aacttggtct gacagttacc
     4381 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     4441 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     4501 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     4561 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     4621 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     4681 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     4741 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     4801 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     4861 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     4921 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     4981 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     5041 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     5101 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     5161 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     5221 aatgttgaat actcatactc ttcctcaggt tactcatata tactttagat tgatttaaaa
     5281 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     5341 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     5401 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     5461 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     5521 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     5581 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     5641 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     5701 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     5761 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     5821 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     5881 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     5941 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     6001 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     6061 cctgcgttat cccctgattc tgtggataac cgtattaccg cc
//
