LOCUS       sequence-325133-        5049 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3x NFAT binding sequence upstream of a luciferase reporter; can be 
            used to assay signal transduction through Ca2+, calcineurin and NFAT
            in any tissue.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5049)
  AUTHORS   Clipstone NA, Crabtree GR
  TITLE     Identification of calcineurin as a key signalling enzyme in 
            T-lymphocyte activation.
  JOURNAL   Nature. 1992 Jun 25. 357(6380):695-7.
  PUBMED    1377362
REFERENCE   2  (bases 1 to 5049)
  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: "Nature. 
            1992 Jun 25. 357(6380):695-7."
COMMENT     Sequence Label: pGL3-NFAT luciferase
FEATURES             Location/Qualifiers
     source          1..5049
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1105..1123
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     regulatory      1186..1195
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1227..1340)
                     /label=minimal IL-2 promoter
                     /label=IL-2 promoter
                     /note="minimal promoter from the human interleukin-2 gene"
     protein_bind    1348..1377
                     /label=NFAT binding site
                     /bound_moiety="NFAT transcription factor"
                     /note="NFAT binding site from the human interleukin 2 
                     (IL-2) gene (Mattila et al., 1990)"
     protein_bind    1378..1407
                     /label=NFAT binding site
                     /bound_moiety="NFAT transcription factor"
                     /note="NFAT binding site from the human interleukin 2 
                     (IL-2) gene (Mattila et al., 1990)"
     protein_bind    1408..1437
                     /label=NFAT binding site
                     /bound_moiety="NFAT transcription factor"
                     /note="NFAT binding site from the human interleukin 2 
                     (IL-2) gene (Mattila et al., 1990)"
     misc_feature    1515..1606
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(1547..1566)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    1620..1668
                     /note="synthetic polyadenylation signal"
     rep_origin      complement(1799..2254)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1936..1957)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2148..2167
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2281..2385
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2386..2454,2455..3246)
                     /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(2604..2623)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3417..4005
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3906..3925
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4159..4176
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    4424..4545
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4461..4480)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4515..4534
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(join(4586..5049,1..1189))
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
ORIGIN
        1 tcataggacc tctcacacac agttcgcctc tttgattaac gcccagcgtt ttcccggtat
       61 ccagatccac aaccttcgct tcaaaaaatg gaacaacttt accgaccgcg cccggtttat
      121 catccccctc gggtgtaatc agaatagctg atgtagtctc agtgagccca tatccttgcc
      181 tgatacctgg cagatggaac ctcttggcaa ccgcttcccc gacttcctta gagaggggag
      241 cgccaccaga agcaatttcg tgtaaattag ataaatcgta tttgtcaatc agagtgcttt
      301 tggcgaagaa ggagaatagg gttggcacca gcagcgcact ttgaatcttg taatcctgaa
      361 ggctcctcag aaacagctct tcttcaaatc tatacattaa gacgactcga aatccacata
      421 tcaaatatcc gagtgtagta aacattccaa aaccgtgatg gaatggaaca acacttaaaa
      481 tcgcagtatc cggaatgatt tgattgccaa aaataggatc tctggcatgc gagaatctca
      541 cgcaggcagt tctatgaggc agagcgacac ctttaggcag accagtagat ccagaggagt
      601 tcatgatcag tgcaattgtc ttgtccctat cgaaggactc tggcacaaaa tcgtattcat
      661 taaaaccggg aggtagatga gatgtgacga acgtgtacat cgactgaaat ccctggtaat
      721 ccgttttaga atccatgata ataatttttt ggatgattgg gagctttttt tgcacgttca
      781 aaattttttg caaccccttt ttggaaacga acaccacggt aggctgcgaa atgcccatac
      841 tgttgagcaa ttcacgttca ttataaatgt cgttcgcggg cgcaactgca actccgataa
      901 ataacgcgcc caacaccggc ataaagaatt gaagagagtt ttcactgcat acgacgattc
      961 tgtgatttgt attcagccca tatcgtttca tagcttctgc caaccgaacg gacatttcga
     1021 agtactcagc gtaagtgatg tccacctcga tatgtgcatc tgtaaaagca attgttccag
     1081 gaaccagggc gtatctcttc atagccttat gcagttgctc tccagcggtt ccatcttcca
     1141 gcggatagaa tggcgccggg cctttcttta tgtttttggc gtcttccatg gtggctttac
     1201 caacagtacc ggaatgccaa gcttgggcag gagttgaggt tactgtgagt agtgattaaa
     1261 gagagtgata gggaactctt gaacaagaga tgcaatttat actgttaatt ctggaaaaat
     1321 attatggggg tgtcaaaatg tcctcgagga ggaaaaactg tttcatacag aaggcgtgga
     1381 ggaaaaactg tttcatacag aaggcgtacg ccttctgtat gaaacagttt ttcctcctcg
     1441 agggggggtg gggatacaaa agtaactcag aaaacatcgg ggctagcacg cgtaagagct
     1501 cggtacctat cgatagagaa atgttctggc acctgcactt gcactgggga cagcctattt
     1561 tgctagtttg ttttgtttcg ttttgttttg atggagagcg tatgttagta ctatcgattc
     1621 acacaaaaaa ccaacacaca gatgtaatga aaataaagat attttattgc ggccgctcca
     1681 agtacctccc gtaccttaat attacttact tatcatggta gcttgggctg gcgtaatagc
     1741 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcaa
     1801 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     1861 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     1921 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     1981 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     2041 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     2101 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     2161 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     2221 cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg
     2281 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2341 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     2401 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     2461 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     2521 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     2581 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     2641 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     2701 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     2761 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     2821 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     2881 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     2941 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3001 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3061 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3121 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3181 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3241 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3301 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3361 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3421 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3481 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     3541 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     3601 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     3661 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     3721 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     3781 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     3841 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     3901 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     3961 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4021 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4081 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4141 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg ctgccggcac
     4201 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     4261 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgacggat
     4321 ccttatcgat tttaccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct
     4381 ccccctgaac ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc
     4441 ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc
     4501 actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgctcgaa
     4561 gcggccggcc gccccgactc tagaattaca cggcgatctt tccgcccttc ttggccttta
     4621 tgaggatctc tctgattttt cttgcgtcga gttttccggt aagacctttc ggtacttcgt
     4681 ccacaaacac aactcctccg cgcaactttt tcgcggttgt tacttgactg gcgacgtaat
     4741 ccacgatctc tttttccgtc atcgtctttc cgtgctccaa aacaacaacg gcggcgggaa
     4801 gttcaccggc gtcatcgtcg ggaagacctg cgacacctgc gtcgaagatg ttggggtgtt
     4861 ggagcaagat ggattccaat tcagcgggag ccacctgata gcctttgtac ttaatcagag
     4921 acttcaggcg gtcaacgatg aagaagtgtt cgtcttcgtc ccagtaagct atgtctccag
     4981 aatgtagcca tccatccttg tcaatcaagg cgttggtcgc ttccggattg tttacataac
     5041 cggacataa
//
