LOCUS       sequence-236404-        8038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac-based expression of rtTA-M2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8038)
  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 8038)
  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: KA1775_pPBCAG-rtTAM2-IH
FEATURES             Location/Qualifiers
     source          1..8038
                     /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)"
     enhancer        972..1355
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1360..1637
                     /label=chicken beta-actin promoter
     intron          1638..2654
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2578..2600
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2662..2681
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(2721..3338,3339..3344,3345..3380,3381..3383,3384..3419,
                     3420..3422,3423..3458,3459..3467)
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    join(3508..4082,4083..4085,4086..4094)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3675..3692)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3902..3921
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4095..5120
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(5423..5439)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5447..5463
                     /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(5471..5477,5478..5495,5496..5501))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5516..5537
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   5764..5826
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(5850..5868)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5889..5905)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5913..5929
                     /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(5937..5943,5944..5961,5962..5967))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5982..6003
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6120..6137)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6291..6879)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6371..6390)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7050..7841,7842..7910))
                     /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     7673..7692
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7911..8015)
                     /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 tagatatcgt cgacattgat tattgactag ctagttatta atagtaatca attacggggt
     1021 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     1081 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     1141 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     1201 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     1261 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     1321 agtacatcta cgtattagtc atcgctatta ccatgatggt cgaggtgagc cccacgttct
     1381 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
     1441 aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg
     1501 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
     1561 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     1621 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     1681 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     1741 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
     1801 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
     1861 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc
     1921 ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg
     1981 aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag
     2041 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc
     2101 tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg
     2161 ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg
     2221 gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg
     2281 gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     2341 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg
     2401 ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga
     2461 aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc
     2521 agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     2581 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct
     2641 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
     2701 aagaattcct cgaggccacc atgtctagac tggacaagag caaagtcata aacggcgctc
     2761 tggaattact caatggagtc ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc
     2821 tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg
     2881 ccctgccaat cgagatgctg gacaggcatc atacccactt ctgccccctg gaaggcgagt
     2941 catggcaaga ctttctgcgg aacaacgcca agtcattccg ctgtgctctc ctctcacatc
     3001 gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg
     3061 aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca ctgtacgctc
     3121 tgtccgccgt gggccacttt acactgggct gcgtattgga ggaacaggag catcaagtag
     3181 caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg agacaagcaa
     3241 ttgagctgtt cgaccggcag ggagccgaac ctgccttcct tttcggcctg gaactaatca
     3301 tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccggccgac gcccttgacg
     3361 attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt gatatgctgc
     3421 ctgctgacgc tcttgacgat tttgaccttg acatgctccc cgggtaagcg gccgctcgat
     3481 aagcttgata tcgaattccg cccccccccc ctctccctcc ccccccccta acgttactgg
     3541 ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt
     3601 gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc
     3661 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc
     3721 agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg
     3781 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc
     3841 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa
     3901 atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg
     3961 tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa
     4021 aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata
     4081 atatggccac aaccatgaaa aagcctgaac tcaccgcgac gtctgtcgag aagtttctga
     4141 tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa gaatctcgtg
     4201 ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg
     4261 gtttctacaa agatcgttat gttcatcggc actttgcatc ggccgcgctc ccgattccgg
     4321 aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac
     4381 agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg
     4441 cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg ttcggcccat
     4501 tcggaccgca aggaatcggt caatacacta cgtggcgtga tttcatatgc gcgattgctg
     4561 atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc
     4621 aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg cacctcgtgc
     4681 acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca gcggtcattg
     4741 actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc ttcttctgga
     4801 ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg catccggagc
     4861 ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac caactctatc
     4921 agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa
     4981 tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga agcgcggccg
     5041 tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc cccagcactc
     5101 gtccgagggc aaaggaatag tcgagaaatt gatgatctat taagcaataa agacgtccac
     5161 taaaatggaa gtttttcctg tcatactttg ttaagaaggg tgagaacaga gtacctacat
     5221 tttgaatgga aggattggag ctacgggggt gggggtgggg tgggattaga taaatgcctg
     5281 ctctttactg aaggctcttt actattgctt tatgataatg tttcatagtt ggatatcata
     5341 atttaaacaa gcaaaaccaa attaagggcc agctcattcc tccactcacg atctatagat
     5401 ccactagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     5461 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     5521 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5581 cgtgccagcg gatcctttgt tactttatag aagaaatttt gagtttttgt ttttttttaa
     5641 taaataaata aacataaata aattgtttgt tgaatttatt attagtatgt aagtgtaaat
     5701 ataataaaac ttaatatcta ttcaaattaa taaataaacc tcgatataca gaccgataaa
     5761 acacatgcgt caattttacg catgattatc tttaacgtac gtcacaatat gattatcttt
     5821 ctagggttaa agagctccag cttttgttcc ctttagtgag ggttaatttc gagcttggcg
     5881 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5941 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     6001 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     6061 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     6121 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     6181 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     6241 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6301 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6361 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6421 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6481 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6541 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6601 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6661 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6721 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6781 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6841 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6901 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6961 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     7021 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7081 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7141 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7201 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7261 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7321 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7381 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7441 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7501 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7561 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7621 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7681 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7741 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7801 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7861 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7921 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7981 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
//
