LOCUS       sequence-239459-        8007 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 8007)
  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 8007)
  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..8007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          134..1150
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1074..1096
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1158..1177
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(1217..1834,1835..1840,1841..1876,1877..1879,1880..1915,
                     1916..1918,1919..1954,1955..1963)
                     /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(2004..2578,2579..2581,2582..2590)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2171..2188)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2398..2417
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2591..3616
                     /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(3919..3935)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3943..3959
                     /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(3967..3973,3974..3991,3992..3997))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4012..4033
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   4260..4322
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(4346..4364)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4385..4401)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4409..4425
                     /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(4433..4439,4440..4457,4458..4463))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4478..4499
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4616..4633)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4787..5375)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4867..4886)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5546..6337,6338..6406))
                     /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     6169..6188
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6407..6511)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6537..6992)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6674..6695)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6886..6905
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7119..7141
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7133..7150
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7134..7150
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7157..7176
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7157..7175
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   7194..7228
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        7506..7889
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc
       61 gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag
      121 cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc
      181 ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga
      241 cggcccttct cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct
      301 gtggctgcgt gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc
      361 ggggggtgcg tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg
      421 cggctgtgag cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg
      481 gagcgcggcc gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg
      541 cgtgcggggt gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca
      601 accccccctg cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct
      661 ccgtacgggg cgtggcgcgg ggctcgccgt gccgggcggg gggtggcggc aggtgggggt
      721 gccgggcggg gcggggccgc ctcgggccgg ggagggctcg ggggaggggc gcggcggccc
      781 ccggagcgcc ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc
      841 gtgcgagagg gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag
      901 gcgccgccgc accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga
      961 aatgggcggg gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc
     1021 tcggggctgt ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg
     1081 cttctggcgt gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt
     1141 tttcctacag ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga
     1201 attcctcgag gccaccatgt ctagactgga caagagcaaa gtcataaacg gcgctctgga
     1261 attactcaat ggagtcggta tcgaaggcct gacgacaagg aaactcgctc aaaagctggg
     1321 agttgagcag cctaccctgt actggcacgt gaagaacaag cgggccctgc tcgatgccct
     1381 gccaatcgag atgctggaca ggcatcatac ccacttctgc cccctggaag gcgagtcatg
     1441 gcaagacttt ctgcggaaca acgccaagtc attccgctgt gctctcctct cacatcgcga
     1501 cggggctaaa gtgcatctcg gcacccgccc aacagagaaa cagtacgaaa ccctggaaaa
     1561 tcagctcgcg ttcctgtgtc agcaaggctt ctccctggag aacgcactgt acgctctgtc
     1621 cgccgtgggc cactttacac tgggctgcgt attggaggaa caggagcatc aagtagcaaa
     1681 agaggaaaga gagacaccta ccaccgattc tatgccccca cttctgagac aagcaattga
     1741 gctgttcgac cggcagggag ccgaacctgc cttccttttc ggcctggaac taatcatatg
     1801 tggcctggag aaacagctaa agtgcgaaag cggcgggccg gccgacgccc ttgacgattt
     1861 tgacttagac atgctcccag ccgatgccct tgacgacttt gaccttgata tgctgcctgc
     1921 tgacgctctt gacgattttg accttgacat gctccccggg taagcggccg ctcgataagc
     1981 ttgatatcga attccgcccc ccccccctct ccctcccccc cccctaacgt tactggccga
     2041 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     2101 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     2161 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     2221 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     2281 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     2341 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     2401 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     2461 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa
     2521 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat
     2581 ggccacaacc atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga
     2641 aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt
     2701 cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt
     2761 ctacaaagat cgttatgttc atcggcactt tgcatcggcc gcgctcccga ttccggaagt
     2821 gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg
     2881 tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga
     2941 ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg
     3001 accgcaagga atcggtcaat acactacgtg gcgtgatttc atatgcgcga ttgctgatcc
     3061 ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc
     3121 tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc
     3181 ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg
     3241 gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc
     3301 gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc
     3361 aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag
     3421 cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt
     3481 ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg
     3541 gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc
     3601 gagggcaaag gaatagtcga gaaattgatg atctattaag caataaagac gtccactaaa
     3661 atggaagttt ttcctgtcat actttgttaa gaagggtgag aacagagtac ctacattttg
     3721 aatggaagga ttggagctac gggggtgggg gtggggtggg attagataaa tgcctgctct
     3781 ttactgaagg ctctttacta ttgctttatg ataatgtttc atagttggat atcataattt
     3841 aaacaagcaa aaccaaatta agggccagct cattcctcca ctcacgatct atagatccac
     3901 tagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3961 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     4021 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4081 ccagcggatc ctttgttact ttatagaaga aattttgagt ttttgttttt ttttaataaa
     4141 taaataaaca taaataaatt gtttgttgaa tttattatta gtatgtaagt gtaaatataa
     4201 taaaacttaa tatctattca aattaataaa taaacctcga tatacagacc gataaaacac
     4261 atgcgtcaat tttacgcatg attatcttta acgtacgtca caatatgatt atctttctag
     4321 ggttaaagag ctccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat
     4381 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4441 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4501 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4561 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4621 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4681 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4741 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4801 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4861 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4921 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4981 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5041 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5101 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5161 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5221 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5281 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5341 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5401 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5461 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5521 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5581 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5641 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5701 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5761 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5821 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5881 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5941 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6001 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6061 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6121 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6181 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6241 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6301 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6361 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6421 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6481 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     6541 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     6601 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     6661 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     6721 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     6781 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     6841 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     6901 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     6961 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg
     7021 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg
     7081 tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac
     7141 gacggccagt gaattgtaat acgactcact atagggcgaa ttgggtacct taaccctaga
     7201 aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat attgctctct ctttctaaat
     7261 agcgcgaatc cgtcgctgtg catttaggac atctcagtcg ccgcttggag ctcccgtgag
     7321 gcgtgcttgt caatgcggta agtgtcactg attttgaact ataacgaccg cgtgagtcaa
     7381 aatgacgcat gattatcttt tacgtgactt ttaagattta actcatacga taattatatt
     7441 gttatttcat gttctactta cgtgataact tattatatat atattttctt gttatagata
     7501 tcgtcgacat tgattattga ctagctagtt attaatagta atcaattacg gggtcattag
     7561 ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     7621 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     7681 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     7741 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     7801 ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     7861 tctacgtatt agtcatcgct attaccatga tggtcgaggt gagccccacg ttctgcttca
     7921 ctctccccat ctcccccccc tccccacccc caattttgta tttatttatt ttttaattat
     7981 tttgtgcagc gatgggggcg ggggggg
//
