LOCUS       sequence-227447-        7697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ePiggyBac mammalian expression plasmid for CymR-Halo fusion.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7697)
  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 7697)
  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-Halox2_DEx4
FEATURES             Location/Qualifiers
     source          1..7697
                     /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             4620..5510
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             5517..6407
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     protein_bind    complement(6442..6466)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    6649..6770
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6686..6705)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6740..6759
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6956..7018
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7053..7072)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7054..7072)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7082..7099)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7082..7098)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7091..7113)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7240..7695
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7327..7346)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7537..7558
                     /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 tggcagaaat cggtactggc tttccattcg acccccatta tgtggaagtc ctgggcgagc
     4681 gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc ctgcacggta
     4741 acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg acccatcgct
     4801 gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg ggttatttct
     4861 tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg gaagaggtcg
     4921 tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag cgcaatccag
     4981 agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc tgggacgaat
     5041 ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc ggccgcaagc
     5101 tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc gtccgcccgc
     5161 tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt gaccgcgagc
     5221 cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac atcgtcgcgc
     5281 tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg ctgttctggg
     5341 gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa agcctgccta
     5401 actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac aacccggacc
     5461 tgatcggcag cgagatcgcg cgctggctgt ctactctgga gatttccggc ggatctatgg
     5521 cagaaatcgg tactggcttt ccattcgacc cccattatgt ggaagtcctg ggcgagcgca
     5581 tgcactacgt cgatgttggt ccgcgcgatg gcacccctgt gctgttcctg cacggtaacc
     5641 cgacctcctc ctacgtgtgg cgcaacatca tcccgcatgt tgcaccgacc catcgctgca
     5701 ttgctccaga cctgatcggt atgggcaaat ccgacaaacc agacctgggt tatttcttcg
     5761 acgaccacgt ccgcttcatg gatgccttca tcgaagccct gggtctggaa gaggtcgtcc
     5821 tggtcattca cgactggggc tccgctctgg gtttccactg ggccaagcgc aatccagagc
     5881 gcgtcaaagg tattgcattt atggagttca tccgccctat cccgacctgg gacgaatggc
     5941 cagaatttgc ccgcgagacc ttccaggcct tccgcaccac cgacgtcggc cgcaagctga
     6001 tcatcgatca gaacgttttt atcgagggta cgctgccgat gggtgtcgtc cgcccgctga
     6061 ctgaagtcga gatggaccat taccgcgagc cgttcctgaa tcctgttgac cgcgagccac
     6121 tgtggcgctt cccaaacgag ctgccaatcg ccggtgagcc agcgaacatc gtcgcgctgg
     6181 tcgaagaata catggactgg ctgcaccagt cccctgtccc gaagctgctg ttctggggca
     6241 ccccaggcgt tctgatccca ccggccgaag ccgctcgcct ggccaaaagc ctgcctaact
     6301 gcaaggctgt ggacatcggc ccgggtctga atctgctgca agaagacaac ccggacctga
     6361 tcggcagcga gatcgcgcgc tggctgtcta ctctggagat ttccggctaa gtgcctaggc
     6421 ggccgcactc gagatatcta gacccagctt tcttgtacaa agtggtgatc cttatttatt
     6481 gatccttatt tattgtagct tatttattgc tacttattta ttgatctgcg gccgcgactc
     6541 tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca
     6601 cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt
     6661 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     6721 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta aggaattcga
     6781 taaaagtttt gttactttat agaagaaatt ttgagttttt gttttttttt aataaataaa
     6841 taaacataaa taaattgttt gttgaattta ttattagtat gtaagtgtaa atataataaa
     6901 acttaatatc tattcaaatt aataaataaa cctcgatata cagaccgata aaacacatgc
     6961 gtcaatttta cgcatgatta tctttaacgt acgtcacaat atgattatct ttctagggtt
     7021 aatctagctg cggcgcgccg gtacccaatt cgccctatag tgagtcgtat tacgcgcgct
     7081 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     7141 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     7201 gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat
     7261 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     7321 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     7381 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     7441 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     7501 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     7561 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     7621 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     7681 taacgcttac aatttag
//
