LOCUS       sequence-239662-        6054 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac-based Dox-inducible expression (very weak).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6054)
  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 6054)
  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: KA2896_pPB-TREtight-Tkmin-cHA-IRESH2BmKO2
FEATURES             Location/Qualifiers
     source          1..6054
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..68
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     60..77
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     61..77
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     84..103
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        84..102
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   121..155
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    521..539
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    557..575
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    592..610
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    628..646
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    664..682
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    699..717
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    735..753
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        784..846
                     /label=Mini-TK promoter
                     /note="minimal herpes simplex virus (HSV) thymidine kinase 
                     promoter"
     CDS             881..907
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(950..1524,1525..1527,1528..1536)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1117..1134)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1344..1363
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1537..1914
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      1927..1936
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1933..2586
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     primer_bind     complement(2623..2640)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2629..2853
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2900..2916)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2924..2940
                     /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(2948..2954,2955..2972,2973..2978))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2993..3014
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   3241..3303
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(3327..3345)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3366..3382)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3390..3406
                     /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(3414..3420,3421..3438,3439..3444))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3459..3480
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3597..3614)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3768..4356)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3848..3867)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4527..5318,5319..5387))
                     /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     5150..5169
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5388..5492)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5518..5973)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5655..5676)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5867..5886
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
       61 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattg ggtaccttaa
      121 ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt gctctctctt
      181 tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg cttggagctc
      241 ccgtgaggcg tgcttgtcaa tgaggtaagt gtcactgatt ttgaactata acgaccgcgt
      301 gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact catacgataa
      361 ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata ttttcttgtt
      421 atagatatca taatttaaac aagcaaaacc aaattaaggg ccagctcatt cctcccactc
      481 atgatctata gatcttctag tgatttatag gtctcgcaga tccctatcag tgatagagaa
      541 cgtatgtcga gtttactccc tatcagtgat agagaacgat gtcgagttta ctccctatca
      601 gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacgt atgtcgagtt
      661 tactccctat cagtgataga gaacgtatgt cgagtttatc cctatcagtg atagagaacg
      721 tatgtcgagt ttactcccta tcagtgatag agatgataga gacctataaa tcccgcggcc
      781 atgttcgcat attaaggtga cgcgtgtggc ctcgaacacc gagcgaccct gcagcgaccc
      841 gcttaagaat tcctcgagac gcgtgactag tgccatggcc tacccatacg acgtgccaga
      901 ctacgcctag cggccgctcg ataagcttga tatcgaattc cgcccccccc ccctctccct
      961 cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct
     1021 atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc
     1081 ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc
     1141 tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg
     1201 tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa
     1261 agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt
     1321 ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg
     1381 atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta
     1441 catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt
     1501 tcctttgaaa aacacgatga taatatggcc acaaccatgc cagagccagc gaagtctgct
     1561 cccgccccga aaaagggctc caagaaggcg gtgactaagg cgcagaagaa agacggcaag
     1621 aagcgcaagc gcagccgcaa ggagagctat tccatctatg tgtacaaggt tctgaagcag
     1681 gtccaccctg acaccggcat ttcgtccaag gccatgggca tcatgaattc gtttgtgaac
     1741 gacattttcg agcgcatcgc aggtgaggct tcccgcctgg cgcattacaa caagcgctcg
     1801 accatcacct ccagggagat ccagacggcc gtgcgcctgc tgctgcctgg ggagttggcc
     1861 aagcacgccg tgtccgaggg tactaaggcc gtcaccaagt acaccagcgc taagctcgag
     1921 actagtgcca ccatggtgag tgtgattaaa ccagagatga agatgaggta ctacatggac
     1981 ggctccgtca atgggcatga gttcacaatt gaaggtgaag gcacaggcag accttacgag
     2041 ggacatcaag agatgacact acgcgtcaca atggccgagg gcgggccaat gcctttcgcg
     2101 tttgacttag tgtcacacgt gttctgttac ggccacagag tatttactaa atatccagaa
     2161 gagataccag actatttcaa acaagcattt cctgaaggcc tgtcatggga aaggtcgttg
     2221 gagttcgaag atggtgggtc cgcttcagtc agtgcgcata taagccttag aggaaacacc
     2281 ttctaccaca aatccaaatt tactggggtt aactttcctg ccgatggtcc tatcatgcaa
     2341 aaccaaagtg ttgattggga gccatcaacc gagaaaatta ctgccagcga cggagttctg
     2401 aagggtgatg ttacgatgta cctaaaactt gaaggaggcg gcaatcacaa atgccaaatg
     2461 aagactactt acaaggcggc aaaagagatt cttgaaatgc caggagacca ttacatcggc
     2521 catcgcctcg tcaggaaaac cgaaggcaac attactgagc aggtagaaga tgcagtagct
     2581 cattccgcta gctgaatcga tgatctagag ctcgctgatc agcctcgact gtgccttcta
     2641 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2701 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2761 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     2821 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga acctgcagcc
     2881 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     2941 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3001 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3061 gccagcggat cctttgttac tttatagaag aaattttgag tttttgtttt tttttaataa
     3121 ataaataaac ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata
     3181 ataaaactta atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca
     3241 catgcgtcaa ttttacgcat gattatcttt aacgtacgtc acaatatgat tatctttcta
     3301 gggttaaaga gctccagctt ttgttccctt tagtgagggt taatttcgag cttggcgtaa
     3361 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3421 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3481 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3541 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3601 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     3661 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     3721 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     3781 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     3841 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     3901 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     3961 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4021 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4081 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4141 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4201 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4261 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4321 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4381 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4441 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4501 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4561 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4621 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     4681 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     4741 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     4801 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     4861 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     4921 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     4981 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5041 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5101 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5161 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5221 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5281 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacggg aaggcaaaat
     5341 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5401 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5461 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     5521 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     5581 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     5641 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     5701 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     5761 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     5821 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     5881 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     5941 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     6001 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaag
//
