LOCUS       sequence-239664-        6061 bp    DNA     circular SYN 15-APR-2025
DEFINITION  piggyBac-based Dox-inducible expression (moderate).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6061)
  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 6061)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: KA2897_pPB-TREtight-UBCmin-cHA-IRESH2BmKO2
FEATURES             Location/Qualifiers
     source          1..6061
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     43..65
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     57..74
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     58..74
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     81..100
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        81..99
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   118..152
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    518..536
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    554..572
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    589..607
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    625..643
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    661..679
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    696..714
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    732..750
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             885..911
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(954..1528,1529..1531,1532..1540)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1121..1138)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1348..1367
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1541..1918
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
     regulatory      1931..1940
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1937..2590
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     primer_bind     complement(2627..2644)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2633..2857
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2904..2920)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    2928..2944
                     /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(2952..2958,2959..2976,2977..2982))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2997..3018
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   3245..3307
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(3331..3349)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3370..3386)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3394..3410
                     /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(3418..3424,3425..3442,3443..3448))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3463..3484
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3601..3618)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3772..4360)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3852..3871)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4531..5322,5323..5391))
                     /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     5154..5173
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5392..5496)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5522..5977)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5659..5680)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5871..5890
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
       61 aaacgacggc cagtgaattg taatacgact cactataggg cgaattgggt accttaaccc
      121 tagaaagata gtctgcgtaa aattgacgca tgcattcttg aaatattgct ctctctttct
      181 aaatagcgcg aatccgtcgc tgtgcattta ggacatctca gtcgccgctt ggagctcccg
      241 tgaggcgtgc ttgtcaatga ggtaagtgtc actgattttg aactataacg accgcgtgag
      301 tcaaaatgac gcatgattat cttttacgtg acttttaaga tttaactcat acgataatta
      361 tattgttatt tcatgttcta cttacgtgat aacttattat atatatattt tcttgttata
      421 gatatcataa tttaaacaag caaaaccaaa ttaagggcca gctcattcct cccactcatg
      481 atctatagat cttctagtga tttataggtc tcgcagatcc ctatcagtga tagagaacgt
      541 atgtcgagtt tactccctat cagtgataga gaacgatgtc gagtttactc cctatcagtg
      601 atagagaacg tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttac
      661 tccctatcag tgatagagaa cgtatgtcga gtttatccct atcagtgata gagaacgtat
      721 gtcgagttta ctccctatca gtgatagaga tgatagagac ctataaatcc cgcggccatg
      781 gcggtgaacg ccgatgatta tataaggacg cgccgggtgt ggcacagcta gttccgtcgc
      841 agccgggttt gaattcctcg agacgcgtga ctagtgccat ggcctaccca tacgacgtgc
      901 cagactacgc ctagcggccg ctcgataagc ttgatatcga attccgcccc ccccccctct
      961 ccctcccccc cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt
     1021 gtctatatgt tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct
     1081 ggccctgtct tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa
     1141 ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg
     1201 tctgtagcga ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc
     1261 caaaagccac gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg
     1321 agttggatag ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg
     1381 aaggatgccc agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc
     1441 tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg
     1501 gttttccttt gaaaaacacg atgataatat ggccacaacc atgccagagc cagcgaagtc
     1561 tgctcccgcc ccgaaaaagg gctccaagaa ggcggtgact aaggcgcaga agaaagacgg
     1621 caagaagcgc aagcgcagcc gcaaggagag ctattccatc tatgtgtaca aggttctgaa
     1681 gcaggtccac cctgacaccg gcatttcgtc caaggccatg ggcatcatga attcgtttgt
     1741 gaacgacatt ttcgagcgca tcgcaggtga ggcttcccgc ctggcgcatt acaacaagcg
     1801 ctcgaccatc acctccaggg agatccagac ggccgtgcgc ctgctgctgc ctggggagtt
     1861 ggccaagcac gccgtgtccg agggtactaa ggccgtcacc aagtacacca gcgctaagct
     1921 cgagactagt gccaccatgg tgagtgtgat taaaccagag atgaagatga ggtactacat
     1981 ggacggctcc gtcaatgggc atgagttcac aattgaaggt gaaggcacag gcagacctta
     2041 cgagggacat caagagatga cactacgcgt cacaatggcc gagggcgggc caatgccttt
     2101 cgcgtttgac ttagtgtcac acgtgttctg ttacggccac agagtattta ctaaatatcc
     2161 agaagagata ccagactatt tcaaacaagc atttcctgaa ggcctgtcat gggaaaggtc
     2221 gttggagttc gaagatggtg ggtccgcttc agtcagtgcg catataagcc ttagaggaaa
     2281 caccttctac cacaaatcca aatttactgg ggttaacttt cctgccgatg gtcctatcat
     2341 gcaaaaccaa agtgttgatt gggagccatc aaccgagaaa attactgcca gcgacggagt
     2401 tctgaagggt gatgttacga tgtacctaaa acttgaagga ggcggcaatc acaaatgcca
     2461 aatgaagact acttacaagg cggcaaaaga gattcttgaa atgccaggag accattacat
     2521 cggccatcgc ctcgtcagga aaaccgaagg caacattact gagcaggtag aagatgcagt
     2581 agctcattcc gctagctgaa tcgatgatct agagctcgct gatcagcctc gactgtgcct
     2641 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     2701 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     2761 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     2821 aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaacctgc
     2881 agcccaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     2941 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3001 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3061 tcgtgccagc ggatcctttg ttactttata gaagaaattt tgagtttttg ttttttttta
     3121 ataaataaat aaacataaat aaattgtttg ttgaatttat tattagtatg taagtgtaaa
     3181 tataataaaa cttaatatct attcaaatta ataaataaac ctcgatatac agaccgataa
     3241 aacacatgcg tcaattttac gcatgattat ctttaacgta cgtcacaata tgattatctt
     3301 tctagggtta aagagctcca gcttttgttc cctttagtga gggttaattt cgagcttggc
     3361 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3421 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     3481 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     3541 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     3601 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     3661 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     3721 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     3781 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3841 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3901 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3961 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4021 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4081 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4141 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4201 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4261 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4321 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4381 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4441 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     4501 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     4561 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     4621 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     4681 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     4741 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     4801 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4861 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4921 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4981 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5041 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5101 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5161 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5221 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5281 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa cgggaaggca
     5341 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5401 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5461 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5521 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     5581 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     5641 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     5701 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     5761 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     5821 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     5881 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     5941 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
     6001 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
     6061 g
//
