LOCUS       sequence-239657-        6696 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 6696)
  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 6696)
  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: KA1123_pPB-TREtight-cHA-IRESH2BmKO2
FEATURES             Location/Qualifiers
     source          1..6696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     64..86
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     78..95
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     79..95
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     102..121
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        102..120
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   139..173
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        523..837
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    531..549
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    567..585
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    602..620
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    638..656
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    674..692
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    709..727
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    745..763
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     810..834
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          881..1453
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             1541..1567
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(1610..2184,2185..2187,2188..2196)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1777..1794)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2004..2023
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2197..2574
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      2587..2596
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2593..3246
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     primer_bind     complement(3283..3300)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3289..3513
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3560..3576)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3584..3600
                     /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(3608..3614,3615..3632,3633..3638))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3653..3674
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   3901..3963
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(3987..4005)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4026..4042)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4050..4066
                     /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(4074..4080,4081..4098,4099..4104))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4119..4140
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4257..4274)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4428..5016)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4508..4527)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5187..5978,5979..6047))
                     /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     5810..5829
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6048..6152)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6178..6633)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6315..6336)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6527..6546
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
       61 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt gtaatacgac tcactatagg
      121 gcgaattggg taccttaacc ctagaaagat agtctgcgta aaattgacgc atgcattctt
      181 gaaatattgc tctctctttc taaatagcgc gaatccgtcg ctgtgcattt aggacatctc
      241 agtcgccgct tggagctccc gtgaggcgtg cttgtcaatg aggtaagtgt cactgatttt
      301 gaactataac gaccgcgtga gtcaaaatga cgcatgatta tcttttacgt gacttttaag
      361 atttaactca tacgataatt atattgttat ttcatgttct acttacgtga taacttatta
      421 tatatatatt ttcttgttat agatatcata atttaaacaa gcaaaaccaa attaagggcc
      481 agctcattcc tcccactcat gatctataga tctggatccg tcgagtttac tccctatcag
      541 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgat gtcgagttta
      601 ctccctatca gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt
      661 atgtcgagtt tactccctat cagtgataga gaacgtatgt cgagtttatc cctatcagtg
      721 atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgaggtagg
      781 cgtgtacggt gggaggccta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      841 agaattcgag ctcggtaccc gggatcctga gaacttcagg gtgagtttgg ggacccttga
      901 ttgttctttc tttttcgcta ttgtaaaatt catgttatat ggagggggca aagttttcag
      961 ggtgttgttt agaatgggaa gatgtccctt gtatcaccat ggaccctcat gataattttg
     1021 tttctttcac tttctactct gttgacaacc attgtctcct cttattttct tttcattttc
     1081 tgtaactttt tcgttaaact ttagcttgca tttgtaacga atttttaaat tcacttttgt
     1141 ttatttgtca gattgtaagt actttctcta atcacttttt tttcaaggca atcagggtat
     1201 attatattgt acttcagcac agttttagag aacaattgtt ataattaaat gataaggtag
     1261 aatatttctg catataaatt ctggctggcg tggaaatatt cttattggta gaaacaacta
     1321 caccctggtc atcatcctgc ctttctcttt atggttacaa tgatatacac tgtttgagat
     1381 gaggataaaa tactctgagt ccaaaccggg cccctctgct aaccatgttc atgccttctt
     1441 ctctttccta cagctcctgg gcaacgtgct ggttgttgtg ctgtctcatc attttggcaa
     1501 agaattcctc gagatctcac gcgtgactag tgccatggcc tacccatacg acgtgccaga
     1561 ctacgcctag cggccgctcg ataagcttga tatcgaattc cgcccccccc ccctctccct
     1621 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     1681 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     1741 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     1801 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     1861 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     1921 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     1981 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     2041 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta
     2101 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     2161 tcctttgaaa aacacgatga taatatggcc acaaccatgc cagagccagc gaagtctgct
     2221 cccgccccga aaaagggctc caagaaggcg gtgactaagg cgcagaagaa agacggcaag
     2281 aagcgcaagc gcagccgcaa ggagagctat tccatctatg tgtacaaggt tctgaagcag
     2341 gtccaccctg acaccggcat ttcgtccaag gccatgggca tcatgaattc gtttgtgaac
     2401 gacattttcg agcgcatcgc aggtgaggct tcccgcctgg cgcattacaa caagcgctcg
     2461 accatcacct ccagggagat ccagacggcc gtgcgcctgc tgctgcctgg ggagttggcc
     2521 aagcacgccg tgtccgaggg tactaaggcc gtcaccaagt acaccagcgc taagctcgag
     2581 actagtgcca ccatggtgag tgtgattaaa ccagagatga agatgaggta ctacatggac
     2641 ggctccgtca atgggcatga gttcacaatt gaaggtgaag gcacaggcag accttacgag
     2701 ggacatcaag agatgacact acgcgtcaca atggccgagg gcgggccaat gcctttcgcg
     2761 tttgacttag tgtcacacgt gttctgttac ggccacagag tatttactaa atatccagaa
     2821 gagataccag actatttcaa acaagcattt cctgaaggcc tgtcatggga aaggtcgttg
     2881 gagttcgaag atggtgggtc cgcttcagtc agtgcgcata taagccttag aggaaacacc
     2941 ttctaccaca aatccaaatt tactggggtt aactttcctg ccgatggtcc tatcatgcaa
     3001 aaccaaagtg ttgattggga gccatcaacc gagaaaatta ctgccagcga cggagttctg
     3061 aagggtgatg ttacgatgta cctaaaactt gaaggaggcg gcaatcacaa atgccaaatg
     3121 aagactactt acaaggcggc aaaagagatt cttgaaatgc caggagacca ttacatcggc
     3181 catcgcctcg tcaggaaaac cgaaggcaac attactgagc aggtagaaga tgcagtagct
     3241 cattccgcta gctgaatcga tgatctagag ctcgctgatc agcctcgact gtgccttcta
     3301 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3361 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3421 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3481 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga acctgcagcc
     3541 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3601 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3661 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3721 gccagcggat cctttgttac tttatagaag aaattttgag tttttgtttt tttttaataa
     3781 ataaataaac ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata
     3841 ataaaactta atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca
     3901 catgcgtcaa ttttacgcat gattatcttt aacgtacgtc acaatatgat tatctttcta
     3961 gggttaaaga gctccagctt ttgttccctt tagtgagggt taatttcgag cttggcgtaa
     4021 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4081 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4141 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4201 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4261 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4321 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4381 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4441 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4501 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4561 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4621 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4681 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4741 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4801 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4861 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4921 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4981 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5041 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5101 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5161 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5221 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5281 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5341 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5401 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5461 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5521 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5581 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5641 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5701 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5761 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5821 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5881 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5941 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacggg aaggcaaaat
     6001 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6061 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6121 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     6181 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     6241 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     6301 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     6361 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     6421 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     6481 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     6541 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     6601 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     6661 gttgggaagg gcgatcggtg cgggcctctt cgctat
//
