LOCUS       sequence-239470-        6833 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 6833)
  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 6833)
  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: KA1063_pPB-hCMV*1-cHA-IRESH2BmKO2
FEATURES             Location/Qualifiers
     source          1..6833
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   660..694
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    join(1075..1093,1094..1116,1117..1135,1136..1158,1159..1177,
                     1178..1200,1201..1219,1220..1242,1243..1261,1262..1284,
                     1285..1303,1304..1326,1327..1345)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        1378..1416
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1413..1437
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          1539..2111
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             2199..2225
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(2268..2842,2843..2845,2846..2854)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2435..2452)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2662..2681
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2855..3232
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      3245..3254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3251..3904
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     primer_bind     complement(3941..3958)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3947..4171
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4218..4234)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4242..4258
                     /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(4266..4272,4273..4290,4291..4296))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4311..4332
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   4559..4621
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(4645..4663)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4684..4700)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4708..4724
                     /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(4732..4738,4739..4756,4757..4762))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4777..4798
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4915..4932)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5086..5674)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5166..5185)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5845..6636,6637..6705))
                     /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     6468..6487
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6706..6810)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccttaac
      661 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt
      721 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc
      781 cgtgaggcgt gcttgtcaat gaggtaagtg tcactgattt tgaactataa cgaccgcgtg
      841 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat
      901 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta
      961 tagatatcat aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca
     1021 tgatctatag atctggatcc gtcgacaacg gcaagcttac atcgagttta ccactcccta
     1081 tcagtgatag agaaaagtga aagtcgagtt taccactccc tatcagtgat agagaaaagt
     1141 gaaagtcgag tttaccactc cctatcagtg atagagaaaa gtgaaagtcg agtttaccac
     1201 tccctatcag tgatagagaa aagtgaaagt cgagtttacc actccctatc agtgatagag
     1261 aaaagtgaaa gtcgagttta ccactcccta tcagtgatag agaaaagtga aagtcgagtt
     1321 taccactccc tatcagtgat agagaaaagt gaaagtcgag ctcggtaccc gggtcgaggt
     1381 aggcgtgtac ggtgggaggc ctatataagc agagctcgtt tagtgaaccg tcagatcgcc
     1441 tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg atccagcctc
     1501 cgcgggactc agacacccgg gatcctgaga acttcagggt gagtttgggg acccttgatt
     1561 gttctttctt tttcgctatt gtaaaattca tgttatatgg agggggcaaa gttttcaggg
     1621 tgttgtttag aatgggaaga tgtcccttgt atcaccatgg accctcatga taattttgtt
     1681 tctttcactt tctactctgt tgacaaccat tgtctcctct tattttcttt tcattttctg
     1741 taactttttc gttaaacttt agcttgcatt tgtaacgaat ttttaaattc acttttgttt
     1801 atttgtcaga ttgtaagtac tttctctaat cacttttttt tcaaggcaat cagggtatat
     1861 tatattgtac ttcagcacag ttttagagaa caattgttat aattaaatga taaggtagaa
     1921 tatttctgca tataaattct ggctggcgtg gaaatattct tattggtaga aacaactaca
     1981 ccctggtcat catcctgcct ttctctttat ggttacaatg atatacactg tttgagatga
     2041 ggataaaata ctctgagtcc aaaccgggcc cctctgctaa ccatgttcat gccttcttct
     2101 ctttcctaca gctcctgggc aacgtgctgg ttgttgtgct gtctcatcat tttggcaaag
     2161 aattcctcga gatctcacgc gtgactagtg ccatggccta cccatacgac gtgccagact
     2221 acgcctagcg gccgctcgat aagcttgata tcgaattccg cccccccccc ctctccctcc
     2281 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat
     2341 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct
     2401 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg
     2461 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta
     2521 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag
     2581 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg
     2641 atagttgtgg aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat
     2701 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca
     2761 tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc
     2821 ctttgaaaaa cacgatgata atatggccac aaccatgcca gagccagcga agtctgctcc
     2881 cgccccgaaa aagggctcca agaaggcggt gactaaggcg cagaagaaag acggcaagaa
     2941 gcgcaagcgc agccgcaagg agagctattc catctatgtg tacaaggttc tgaagcaggt
     3001 ccaccctgac accggcattt cgtccaaggc catgggcatc atgaattcgt ttgtgaacga
     3061 cattttcgag cgcatcgcag gtgaggcttc ccgcctggcg cattacaaca agcgctcgac
     3121 catcacctcc agggagatcc agacggccgt gcgcctgctg ctgcctgggg agttggccaa
     3181 gcacgccgtg tccgagggta ctaaggccgt caccaagtac accagcgcta agctcgagac
     3241 tagtgccacc atggtgagtg tgattaaacc agagatgaag atgaggtact acatggacgg
     3301 ctccgtcaat gggcatgagt tcacaattga aggtgaaggc acaggcagac cttacgaggg
     3361 acatcaagag atgacactac gcgtcacaat ggccgagggc gggccaatgc ctttcgcgtt
     3421 tgacttagtg tcacacgtgt tctgttacgg ccacagagta tttactaaat atccagaaga
     3481 gataccagac tatttcaaac aagcatttcc tgaaggcctg tcatgggaaa ggtcgttgga
     3541 gttcgaagat ggtgggtccg cttcagtcag tgcgcatata agccttagag gaaacacctt
     3601 ctaccacaaa tccaaattta ctggggttaa ctttcctgcc gatggtccta tcatgcaaaa
     3661 ccaaagtgtt gattgggagc catcaaccga gaaaattact gccagcgacg gagttctgaa
     3721 gggtgatgtt acgatgtacc taaaacttga aggaggcggc aatcacaaat gccaaatgaa
     3781 gactacttac aaggcggcaa aagagattct tgaaatgcca ggagaccatt acatcggcca
     3841 tcgcctcgtc aggaaaaccg aaggcaacat tactgagcag gtagaagatg cagtagctca
     3901 ttccgctagc tgaatcgatg atctagagct cgctgatcag cctcgactgt gccttctagt
     3961 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     4021 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     4081 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     4141 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac ctgcagccca
     4201 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     4261 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4321 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4381 cagcggatcc tttgttactt tatagaagaa attttgagtt tttgtttttt tttaataaat
     4441 aaataaacat aaataaattg tttgttgaat ttattattag tatgtaagtg taaatataat
     4501 aaaacttaat atctattcaa attaataaat aaacctcgat atacagaccg ataaaacaca
     4561 tgcgtcaatt ttacgcatga ttatctttaa cgtacgtcac aatatgatta tctttctagg
     4621 gttaaagagc tccagctttt gttcccttta gtgagggtta atttcgagct tggcgtaatc
     4681 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4741 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4801 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     4861 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     4921 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     4981 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5041 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5101 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5161 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5221 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5281 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5341 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5401 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5461 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5521 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5581 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5641 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5701 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5761 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5821 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5881 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5941 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6001 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6061 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6121 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6181 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6241 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6301 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6361 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6421 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6481 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6541 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6601 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacgggaa ggcaaaatgc
     6661 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6721 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6781 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac
//
