LOCUS       sequence-236411-        6044 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 6044)
  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 6044)
  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..6044
                     /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    1060..1078
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1096..1114
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1131..1149
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1167..1185
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1203..1221
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1238..1256
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1274..1292
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        1327..1358
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     CDS             1418..1444
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(1480..2053,2054..2056,2057..2065)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1647..1664)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1874..1893
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2066..2443
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      2456..2465
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2462..3115
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     primer_bind     complement(3152..3169)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3158..3382
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3429..3445)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3453..3469
                     /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(3477..3483,3484..3501,3502..3507))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3522..3543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   3770..3832
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(3856..3874)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3895..3911)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3919..3935
                     /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(3943..3949,3950..3967,3968..3973))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3988..4009
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4126..4143)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4297..4885)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4377..4396)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5056..5847,5848..5916))
                     /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     5679..5698
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5917..6021)
                     /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 atcttctagt gatttatagg tctcgcagat ccctatcagt gatagagaac
     1081 gtatgtcgag tttactccct atcagtgata gagaacgatg tcgagtttac tccctatcag
     1141 tgatagagaa cgtatgtcga gtttactccc tatcagtgat agagaacgta tgtcgagttt
     1201 actccctatc agtgatagag aacgtatgtc gagtttatcc ctatcagtga tagagaacgt
     1261 atgtcgagtt tactccctat cagtgataga gatgatagag acctataaat cccgcggcca
     1321 tgacactaga gggtatataa tggaagctcg acttccagct tggcaatccg gtactgttgg
     1381 taagaattcc tcgagacgcg tgactagtgc catggcctac ccatacgacg tgccagacta
     1441 cgcctagcgg ccgctcgata agcttgatat cgaattccgc ccctctccct cccccccccc
     1501 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     1561 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     1621 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     1681 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
     1741 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     1801 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     1861 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     1921 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta
     1981 gtcgaggtta aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa
     2041 acacgatgat aatatggcca caaccatgcc agagccagcg aagtctgctc ccgccccgaa
     2101 aaagggctcc aagaaggcgg tgactaaggc gcagaagaaa gacggcaaga agcgcaagcg
     2161 cagccgcaag gagagctatt ccatctatgt gtacaaggtt ctgaagcagg tccaccctga
     2221 caccggcatt tcgtccaagg ccatgggcat catgaattcg tttgtgaacg acattttcga
     2281 gcgcatcgca ggtgaggctt cccgcctggc gcattacaac aagcgctcga ccatcacctc
     2341 cagggagatc cagacggccg tgcgcctgct gctgcctggg gagttggcca agcacgccgt
     2401 gtccgagggt actaaggccg tcaccaagta caccagcgct aagctcgaga ctagtgccac
     2461 catggtgagt gtgattaaac cagagatgaa gatgaggtac tacatggacg gctccgtcaa
     2521 tgggcatgag ttcacaattg aaggtgaagg cacaggcaga ccttacgagg gacatcaaga
     2581 gatgacacta cgcgtcacaa tggccgaggg cgggccaatg cctttcgcgt ttgacttagt
     2641 gtcacacgtg ttctgttacg gccacagagt atttactaaa tatccagaag agataccaga
     2701 ctatttcaaa caagcatttc ctgaaggcct gtcatgggaa aggtcgttgg agttcgaaga
     2761 tggtgggtcc gcttcagtca gtgcgcatat aagccttaga ggaaacacct tctaccacaa
     2821 atccaaattt actggggtta actttcctgc cgatggtcct atcatgcaaa accaaagtgt
     2881 tgattgggag ccatcaaccg agaaaattac tgccagcgac ggagttctga agggtgatgt
     2941 tacgatgtac ctaaaacttg aaggaggcgg caatcacaaa tgccaaatga agactactta
     3001 caaggcggca aaagagattc ttgaaatgcc aggagaccat tacatcggcc atcgcctcgt
     3061 caggaaaacc gaaggcaaca ttactgagca ggtagaagat gcagtagctc attccgctag
     3121 ctgaatcgat gatctagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     3181 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     3241 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     3301 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     3361 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa cctgcagccc aagcttggcg
     3421 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     3481 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     3541 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc
     3601 ctttgttact ttatagaaga aattttgagt ttttgttttt ttttaataaa taaataaaca
     3661 taaataaatt gtttgttgaa tttattatta gtatgtaagt gtaaatataa taaaacttaa
     3721 tatctattca aattaataaa taaacctcga tatacagacc gataaaacac atgcgtcaat
     3781 tttacgcatg attatcttta acgtacgtca caatatgatt atctttctag ggttaaagag
     3841 ctccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat catggtcata
     3901 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3961 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4021 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4081 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4141 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4201 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4261 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     4321 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     4381 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     4441 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     4501 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     4561 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     4621 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     4681 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     4741 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     4801 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     4861 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     4921 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     4981 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5041 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5101 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5161 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5221 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     5281 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     5341 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     5401 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     5461 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     5521 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     5581 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     5641 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     5701 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     5761 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     5821 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     5881 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     5941 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6001 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccac
//
