LOCUS       sequence-227441-        7827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ePiggyBac mammalian expression plasmid for TetR-tdTom fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7827)
  AUTHORS   Alexander JM, Guan J, Li B, Maliskova L, Song M, Shen Y, Huang B, 
            Lomvardas S, Weiner OD
  TITLE     Live-cell imaging reveals enhancer-dependent Sox2 transcription in 
            the absence of enhancer proximity.
  JOURNAL   Elife. 2019 May 24;8. pii: 41769. doi: 10.7554/eLife.41769.
  PUBMED    31124784
REFERENCE   2  (bases 1 to 7827)
  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"; journalName: "Elife."; 
            date: "2019-05-24"; volume: "8. pii: 41769. doi"; pages: " 
            10.7554/eLife.41769"
COMMENT     Sequence Label: epB_CAG_TetRFlag-nls-tdTom_DEx4
FEATURES             Location/Qualifiers
     source          1..7827
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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     complement(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1902..1919
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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)."
     primer_bind     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2169..2189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   2237..2271
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        2556..2935
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2937..3214
                     /label=chicken beta-actin promoter
     intron          3215..4231
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4155..4177
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4239..4258
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    4298..4322
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             4384..5007
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MMSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWH
                     VKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGT
                     RPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPT
                     TDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(4460..4479)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             5008..5031
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             5110..6540
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGRYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     protein_bind    complement(6572..6596)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    6779..6900
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6816..6835)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6870..6889
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   7086..7148
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7183..7202)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7184..7202)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7212..7229)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7212..7228)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7221..7243)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7370..7825
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7457..7476)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7667..7688
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggagctccac cgcggggcgc
     2221 gccgcagcta gattaaccct agaaagatag tctgcgtaaa attgacgcat gcattcttga
     2281 aatattgctc tctctttcta aatagcgcga atccgtcgct gtgcatttag gacatctcag
     2341 tcgccgcttg gagctcccgt gaggcgtgct tgtcaatgcg gtaagtgtca ctgattttga
     2401 actataacaa ccgcgtgagt caaaatgacg catgattatc ttttacgtga cttttaagat
     2461 ttaactcata cgataattat attgttattt cgtgttctac ttacgtgata acttattata
     2521 tatatatttt cttgttatag atatccttct cgagcgacat tgattattga ctagttatta
     2581 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     2641 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     2701 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     2761 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     2821 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     2881 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga
     2941 ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt
     3001 gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg ggggggggcg
     3061 cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga gaggtgcggc
     3121 ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc ggcggcggcg
     3181 gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct gccttcgccc
     3241 cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact
     3301 cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta
     3361 atgacggctt gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc
     3421 tttgtgcggg gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc
     3481 gtgcggctcc gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc
     3541 gctccgcagt gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg
     3601 ctgcgagggg aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt
     3661 gggcgcgtcg gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg
     3721 cccggcttcg ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg
     3781 ggggtggcgg caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc
     3841 gggggagggg cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag
     3901 ccattgcctt ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg
     3961 tgcggagccg aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg
     4021 gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg
     4081 tccccttctc cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg
     4141 acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa
     4201 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct
     4261 gtctcatcat tttggcaaag aattgctcga ggatatcaca agtttgtaca aaaaagcagg
     4321 ctttaaagga accaattcag tcgactggat ccggtaccga attcgcggcc gcctagctaa
     4381 accatgatgt ctagattaga taaaagtaaa gtgattaaca gcgcattaga gctgcttaat
     4441 gaggtcggaa tcgaaggttt aacaacccgt aaactcgccc agaagctagg tgtagagcag
     4501 cctacattgt attggcatgt aaaaaataag cgggctttgc tcgacgcctt agccattgag
     4561 atgttagata ggcaccatac tcacttttgc cctttagaag gggaaagctg gcaagatttt
     4621 ttacgtaata acgctaaaag ttttagatgt gctttactaa gtcatcgcga tggagcaaaa
     4681 gtacatttag gtacacggcc tacagaaaaa cagtatgaaa ctctcgaaaa tcaattagcc
     4741 tttttatgcc aacaaggttt ttcactagag aatgcattat atgcactcag cgctgtgggg
     4801 cattttactt taggttgcgt attggaagat caagagcatc aagtcgctaa agaagaaagg
     4861 gaaacaccta ctactgatag tatgccgcca ttattacgac aagctatcga attatttgat
     4921 caccaaggtg cagagccagc cttcttattc ggccttgaat tgatcatatg cggattagaa
     4981 aaacaactta aatgtgaaag tgggtctgat tataaagatg atgatgataa accggataag
     5041 gcacgtcgtc cagcagtaag atcaccacca ctaggaactc gtaagcgaaa gcgtccttct
     5101 cggggtctga tggtgagcaa gggcgaggag gtcatcaaag agttcatgcg cttcaaggtg
     5161 cgcatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc
     5221 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc
     5281 gcctgggaca tcctgtcccc ccagttcatg tacggctcca aggcgtacgt gaagcacccc
     5341 gccgacatcc ccgattacaa gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg
     5401 atgaacttcg aggacggcgg tctggtgacc gtgacccagg actcctccct gcaggacggc
     5461 acgctgatct acaaggtgaa gatgcgcggc accaacttcc cccccgacgg ccccgtaatg
     5521 cagaagaaga ccatgggctg ggaggcctcc accgagcgcc tgtacccccg cgacggcgtg
     5581 ctgaagggcg agatccacca ggccctgaag ctgaaggacg gcggccacta cctggtggag
     5641 ttcaagacca tctacatggc caagaagccc gtgcaactgc ccggctacta ctacgtggac
     5701 accaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc
     5761 tccgagggcc gccaccacct gttcctgggg catggcaccg gcagcaccgg cagcggcagc
     5821 tccggcaccg cctcctccga ggacaacaac atggccgtca tcaaagagtt catgcgcttc
     5881 aaggtgcgca tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag
     5941 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg
     6001 cccttcgcct gggacatcct gtccccccag ttcatgtacg gctccaaggc gtacgtgaag
     6061 caccccgccg acatccccga ttacaagaag ctgtccttcc ccgagggctt caagtgggag
     6121 cgcgtgatga acttcgagga cggcggtctg gtgaccgtga cccaggactc ctccctgcag
     6181 gacggcacgc tgatctacaa ggtgaagatg cgcggcacca acttcccccc cgacggcccc
     6241 gtaatgcaga agaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac
     6301 ggcgtgctga agggcgagat ccaccaggcc ctgaagctga aggacggcgg ccgctacctg
     6361 gtggagttca agaccatcta catggccaag aagcccgtgc aactgcccgg ctactactac
     6421 gtggacacca agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     6481 gagcgctccg agggccgcca ccacctgttc ctgtacggca tggacgagct gtacaagtaa
     6541 gtgcctaggc ggccgcactc gagatatcta gacccagctt tcttgtacaa agtggtgatc
     6601 cttatttatt gatccttatt tattgtagct tatttattgc tacttattta ttgatctgcg
     6661 gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     6721 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     6781 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     6841 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     6901 aggaattcga taaaagtttt gttactttat agaagaaatt ttgagttttt gttttttttt
     6961 aataaataaa taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa
     7021 atataataaa acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata
     7081 aaacacatgc gtcaatttta cgcatgatta tctttaacgt acgtcacaat atgattatct
     7141 ttctagggtt aatctagctg cggcgcgccg gtacccaatt cgccctatag tgagtcgtat
     7201 tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     7261 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     7321 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt
     7381 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     7441 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     7501 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     7561 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     7621 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     7681 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     7741 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     7801 aacaaaatat taacgcttac aatttag
//
