LOCUS       sequence-385070-        6591 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E1b promoter for transgenic enhancer assays in zebrafish.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6591)
  AUTHORS   Begeman IJ, Emery B, Kurth A, Kang J
  TITLE     Regeneration and developmental enhancers are differentially 
            compatible with minimal promoters.
  JOURNAL   Dev Biol. 2022 Sep 24;492:47-58. doi: 10.1016/j.ydbio.2022.09.007.
  PUBMED    36167150
REFERENCE   2  (bases 1 to 6591)
  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: "Dev Biol.";
            date: "2022-09-24"; volume: "492:47-58. doi"; pages: " 
            10.1016/j.ydbio.2022.09.007"
COMMENT     Sequence Label: Tol2-ISceI-E1b:EGFP;cryaa:mCherry-ISceI-Tol2
FEATURES             Location/Qualifiers
     source          1..6591
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     177..196
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     436..457
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      499..508
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     intron          582..1154
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     misc_feature    join(1340..1502,1503..1514)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(1529..1548)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1530..1548)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1558..1575)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1558..1574)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1567..1589)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1716..2171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1803..1822)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2013..2034
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2197..2301
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2302..2370,2371..3162)
                     /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(2520..2539)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3333..3921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3822..3841
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4075..4092
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4209..4230
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4245..4250,4251..4268,4269..4275)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4283..4299
                     /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     4288..4310
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4307..4323
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4307..4323
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4342..4362
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4344..4362
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     4399..4415
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4399..4415
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(4449..4460,4461..4648)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     regulatory      5345..5354
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             5351..6061
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEDDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGHHSTGGMDELYK"
     primer_bind     complement(5496..5514)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     5764..5783
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5958..5981
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    6184..6318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6221..6240)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6275..6294
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(6374..6591,1..496,497..499,500..502))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPILVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(6419..6440)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc
       61 tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc
      121 ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct
      181 tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg
      241 cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc
      301 cgtaggtcag ggtggtcacg aggatgggcc agggcacggg cagcttgccg gtggtgcaga
      361 tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga
      421 acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca
      481 gctcctcgcc cttgctcacc atggtggcga ccggtggatc cgtcgaggaa ttctttgcca
      541 aaatgatgag acagcacaac aaccagcacg ttgcccagga gctgtaggaa agagaagaag
      601 gcatgaacat ggttagcaga ggggcccggt ttggactcag agtattttat cctcatctca
      661 aacagtgtat atcattgtaa ccataaagag aaaggcagga tgatgaccag ggtgtagttg
      721 tttctaccaa taagaatatt tccacgccag ccagaattta tatgcagaaa tattctacct
      781 tatcatttaa ttataacaat tgttctctaa aactgtgctg aagtacaata taatataccc
      841 tgattgcctt gaaaaaaaag tgattagaga aagtacttac aatctgacaa ataaacaaaa
      901 gtgaatttaa aaattcgtta caaatgcaag ctaaagttta acgaaaaagt tacagaaaat
      961 gaaaagaaaa taagaggaga caatggttgt caacagagta gaaagtgaaa gaaacaaaat
     1021 tatcatgagg gtccatggtg atacaaggga catcttccca ttctaaacaa caccctgaaa
     1081 actttgcccc ctccatataa catgaatttt acaatagcga aaaagaaaga acaatcaagg
     1141 gtccccaaac tcaccctgaa gttctcagga tcggtcgaaa ttcgtgtgga ggagctcaaa
     1201 gtgaggctga gacgcgatgg gatccattat ataccctcta gagtcgaaga tctgcaggat
     1261 ccgtttaaac atcgatggcg cctaggctag ctcgagatta ccctgttatc cctaaagctt
     1321 caagaatctc tagttttctt tcttgctttt acttttactt ccttaatact caagtacaat
     1381 tttaatggag tactttttta cttttactca agtaagattc tagccagata cttttacttt
     1441 taattgagta aaattttccc taagtacttg tactttcact tgagtaaaat ttttgagtac
     1501 tttttacacc tctgtggtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact
     1561 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct
     1621 tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc
     1681 ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag
     1741 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     1801 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     1861 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     1921 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     1981 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2041 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2101 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2161 gcttacaatt taggtggcat ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2221 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa
     2281 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt
     2341 tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat
     2401 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag
     2461 atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg
     2521 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata
     2581 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat
     2641 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc
     2701 aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg
     2761 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac
     2821 gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact
     2881 ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa
     2941 gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct
     3001 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc
     3061 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
     3121 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
     3181 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     3241 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     3301 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     3361 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     3421 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt
     3481 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     3541 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     3601 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     3661 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     3721 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     3781 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     3841 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     3901 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     3961 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     4021 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     4081 tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     4141 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     4201 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     4261 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     4321 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctc
     4381 caccgcggtg gcggccgctc tagaactagt ggatctgctg ggcttgctga aggtaggggg
     4441 tcaagaacca gaggtgtaaa gtacttgagt aattttactt gattactgta cttaagtatt
     4501 atttttgggg atttttactt tacttgagta caattaaaaa tcaatacttt tacttttact
     4561 taattacatt tttttagaaa aaaaagtact ttttactcct tacaatttta tttacagtca
     4621 aaaagtactt attttttgga gatcacttca ttctattttc ccttgctatt accaaaccaa
     4681 ttgattaccc tgttatccct acgggcccaa aattgtgaaa tgcacagaat gtacatatac
     4741 aatacatttc ctacaataat gctctctttt ctattaatag tgtgcattca gtgcaggatg
     4801 ttatgtattt attttctgta gtgcaaactt tttcaaagaa ttttccagtc taagcttttc
     4861 agtcaagaaa aaaaaaaagc gattgattat ctgtgatatt taaaaaaatt agctcttaaa
     4921 tctggtgaac caagtggcca caacacatta aatactgacg ttctacagcc atgtagcttc
     4981 atctggtctg gtttgttttg gcaggcactt attttccggt gatagttggc cacaagagta
     5041 ttgtctgagc cattcagtgc tagactgtca ttctcaggtc agagtccacc ccgctgattc
     5101 tgctgacaac actgttccca ccatgagata atgccattcc agagagatcc atttgtaagc
     5161 ccctctttct gcagcacagg tatataacca ggggtctgcc tccactaagg ccggcacaca
     5221 tcatttgggg atctttgtac tgtactagcc tctattagcc tcctacttgc gttcagttca
     5281 catcttttgt tcatcaggtg caataagtta cagtaaggag ttaccaggtc tgacattacc
     5341 ggtcgccacc atggtgagca agggcgagga tgacaacatg gccatcatca aggagttcat
     5401 gcgcttcaag gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga
     5461 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggcgg
     5521 ccccctgccc ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta
     5581 cgtgaagcac cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa
     5641 gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc
     5701 cctgcaggac ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga
     5761 cggccccgta atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc
     5821 cgaggacggc gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca
     5881 ctacgacgcc gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc
     5941 ctacaacgtc aacatcaagc tggacatcac ctcccacaac gaggactaca ccatcgtgga
     6001 acagtacgag cgcgccgagg gccaccactc caccggcggc atggacgagc tgtacaagta
     6061 aagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt tttacttgct
     6121 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     6181 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     6241 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     6301 tcttatcatg tctggatcat catcgatcta actcttcccg ggtcgatgaa ttaattcgca
     6361 tgctttaact tccttacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     6421 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact
     6481 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     6541 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg a
//
