LOCUS       sequence-489484-        5099 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  Expression of a DsRed-Express2 reporter under an NFAT-responsive 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5099)
  AUTHORS   Edelstein HI, Cosio A, Ezekiel ML, Corcoran WK, Morris AH, Leonard 
            JN
  TITLE     Conversion of natural cytokine receptors into orthogonal synthetic 
            biosensors.
  JOURNAL   Nat Chem Biol. 2025 Aug 22:10.1038/s41589-025-01986-1. doi: 
            10.1038/s41589-025-01986-1.
  PUBMED    40846994
REFERENCE   2  (bases 1 to 5099)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2025 Aug 22:10.1038/s41589-025-01986-1. doi: 
            10.1038/s41589-025-01986-1."
COMMENT     Sequence Label: L4552 x6NFATRE (NFAT) Ben DsRed-Express2 in TUPV1
FEATURES             Location/Qualifiers
     source          1..5099
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    68..97
                     /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    98..127
                     /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    128..157
                     /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    158..187
                     /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    188..217
                     /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)"
     regulatory      278..287
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             284..961
                     /codon_start=1
                     /product="noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2008)"
                     /label=DsRed-Express2
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTFIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERLYPRDGVLKGEIHK
                     ALKLKGGGHYLVEFKSIYMAKKPVKLPGYYYVDSKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     complement(464..484)
                     /label=DsRed1-N
                     /note="DsRed1, reverse primer"
     primer_bind     876..899
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1006..1023)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1012..1236
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2005..2433
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2092..2111)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2302..2323
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2456..2652
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2503..2638
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2565..2584
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     polyA_signal    2772..2893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2809..2828)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2863..2882
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2942..2958)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2942..2958)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2955..2977)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2966..2982
                     /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(2990..2996,2997..3014,3015..3020))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3035..3056
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3173..3190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3344..3932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3424..3443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4103..4894,4895..4963))
                     /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     4726..4745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4964..5068)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatcgaag acggtgccaa ggtcttgaca aacgagtgct agtacaagat
       61 ctgcgaggga ggaaaaactg tttcatacag aaggcgtgga ggaaaaactg tttcatacag
      121 aaggcgtgga ggaaaaactg tttcatacag aaggcgtgga ggaaaaactg tttcatacag
      181 aaggcgtgga ggaaaaactg tttcatacag aaggcgtacg ctctctagag ggtatataat
      241 gggggccact agtctactac cagagctcat cgctagcgcc accatggata gcactgagaa
      301 cgtcatcaag cccttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga
      361 gttcgagatc gagggcgagg gcgagggcaa gccctacgag ggcacccaga ccgccaagct
      421 ccaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcca
      481 gtacggctcc aaggtgtacg tgaagcaccc cgccgacatc cccgactaca agaagctgtc
      541 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac
      601 cgtgacccag gactcctccc tccaggacgg caccttcatc taccacgtga agttcatcgg
      661 cgtgaacttc ccctccgacg gccccgtaat gcagaagaaa actctgggct gggagccctc
      721 caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccaca aggcgctgaa
      781 gctgaagggc ggcggccact acctggtgga gttcaagtca atctacatgg ccaagaagcc
      841 cgtgaagctg cccggctact actacgtgga ctccaagctg gacatcacct cccacaacga
      901 ggactacacc gtggtggagc agtacgagcg cgccgaggcc cgccaccacc tgttccagta
      961 ggcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     1021 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     1081 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     1141 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagtca
     1201 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcaggt cgagggtaca
     1261 tgcgccttac tccttgtgag cgcatcgcga taagctctag agggacagcc cccccccaaa
     1321 gcccccaggg atgtaattac gtccctcccc cgctaggggg cagcagcgag ccgcccgggg
     1381 ctccgctccg gtccggcgct ccccccgcat ccccgagccg gcagcgtgcg gggacagccc
     1441 gggcacgggg aaggtggcac gggatcgctt tcctctgaac gcttctcgct gctctttgag
     1501 cctgcagaca cctgggggga tacggggaaa aagctttagg ctgaaagaga gatttagaat
     1561 gacagaatca tagaacggcc tgcgtacgga tggggctcta gctctagagc ttgatatcga
     1621 attcctgcag ccccgctaga gggacagccc ccccccaaag cccccaggga tgtaattacg
     1681 tccctccccc gctagggggc agcagcgagc cgcccggggc tccgctccgg tccggcgctc
     1741 cccccgcatc cccgagccgg cagcgtgcgg ggacagcccg ggcacgggga aggtggcacg
     1801 ggatcgcttt cctctgaacg cttctcgctg ctctttgagc ctgcagacac ctggggggat
     1861 acggggaaaa agctttaggc tgaaagagag atttagaatg acagaatcat agaacggcct
     1921 gggcgcgcct ggtggcgaac gatcaacaga cgggagaggg caaaagtctt cgctgacagc
     1981 gaccggactc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2041 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2101 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2161 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2221 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     2281 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     2341 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     2401 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     2461 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     2521 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     2581 gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     2641 ggcttttgca aaaagctccc gggagcttgt atatccattt tcggaggttg ggcttcggaa
     2701 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     2761 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     2821 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     2881 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     2941 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3001 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3061 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3121 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3181 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3241 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3301 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     3361 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     3421 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     3481 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     3541 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     3601 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     3661 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3721 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3781 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3841 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3901 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3961 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4021 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4081 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4141 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4201 gggagggctt accatctggc cccagtgctg caatgatacc gcgagatcca cgctcaccgg
     4261 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4321 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     4381 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     4441 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     4501 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     4561 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     4621 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     4681 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4741 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4801 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4861 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4921 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4981 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5041 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
