LOCUS       sequence-239462-        6851 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 6851)
  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 6851)
  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: KA0717_pPB-hCMV*1-cHA-IRESVenus
FEATURES             Location/Qualifiers
     source          1..6851
                     /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             join(2855..2857,2858..2860,2861..3574)
                     /codon_start=1
                     /product="yellow fluorescent protein (YFP) with fast and 
                     efficient maturation (Nagai et al., 2002)"
                     /label=Venus
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2900..2921)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3161..3180)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3508..3529
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3959..3976)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3965..4189
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4236..4252)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4260..4276
                     /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(4284..4290,4291..4308,4309..4314))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4329..4350
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   4577..4639
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(4663..4681)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4702..4718)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4726..4742
                     /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(4750..4756,4757..4774,4775..4780))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4795..4816
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4933..4950)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5104..5692)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5184..5203)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5863..6654,6655..6723))
                     /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     6486..6505
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6724..6828)
                     /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 aaccatggtg agcaagggcg aggagctgtt
     2881 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     2941 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agctgatctg
     3001 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctgg gctacggcct
     3061 gcagtgcttc gcccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
     3121 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     3181 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     3241 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     3301 caacgtctat atcaccgccg acaagcagaa gaacggcatc aaggccaact tcaagatccg
     3361 ccacaacatc gaggacggcg gcgtgcagct cgccgaccac taccagcaga acacccccat
     3421 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgag
     3481 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     3541 gatcactctc ggcatggacg agctgtacaa gtaaagcggc cagcttatcg actcgccgat
     3601 agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagtcgag aaattgatga
     3661 tctattaaac aataaagatg tccacatgga agtttttcct gtcatacttt gttaagaagg
     3721 gtgagaacag agtacctaca ttttgaatgg aaggattgga gctacggggg tgggggtggg
     3781 gtgggattag ataaatgcct gctctttact gaaggctctt tactattgct ttatgataat
     3841 gtttcatagt tggatatcat aatttaaaca agcaaaacca aattaagggc cagctcattc
     3901 ctcccactca tgatctatag atcctaagct tatcgatgat ctagagctcg ctgatcagcc
     3961 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     4021 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     4081 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     4141 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg
     4201 gaaagaacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     4261 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     4321 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     4381 tcgggaaacc tgtcgtgcca gcggatcctt tgttacttta tagaagaaat tttgagtttt
     4441 tgtttttttt taataaataa ataaacataa ataaattgtt tgttgaattt attattagta
     4501 tgtaagtgta aatataataa aacttaatat ctattcaaat taataaataa acctcgatat
     4561 acagaccgat aaaacacatg cgtcaatttt acgcatgatt atctttaacg tacgtcacaa
     4621 tatgattatc tttctagggt taaagagctc cagcttttgt tccctttagt gagggttaat
     4681 ttcgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4741 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4801 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4861 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4921 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4981 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5041 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5101 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5161 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5221 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5281 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5341 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5401 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5461 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5521 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5581 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5641 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5701 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5761 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5821 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5881 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5941 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6001 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6061 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6121 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6181 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6241 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6301 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6361 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6421 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6481 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6541 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6601 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6661 aacgggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6721 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6781 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6841 aaaagtgcca c
//
