LOCUS       sequence-233149-        6598 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ePiggyBac mammalian expression plasmid for CymR-GFP fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6598)
  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 6598)
  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_UbC_CymRV5-nls-GFP_DEx2
FEATURES             Location/Qualifiers
     source          1..6598
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     187..204
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    321..342
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(357..362,363..380,381..387)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    395..411
                     /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     400..422
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     419..435
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     419..435
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     454..474
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        456..474
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     repeat_region   522..556
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        862..2073
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     1910..1929
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     protein_bind    2090..2114
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2176..2784
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     CDS             2785..2826
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             join(2905..2907,2908..2910,2911..3624)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2905..2907,2908..2910,2911..3621)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2950..2971)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3211..3230)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3558..3579
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3656..3680)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3835..3956
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3872..3891)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3926..3945
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4142..4204
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4239..4258)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4240..4258)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4268..4285)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4268..4284)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4277..4299)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4426..4881
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4513..4532)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4723..4744
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4907..5011
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5012..5080,5081..5872)
                     /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(5230..5249)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6043..6598,1..33)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6532..6551
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
       61 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
      121 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
      181 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
      241 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
      301 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
      361 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
      421 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa
      481 aagctggagc tccaccgcgg ggcgcgccgc agctagatta accctagaaa gatagtctgc
      541 gtaaaattga cgcatgcatt cttgaaatat tgctctctct ttctaaatag cgcgaatccg
      601 tcgctgtgca tttaggacat ctcagtcgcc gcttggagct cccgtgaggc gtgcttgtca
      661 atgcggtaag tgtcactgat tttgaactat aacaaccgcg tgagtcaaaa tgacgcatga
      721 ttatctttta cgtgactttt aagatttaac tcatacgata attatattgt tatttcgtgt
      781 tctacttacg tgataactta ttatatatat attttcttgt tatagatatc cttctcgaca
      841 ttgattattg actagagatc tggcctccgc gccgggtttt ggcgcctccc gcgggcgccc
      901 ccctcctcac ggcgagcgct gccacgtcag acgaagggcg cagcgagcgt cctgatcctt
      961 ccgcccggac gctcaggaca gcggcccgct gctcataaga ctcggcctta gaaccccagt
     1021 atcagcagaa ggacatttta ggacgggact tgggtgactc tagggcactg gttttctttc
     1081 cagagagcgg aacaggcgag gaaaagtagt cccttctcgg cgattctgcg gagggatctc
     1141 cgtggggcgg tgaacgccga tgattatata aggacgcgcc gggtgtggca cagctagttc
     1201 cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg
     1261 tgagtagcgg gctgctgggc tggccggggc tttcgtggcc gccgggccgc tcggtgggac
     1321 ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg
     1381 aactgggggt tggggggagc gcagcaaaat ggcggctgtt cccgagtctt gaatggaaga
     1441 cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt gggcggcaag
     1501 aacccaaggt cttgaggcct tcgctaatgc gggaaagctc ttattcgggt gagatgggct
     1561 ggggcaccat ctggggaccc tgacgtgaag tttgtcactg actggagaac tcggtttgtc
     1621 gtctgttgcg ggggcggcag ttatggcggt gccgttgggc agtgcacccg tacctttggg
     1681 agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt tggcttataa tgcagggtgg
     1741 ggccacctgc cggtaggtgt gcggtaggct tttctccgtc gcaggacgca gggttcgggc
     1801 ctagggtagg ctctcctgaa tcgacaggcg ccggacctct ggtgagggga gggataagtg
     1861 aggcgtcagt ttctttggtc ggttttatgt acctatcttc ttaagtagct gaagctccgg
     1921 ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg aagtttttta ggcacctttt
     1981 gaaatgtaat catttgggtc aatatgtaat tttcagtgtt agactagtaa attgtccgct
     2041 aaattctggc cgtttttggc ttttttgtta gacgaagctc gaggatatca caagtttgta
     2101 caaaaaagca ggctttaaag gaaccaattc agtcgactgg atccggtacc gaattcgcgg
     2161 ccgcctagct aaaccatgtc acctaagaga agaactcagg cagagagagc aatggaaact
     2221 caggggaaac tcatcgcagc cgcactcggg gtcctcagag aaaagggcta cgcaggattc
     2281 cggatcgccg atgtgccagg agcagctggt gtctcacgcg gggctcagag ccaccatttc
     2341 cctaccaaac tggagctgct cctggcaaca tttgagtggc tgtatgaaca gatcactgag
     2401 aggagcagag cccggctcgc taagctgaaa ccagaggacg atgtgattca gcagatgctc
     2461 gacgatgcag ccgaattctt tctggacgat gacttcagca tctccctcga tctgattgtg
     2521 gctgcagatc gggaccccgc cctgcgagaa gggattcagc gaaccgtgga gcgcaaccga
     2581 tttgtggtcg aagatatgtg gctcggcgtg ctggtgtcta ggggactgag tagagatgac
     2641 gccgaggaca tcctctggct gattttcaat tccgtgaggg gcctcgctgt cagatctctg
     2701 tggcagaagg acaaagagcg gtttgaacga gtcagaaaca gcacactgga aatcgcacgg
     2761 gagagatacg ctaagttcaa aaggggtaag cctatcccta accctctcct cggtctcgat
     2821 tctacgccgg ataaggcacg tcgtccagca gtaagatcac caccactagg aactcgtaag
     2881 cgaaagcgtc cttctcgggg tctgatggtg agcaagggcg aggagctgtt caccggggtg
     2941 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     3001 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     3061 aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc
     3121 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     3181 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     3241 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     3301 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     3361 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     3421 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     3481 cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc
     3541 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     3601 ggcatggacg agctgtacaa gtaagtgcct aggcggccgc actcgagata tctagaccca
     3661 gctttcttgt acaaagtggt gatccttatt tattgatcct tatttattga tctgcggccg
     3721 cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3781 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3841 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3901 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaagga
     3961 attcgataaa agttttgtta ctttatagaa gaaattttga gtttttgttt ttttttaata
     4021 aataaataaa cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat
     4081 aataaaactt aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac
     4141 acatgcgtca attttacgca tgattatctt taacgtacgt cacaatatga ttatctttct
     4201 agggttaatc tagctgcggc gcgccggtac ccaattcgcc ctatagtgag tcgtattacg
     4261 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     4321 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     4381 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg
     4441 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4501 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4561 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4621 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     4681 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4741 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     4801 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4861 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     4921 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     4981 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     5041 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     5101 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     5161 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     5221 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     5281 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     5341 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     5401 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     5461 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     5521 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     5581 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     5641 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     5701 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     5761 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     5821 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     5881 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5941 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     6001 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     6061 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     6121 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     6181 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     6241 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     6301 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     6361 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     6421 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     6481 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     6541 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttg
//
