LOCUS       sequence-227449-        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"
     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)"
     promoter        2578..3788
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     primer_bind     3625..3644
                     /label=hUBCpro-F
                     /note="Human Ubiquitin C (UbC) promoter, forward primer"
     protein_bind    3805..3829
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3891..4499
                     /codon_start=1
                     /gene="cymR from Pseudomonas putida"
                     /product="cumate repressor (Mullick et al., 2006)"
                     /label=CymR
                     /translation="MSPKRRTQAERAMETQGKLIAAALGVLREKGYAGFRIADVPGAAG
                     VSRGAQSHHFPTKLELLLATFEWLYEQITERSRARLAKLKPEDDVIQQMLDDAAEFFLD
                     DDFSISLDLIVAADRDPALREGIQRTVERNRFVVEDMWLGVLVSRGLSRDDAEDILWLI
                     FNSVRGLAVRSLWQKDKERFERVRNSTLEIARERYAKFKR"
     CDS             4500..4541
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             join(4620..4622,4623..4625,4626..5339)
                     /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(4620..4622,4623..4625,4626..5336)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4665..4686)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4926..4945)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5273..5294
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(5371..5395)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    5550..5671
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5587..5606)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5641..5660
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   5857..5919
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5954..5973)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5955..5973)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5983..6000)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5983..5999)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5992..6014)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6141..6596
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6228..6247)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6438..6459
                     /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 tcgacattga ttattgacta gagatctggc
     2581 ctccgcgccg ggttttggcg cctcccgcgg gcgcccccct cctcacggcg agcgctgcca
     2641 cgtcagacga agggcgcagc gagcgtcctg atccttccgc ccggacgctc aggacagcgg
     2701 cccgctgctc ataagactcg gccttagaac cccagtatca gcagaaggac attttaggac
     2761 gggacttggg tgactctagg gcactggttt tctttccaga gagcggaaca ggcgaggaaa
     2821 agtagtccct tctcggcgat tctgcggagg gatctccgtg gggcggtgaa cgccgatgat
     2881 tatataagga cgcgccgggt gtggcacagc tagttccgtc gcagccggga tttgggtcgc
     2941 ggttcttgtt tgtggatcgc tgtgatcgtc acttggtgag tagcgggctg ctgggctggc
     3001 cggggctttc gtggccgccg ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa
     3061 gggctgtagt ctgggtccgc gagcaaggtt gccctgaact gggggttggg gggagcgcag
     3121 caaaatggcg gctgttcccg agtcttgaat ggaagacgct tgtgaggcgg gctgtgaggt
     3181 cgttgaaaca aggtgggggg catggtgggc ggcaagaacc caaggtcttg aggccttcgc
     3241 taatgcggga aagctcttat tcgggtgaga tgggctgggg caccatctgg ggaccctgac
     3301 gtgaagtttg tcactgactg gagaactcgg tttgtcgtct gttgcggggg cggcagttat
     3361 gcggtgccgt tgggcagtgc acccgtacct ttgggagcgc gcgccctcgt cgtgtcgtga
     3421 cgtcacccgt tctgttggct tataatgcag ggtggggcca cctgccggta ggtgtgcggt
     3481 aggcttttct ccgtcgcagg acgcagggtt cgggcctagg gtaggctctc ctgaatcgac
     3541 aggcgccgga cctctggtga ggggagggat aagtgaggcg tcagtttctt tggtcggttt
     3601 tatgtaccta tcttcttaag tagctgaagc tccggttttg aactatgcgc tcggggttgg
     3661 cgagtgtgtt ttgtgaagtt ttttaggcac cttttgaaat gtaatcattt gggtcaatat
     3721 gtaattttca gtgttagact agtaaattgt ccgctaaatt ctggccgttt ttggcttttt
     3781 tgttagacga agctcgagga tatcacaagt ttgtacaaaa aagcaggctt taaaggaacc
     3841 aattcagtcg actggatccg gtaccgaatt cgcggccgcc tagctaaacc atgtcaccta
     3901 agagaagaac tcaggcagag agagcaatgg aaactcaggg gaaactcatc gcagccgcac
     3961 tcggggtcct cagagaaaag ggctacgcag gattccggat cgccgatgtg ccaggagcag
     4021 ctggtgtctc acgcggggct cagagccacc atttccctac caaactggag ctgctcctgg
     4081 caacatttga gtggctgtat gaacagatca ctgagaggag cagagcccgg ctcgctaagc
     4141 tgaaaccaga ggacgatgtg attcagcaga tgctcgacga tgcagccgaa ttctttctgg
     4201 acgatgactt cagcatctcc ctcgatctga ttgtggctgc agatcgggac cccgccctgc
     4261 gagaagggat tcagcgaacc gtggagcgca accgatttgt ggtcgaagat atgtggctcg
     4321 gcgtgctggt gtctagggga ctgagtagag atgacgccga ggacatcctc tggctgattt
     4381 tcaattccgt gaggggcctc gctgtcagat ctctgtggca gaaggacaaa gagcggtttg
     4441 aacgagtcag aaacagcaca ctggaaatcg cacgggagag atacgctaag ttcaaaaggg
     4501 gtaagcctat ccctaaccct ctcctcggtc tcgattctac gccggataag gcacgtcgtc
     4561 cagcagtaag atcaccacca ctaggaactc gtaagcgaaa gcgtccttct cggggtctga
     4621 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     4681 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     4741 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     4801 tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc
     4861 agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     4921 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     4981 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     5041 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     5101 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     5161 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     5221 acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     5281 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaag
     5341 tgcctaggcg gccgcactcg agatatctag acccagcttt cttgtacaaa gtggtgatcc
     5401 ttatttattg atccttattt attgatctgc ggccgcgact ctagatcata atcagccata
     5461 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     5521 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     5581 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5641 gtggtttgtc caaactcatc aatgtatctt aaggaattcg ataaaagttt tgttacttta
     5701 tagaagaaat tttgagtttt tgtttttttt taataaataa ataaacataa ataaattgtt
     5761 tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat ctattcaaat
     5821 taataaataa acctcgatat acagaccgat aaaacacatg cgtcaatttt acgcatgatt
     5881 atctttaacg tacgtcacaa tatgattatc tttctagggt taatctagct gcggcgcgcc
     5941 ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca
     6001 acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
     6061 tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
     6121 cagcctgaat ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     6181 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     6241 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     6301 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg
     6361 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     6421 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     6481 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     6541 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgctta caatttag
//
