LOCUS       sequence-239659-        6750 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 6750)
  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 6750)
  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: KA1130_pPB-TREtight-cHA-IRESH2BmCherry
FEATURES             Location/Qualifiers
     source          1..6750
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     70..92
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     84..101
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     85..101
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     108..127
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        108..126
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   145..179
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        529..843
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    537..555
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    573..591
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    608..626
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    644..662
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    680..698
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    715..733
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    751..769
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     816..840
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          887..1459
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     CDS             1547..1573
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(1616..2190,2191..2193,2194..2202)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1783..1800)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2010..2029
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2203..2580
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      2593..2602
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2599..3306
                     /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(2744..2762)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3012..3031
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3343..3360)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3349..3573
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3620..3636)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3644..3660
                     /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(3668..3674,3675..3692,3693..3698))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3713..3734
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   3961..4023
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(4047..4065)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4086..4102)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4110..4126
                     /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(4134..4140,4141..4158,4159..4164))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4179..4200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4317..4334)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4488..5076)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4568..4587)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5247..6038,6039..6107))
                     /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     5870..5889
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6108..6212)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6238..6693)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6375..6396)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6587..6606
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc
       61 cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattgtaa tacgactcac
      121 tatagggcga attgggtacc ttaaccctag aaagatagtc tgcgtaaaat tgacgcatgc
      181 attcttgaaa tattgctctc tctttctaaa tagcgcgaat ccgtcgctgt gcatttagga
      241 catctcagtc gccgcttgga gctcccgtga ggcgtgcttg tcaatgaggt aagtgtcact
      301 gattttgaac tataacgacc gcgtgagtca aaatgacgca tgattatctt ttacgtgact
      361 tttaagattt aactcatacg ataattatat tgttatttca tgttctactt acgtgataac
      421 ttattatata tatattttct tgttatagat atcataattt aaacaagcaa aaccaaatta
      481 agggccagct cattcctccc actcatgatc tatagatctg gatccgtcga gtttactccc
      541 tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag aacgatgtcg
      601 agtttactcc ctatcagtga tagagaacgt atgtcgagtt tactccctat cagtgataga
      661 gaacgtatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttatcccta
      721 tcagtgatag agaacgtatg tcgagtttac tccctatcag tgatagagaa cgtatgtcga
      781 ggtaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc
      841 gcctggagaa ttcgagctcg gtacccggga tcctgagaac ttcagggtga gtttggggac
      901 ccttgattgt tctttctttt tcgctattgt aaaattcatg ttatatggag ggggcaaagt
      961 tttcagggtg ttgtttagaa tgggaagatg tcccttgtat caccatggac cctcatgata
     1021 attttgtttc tttcactttc tactctgttg acaaccattg tctcctctta ttttcttttc
     1081 attttctgta actttttcgt taaactttag cttgcatttg taacgaattt ttaaattcac
     1141 ttttgtttat ttgtcagatt gtaagtactt tctctaatca cttttttttc aaggcaatca
     1201 gggtatatta tattgtactt cagcacagtt ttagagaaca attgttataa ttaaatgata
     1261 aggtagaata tttctgcata taaattctgg ctggcgtgga aatattctta ttggtagaaa
     1321 caactacacc ctggtcatca tcctgccttt ctctttatgg ttacaatgat atacactgtt
     1381 tgagatgagg ataaaatact ctgagtccaa accgggcccc tctgctaacc atgttcatgc
     1441 cttcttctct ttcctacagc tcctgggcaa cgtgctggtt gttgtgctgt ctcatcattt
     1501 tggcaaagaa ttcctcgaga tctcacgcgt gactagtgcc atggcctacc catacgacgt
     1561 gccagactac gcctagcggc cgctcgataa gcttgatatc gaattccgcc ccccccccct
     1621 ctccctcccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
     1681 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac
     1741 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
     1801 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa
     1861 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
     1921 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg
     1981 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
     2041 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat
     2101 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg
     2161 tggttttcct ttgaaaaaca cgatgataat atggccacaa ccatgccaga gccagcgaag
     2221 tctgctcccg ccccgaaaaa gggctccaag aaggcggtga ctaaggcgca gaagaaagac
     2281 ggcaagaagc gcaagcgcag ccgcaaggag agctattcca tctatgtgta caaggttctg
     2341 aagcaggtcc accctgacac cggcatttcg tccaaggcca tgggcatcat gaattcgttt
     2401 gtgaacgaca ttttcgagcg catcgcaggt gaggcttccc gcctggcgca ttacaacaag
     2461 cgctcgacca tcacctccag ggagatccag acggccgtgc gcctgctgct gcctggggag
     2521 ttggccaagc acgccgtgtc cgagggtact aaggccgtca ccaagtacac cagcgctaag
     2581 ctcgagacta gtgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag
     2641 gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc
     2701 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
     2761 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc
     2821 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
     2881 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
     2941 gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc
     3001 ccctccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg
     3061 atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac
     3121 ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg
     3181 cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc
     3241 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
     3301 tacaaggcta gctgaatcga tgatctagag ctcgctgatc agcctcgact gtgccttcta
     3361 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     3421 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     3481 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3541 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga acctgcagcc
     3601 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3661 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3721 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3781 gccagcggat cctttgttac tttatagaag aaattttgag tttttgtttt tttttaataa
     3841 ataaataaac ataaataaat tgtttgttga atttattatt agtatgtaag tgtaaatata
     3901 ataaaactta atatctattc aaattaataa ataaacctcg atatacagac cgataaaaca
     3961 catgcgtcaa ttttacgcat gattatcttt aacgtacgtc acaatatgat tatctttcta
     4021 gggttaaaga gctccagctt ttgttccctt tagtgagggt taatttcgag cttggcgtaa
     4081 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4141 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4201 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4261 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4321 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4381 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4441 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4501 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4561 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4621 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4681 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4741 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4801 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4861 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4921 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4981 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5041 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     5101 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5161 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5221 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5281 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5341 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5401 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5461 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5521 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5581 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5641 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5701 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5761 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5821 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5881 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5941 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6001 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacggg aaggcaaaat
     6061 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6121 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6181 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     6241 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     6301 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     6361 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     6421 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     6481 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     6541 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     6601 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     6661 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg ctgcgcaact
     6721 gttgggaagg gcgatcggtg cgggcctctt
//
