LOCUS       sequence-240992-        6887 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac-based Dox-inducible expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6887)
  AUTHORS   Adachi K, Kopp W, Wu G, Heising S, Greber B, Stehling M, 
            Arauzo-Bravo MJ, Boerno ST, Timmermann B, Vingron M, Scholer HR
  TITLE     Esrrb Unlocks Silenced Enhancers for Reprogramming to Naive 
            Pluripotency.
  JOURNAL   Cell Stem Cell. 2018 Aug 2;23(2):266-275.e6. doi: 
            10.1016/j.stem.2018.05.020. Epub 2018 Jun 14.
  PUBMED    29910149
REFERENCE   2  (bases 1 to 6887)
  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: 
            "10.1016/j.stem.2018.05.020"; journalName: "Cell Stem Cell"; date: 
            "2018-08-2- 2"; volume: "23"; issue: "2"; pages: "266-275.e6"
COMMENT     Sequence Label: KA1065_pPB-hCMV*1-cHA-IRESH2BmCherry
FEATURES             Location/Qualifiers
     source          1..6887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    join(209..227,228..250,251..269,270..292,293..311,312..334,
                     335..353,354..376,377..395,396..418,419..437,438..460,
                     461..479)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        512..550
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     547..571
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          673..1245
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             1333..1359
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(1402..1976,1977..1979,1980..1988)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1569..1586)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1796..1815
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1989..2366
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      2379..2388
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2385..3092
                     /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(2530..2548)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2798..2817
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3129..3146)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3135..3359
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3406..3422)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3430..3446
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3454..3460,3461..3478,3479..3484))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3499..3520
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   3747..3809
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(3833..3851)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3872..3888)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3896..3912
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3920..3926,3927..3944,3945..3950))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3965..3986
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4103..4120)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4274..4862)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4354..4373)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5033..5824,5825..5893))
                     /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     5656..5675
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5894..5998)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6024..6479)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6161..6182)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6373..6392
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6606..6628
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6620..6637
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6621..6637
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6644..6663
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6644..6662
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   6681..6715
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 tacgtgactt ttaagattta actcatacga taattatatt gttatttcat gttctactta
       61 cgtgataact tattatatat atattttctt gttatagata tcataattta aacaagcaaa
      121 accaaattaa gggccagctc attcctccca ctcatgatct atagatctgg atccgtcgac
      181 aacggcaagc ttacatcgag tttaccactc cctatcagtg atagagaaaa gtgaaagtcg
      241 agtttaccac tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc
      301 agtgatagag aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga
      361 aagtcgagtt taccactccc tatcagtgat agagaaaagt gaaagtcgag tttaccactc
      421 cctatcagtg atagagaaaa gtgaaagtcg agtttaccac tccctatcag tgatagagaa
      481 aagtgaaagt cgagctcggt acccgggtcg aggtaggcgt gtacggtggg aggcctatat
      541 aagcagagct cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga
      601 cctccataga agacaccggg accgatccag cctccgcggg actcagacac ccgggatcct
      661 gagaacttca gggtgagttt ggggaccctt gattgttctt tctttttcgc tattgtaaaa
      721 ttcatgttat atggaggggg caaagttttc agggtgttgt ttagaatggg aagatgtccc
      781 ttgtatcacc atggaccctc atgataattt tgtttctttc actttctact ctgttgacaa
      841 ccattgtctc ctcttatttt cttttcattt tctgtaactt tttcgttaaa ctttagcttg
      901 catttgtaac gaatttttaa attcactttt gtttatttgt cagattgtaa gtactttctc
      961 taatcacttt tttttcaagg caatcagggt atattatatt gtacttcagc acagttttag
     1021 agaacaattg ttataattaa atgataaggt agaatatttc tgcatataaa ttctggctgg
     1081 cgtggaaata ttcttattgg tagaaacaac tacaccctgg tcatcatcct gcctttctct
     1141 ttatggttac aatgatatac actgtttgag atgaggataa aatactctga gtccaaaccg
     1201 ggcccctctg ctaaccatgt tcatgccttc ttctctttcc tacagctcct gggcaacgtg
     1261 ctggttgttg tgctgtctca tcattttggc aaagaattcc tcgagatctc acgcgtgact
     1321 agtgccatgg cctacccata cgacgtgcca gactacgcct agcggccgct cgataagctt
     1381 gatatcgaat tccgcccccc ccccctctcc ctcccccccc cctaacgtta ctggccgaag
     1441 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc
     1501 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg
     1561 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
     1621 tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
     1681 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
     1741 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
     1801 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
     1861 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
     1921 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
     1981 ccacaaccat gccagagcca gcgaagtctg ctcccgcccc gaaaaagggc tccaagaagg
     2041 cggtgactaa ggcgcagaag aaagacggca agaagcgcaa gcgcagccgc aaggagagct
     2101 attccatcta tgtgtacaag gttctgaagc aggtccaccc tgacaccggc atttcgtcca
     2161 aggccatggg catcatgaat tcgtttgtga acgacatttt cgagcgcatc gcaggtgagg
     2221 cttcccgcct ggcgcattac aacaagcgct cgaccatcac ctccagggag atccagacgg
     2281 ccgtgcgcct gctgctgcct ggggagttgg ccaagcacgc cgtgtccgag ggtactaagg
     2341 ccgtcaccaa gtacaccagc gctaagctcg agactagtgc caccatggtg agcaagggcg
     2401 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
     2461 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     2521 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
     2581 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
     2641 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     2701 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
     2761 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
     2821 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
     2881 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
     2941 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
     3001 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
     3061 actccaccgg cggcatggac gagctgtaca aggctagctg aatcgatgat ctagagctcg
     3121 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3181 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3241 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3301 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3361 ttctgaggcg gaaagaacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc
     3421 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3481 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3541 cgctttccag tcgggaaacc tgtcgtgcca gcggatcctt tgttacttta tagaagaaat
     3601 tttgagtttt tgtttttttt taataaataa ataaacataa ataaattgtt tgttgaattt
     3661 attattagta tgtaagtgta aatataataa aacttaatat ctattcaaat taataaataa
     3721 acctcgatat acagaccgat aaaacacatg cgtcaatttt acgcatgatt atctttaacg
     3781 tacgtcacaa tatgattatc tttctagggt taaagagctc cagcttttgt tccctttagt
     3841 gagggttaat ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3901 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3961 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     4021 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     4081 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     4141 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     4201 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     4261 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     4321 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     4381 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     4441 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     4501 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     4561 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     4621 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     4681 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     4741 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     4801 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     4861 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     4921 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     4981 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5041 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5101 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5161 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5221 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5281 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5341 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5401 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5461 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     5521 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     5581 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     5641 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     5701 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     5761 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     5821 ggtgagcaaa aacgggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     5881 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     5941 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6001 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     6061 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     6121 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     6181 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     6241 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     6301 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     6361 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     6421 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     6481 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     6541 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     6601 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata
     6661 gggcgaattg ggtaccttaa ccctagaaag atagtctgcg taaaattgac gcatgcattc
     6721 ttgaaatatt gctctctctt tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc
     6781 tcagtcgccg cttggagctc ccgtgaggcg tgcttgtcaa tgaggtaagt gtcactgatt
     6841 ttgaactata acgaccgcgt gagtcaaaat gacgcatgat tatcttt
//
