LOCUS       sequence-239660-        6052 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac-based Dox-inducible expression (weak).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6052)
  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 6052)
  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: KA2895_pPB-TREtight-TATAmin-cHA-IRESH2BmKO2
FEATURES             Location/Qualifiers
     source          1..6052
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     95..117
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     109..126
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     110..126
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     133..152
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        133..151
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   170..204
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    570..588
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    606..624
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    641..659
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    677..695
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    713..731
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    748..766
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    784..802
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        837..868
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             928..954
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(997..1571,1572..1574,1575..1583)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1164..1181)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1391..1410
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1584..1961
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      1974..1983
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1980..2633
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     primer_bind     complement(2670..2687)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2676..2900
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2947..2963)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2971..2987
                     /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(2995..3001,3002..3019,3020..3025))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3040..3061
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   3288..3350
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(3374..3392)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3413..3429)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3437..3453
                     /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(3461..3467,3468..3485,3486..3491))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3506..3527
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3644..3661)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3815..4403)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3895..3914)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4574..5365,5366..5434))
                     /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     5197..5216
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5435..5539)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5565..6020)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5702..5723)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5914..5933
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct
       61 gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg
      121 gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccttaac cctagaaaga
      181 tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt ctaaatagcg
      241 cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc cgtgaggcgt
      301 gcttgtcaat gaggtaagtg tcactgattt tgaactataa cgaccgcgtg agtcaaaatg
      361 acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat tatattgtta
      421 tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta tagatatcat
      481 aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca tgatctatag
      541 atcttctagt gatttatagg tctcgcagat ccctatcagt gatagagaac gtatgtcgag
      601 tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag tgatagagaa
      661 cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt actccctatc
      721 agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt atgtcgagtt
      781 tactccctat cagtgataga gatgatagag acctataaat cccgcggcca tgacactaga
      841 gggtatataa tggaagctcg acttccagct tggcaatccg gtactgttgg taagaattcc
      901 tcgagacgcg tgactagtgc catggcctac ccatacgacg tgccagacta cgcctagcgg
      961 ccgctcgata agcttgatat cgaattccgc cccccccccc tctccctccc ccccccctaa
     1021 cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc
     1081 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac
     1141 gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt
     1201 gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg
     1261 caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata
     1321 agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga
     1381 aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt
     1441 accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc
     1501 gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac
     1561 acgatgataa tatggccaca accatgccag agccagcgaa gtctgctccc gccccgaaaa
     1621 agggctccaa gaaggcggtg actaaggcgc agaagaaaga cggcaagaag cgcaagcgca
     1681 gccgcaagga gagctattcc atctatgtgt acaaggttct gaagcaggtc caccctgaca
     1741 ccggcatttc gtccaaggcc atgggcatca tgaattcgtt tgtgaacgac attttcgagc
     1801 gcatcgcagg tgaggcttcc cgcctggcgc attacaacaa gcgctcgacc atcacctcca
     1861 gggagatcca gacggccgtg cgcctgctgc tgcctgggga gttggccaag cacgccgtgt
     1921 ccgagggtac taaggccgtc accaagtaca ccagcgctaa gctcgagact agtgccacca
     1981 tggtgagtgt gattaaacca gagatgaaga tgaggtacta catggacggc tccgtcaatg
     2041 ggcatgagtt cacaattgaa ggtgaaggca caggcagacc ttacgaggga catcaagaga
     2101 tgacactacg cgtcacaatg gccgagggcg ggccaatgcc tttcgcgttt gacttagtgt
     2161 cacacgtgtt ctgttacggc cacagagtat ttactaaata tccagaagag ataccagact
     2221 atttcaaaca agcatttcct gaaggcctgt catgggaaag gtcgttggag ttcgaagatg
     2281 gtgggtccgc ttcagtcagt gcgcatataa gccttagagg aaacaccttc taccacaaat
     2341 ccaaatttac tggggttaac tttcctgccg atggtcctat catgcaaaac caaagtgttg
     2401 attgggagcc atcaaccgag aaaattactg ccagcgacgg agttctgaag ggtgatgtta
     2461 cgatgtacct aaaacttgaa ggaggcggca atcacaaatg ccaaatgaag actacttaca
     2521 aggcggcaaa agagattctt gaaatgccag gagaccatta catcggccat cgcctcgtca
     2581 ggaaaaccga aggcaacatt actgagcagg tagaagatgc agtagctcat tccgctagct
     2641 gaatcgatga tctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     2701 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     2761 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     2821 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     2881 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc tgcagcccaa gcttggcgta
     2941 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3001 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3061 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatcct
     3121 ttgttacttt atagaagaaa ttttgagttt ttgttttttt ttaataaata aataaacata
     3181 aataaattgt ttgttgaatt tattattagt atgtaagtgt aaatataata aaacttaata
     3241 tctattcaaa ttaataaata aacctcgata tacagaccga taaaacacat gcgtcaattt
     3301 tacgcatgat tatctttaac gtacgtcaca atatgattat ctttctaggg ttaaagagct
     3361 ccagcttttg ttccctttag tgagggttaa tttcgagctt ggcgtaatca tggtcatagc
     3421 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3481 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3541 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3601 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     3661 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3721 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3781 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3841 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3901 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     3961 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4021 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4081 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4141 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4201 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4261 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4321 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4381 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4441 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4501 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4561 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4621 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4681 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4741 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4801 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4861 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4921 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     4981 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5041 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5101 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5161 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5221 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5281 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5341 ttactttcac cagcgtttct gggtgagcaa aaacgggaag gcaaaatgcc gcaaaaaagg
     5401 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5461 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5521 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa
     5581 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     5641 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     5701 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     5761 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     5821 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     5881 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     5941 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     6001 tgcgccgcta cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gg
//
