LOCUS       sequence-141736-        5894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GRE-dependent EGFP reporter construct flanked by the minimal Tol2 
            transposon elements.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5894)
  AUTHORS   Benato F, Colletti E, Skobo T, Moro E, Colombo L, Argenton F, Dalla 
            Valle L
  TITLE     A living biosensor model to dynamically trace glucocorticoid 
            transcriptional activity during development and adult life in 
            zebrafish.
  JOURNAL   Mol Cell Endocrinol. 2014 Jul 5;392(1-2):60-72. doi: 
            10.1016/j.mce.2014.04.015. Epub 2014 May 4.
  PUBMED    24796658
REFERENCE   2  (bases 1 to 5894)
  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: "Mol Cell 
            Endocrinol."; date: "2014-07-5"; volume: "392(1-2):60-72. doi"; 
            pages: " 10.1016/j.mce.2014.04.015. Epub 2014 May 4"
COMMENT     Sequence Label: pDestTol2pA2-9xGRE
FEATURES             Location/Qualifiers
     source          1..5894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(144..165)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(514..525,526..688)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     1061..1077
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1097..1117
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     1119..1138
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1119..1137
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1162..1178
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1163..1179
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1487..1632
                     /label=HSV TK promoter
                     /note="herpes simplex virus thymidine kinase promoter"
     primer_bind     complement(1689..1705)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(1742..1760)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    1760..1784
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(1790..1792,1793..1795,1796..2506)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1790..1792,1793..1795,1796..2506)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1835..1856)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2096..2115)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2443..2464
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2568..2592)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    2594..2728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(2791..2811)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(2819..2835)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2851..2985
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    join(3424..3611,3612..3623)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(3657..3673)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(3710..3728)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3749..3765)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3762..3784)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3773..3789
                     /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)."
     promoter        complement(join(3797..3803,3804..3821,3822..3827))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3842..3863
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3980..3997)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4151..4739)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4231..4250)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4910..5701,5702..5770))
                     /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     5533..5552
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5771..5875)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
       61 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
      121 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
      181 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacgc tggctaagaa
      241 ctcatcagcc tccccggtcc atctacccac gtaccaatgc accaattggc cacaatgacg
      301 gctactacat ggtgccattc cttcctcttt ataggaatgg agactacctc ctgtccaaca
      361 aggctcttgg atacgagtac gcctacctgt tggacccagg tcattgcaca acaccagaaa
      421 tgccctctga tctgcaaaag acgtgaatat ctgttcagac acccatatcc actctgttcc
      481 acacaggtca gaggtttgtc caggagttct tgacagaggt gtaaaaagta ctcaaaaatt
      541 ttactcaagt gaaagtacaa gtacttaggg aaaattttac tcaattaaaa gtaaaagtat
      601 ctggctagaa tcttacttga gtaaaagtaa aaaagtactc cattaaaatt gtacttgagt
      661 attaaggaag taaaagtaaa agcaagaaag aaaactagag attcttgttt aagcttttaa
      721 tctcaaaaaa cattaaatga aatgcataca aggttttatc ctgctttaga actgtttgta
      781 tttaattatc aaactataag acagacaatc taatgccagt acacgctact caaagttgta
      841 aaacctcaga tttaacttca gtagaagctg attctcaaaa ttgttagtgt caagcctagc
      901 tcttttgggg ctgaaaagca atcctgcagt gctgaaaagc ctctcacagg cagccgatgc
      961 gggaagaggt gtattagtct tgatagagag gctgcaaata gcaggaaacg tgagcagaga
     1021 ctccctggtg tctgaaacac aggccagatg ggccctcgag caggaaacag ctatgaccat
     1081 gattacgcca agctatcaac tttgtataga aaagttggta atacgactca ctatagggcg
     1141 aattgggtac cgggcccccc ctcgaggtcg acggtatcga taagcttggg ttcgattgga
     1201 tgttctagct acgtcgagct gtacaggatg ttctagctac atcgagctgt acaggatgtt
     1261 ctagctacgt cgagctgtac aggatgttct agctacatcg agctgtacag gatgttctag
     1321 ctacgtcgag ctgtacagga tgttctagct acgtcgagct gtacaggatg ttctagctac
     1381 gtcgagctgt acaggatgtt ctagctacgt cgagctgtac aggatgttct agctacgtcg
     1441 agctgtacag gatgttctag ctaaatcact accgctcgac ctcgagatcc ggcaaacccc
     1501 gcccagcgtc ttgtcattgg cgaattcgaa cacgcagatg cagtcggggc ggcgcggtcc
     1561 gaggtccact tcgcatatta aggtgacgcg tgtggcctcg aacaccgagc gaccctgcag
     1621 cgacccgctt aacagcgtca acagcgtgcc gcagatctga gcttggcatt ccggtactgt
     1681 tggtaaagga tccactagtt ctagagcggc cgccaccgcg gtggagctcc agcttttgtt
     1741 ccctttagtg agggttaatt caagtttgta caaaaaagca ggctggacca tggtgagcaa
     1801 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     1861 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
     1921 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
     1981 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
     2041 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
     2101 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
     2161 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
     2221 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
     2281 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
     2341 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     2401 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     2461 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggcg gtggaagatc
     2521 tgggaattca aggcctctcg agcctctaga ttctgcagcc ctatagcacc cagctttctt
     2581 gtacaaagtg ggggatccag acatgataag atacattgat gagtttggac aaaccacaac
     2641 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     2701 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     2761 ggttcagggg gaggtgtggg aggttttttc caactttatt atacatagtt gataattcac
     2821 tggccgtcgt tttacggtac catcgatgat gatccagaca tgataagata cattgatgag
     2881 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     2941 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     3001 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     3061 ctctacaaat gtggtatggc tgattatgat cctctagatc agatctgcga agatacggcc
     3121 acgggtgctc ttgatcctgt ggctgatttt ggactgtgct gctcgcagct gctgatgaat
     3181 cacatacttc ctccattttc ttccactgat tgactgttat aatttcccta atttccaggt
     3241 caaggtgctg tgcattgtgg taatagatgt gacatgacgt cacttccaaa ggaccaatga
     3301 acatgtctga ccaatttcat ataatgtgaa aacgattttc ataggcagaa taaataacat
     3361 ttaaattaaa ctgggcatca gcgcaattca attggtttgg taatagcaag ggaaaataga
     3421 atgaagtgat ctccaaaaaa taagtacttt ttgactgtaa ataaaattgt aaggagtaaa
     3481 aagtactttt ttttctaaaa aaatgtaatt aagtaaaagt aaaagtattg atttttaatt
     3541 gtactcaagt aaagtaaaaa tccccaaaaa taatacttaa gtacagtaat caagtaaaat
     3601 tactcaagta ctttacacct ctggttcttg accccctacc ttcagcaagc ccagcagatc
     3661 cactagttct agagcggccg ccaccgcggt ggagctccag cttttgttcc ctttagtgag
     3721 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3781 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     3841 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3901 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3961 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4021 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4081 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4141 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4201 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4261 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4321 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4381 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4441 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4501 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4561 agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga
     4621 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4681 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4741 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4801 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4861 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4921 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4981 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5041 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5101 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5161 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5221 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5281 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5341 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5401 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5461 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5521 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5581 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5641 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5701 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5761 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5821 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5881 ttccccgaaa agtg
//
