LOCUS       sequence-306063-        7850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NFAT-driven ZsGreen-1 reporter gene with human CD8ab gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7850)
  AUTHORS   Mann SE, Zhou Z, Landry LG, Anderson AM, Alkanani AK, Fischer J, 
            Peakman M, Mallone R, Campbell K, Michels AW, Nakayama M
  TITLE     Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation 
            Reporter-Based Detection System.
  JOURNAL   Front Immunol. 2020 Apr 9;11:633. doi: 10.3389/fimmu.2020.00633. 
            eCollection 2020.
  PUBMED    32328071
REFERENCE   2  (bases 1 to 7850)
  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: "Front 
            Immunol."; date: "2020-04-9"; volume: "11:633. doi"; pages: " 
            10.3389/fimmu.2020.00633. eCollection 2020"
COMMENT     Sequence Label: 8xNFAT-ZsG-hCD8
FEATURES             Location/Qualifiers
     source          1..7850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             585..760
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    823..1022
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1223..1639
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1569..1591
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1607..1623
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    1673..1702
                     /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    1703..1732
                     /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    1733..1762
                     /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    1763..1792
                     /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    1793..1822
                     /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    1823..1852
                     /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    1853..1882
                     /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    1883..1912
                     /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)"
     promoter        1924..1955
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      1969..1978
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1975..2670
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     promoter        2693..3192
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2728..2751)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3125..3144
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      3204..3213
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3924..3980
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     LTR             4793..5218
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     rep_origin      complement(5388..5976)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5468..5487)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6147..6938,6939..7007))
                     /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     6770..6789
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7008..7112)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7180..7198
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7236..7258)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7358..7377
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7571..7593
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7721..7740
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcaataa aagagcccac
      601 aacccctcac tcggcgcgcc agtcttccga tagactgcgt cgcccgggta cccgtattcc
      661 caataaagcc tcttgctgtt tgcatccgaa tcgtggtctc gctgttcctt gggagggtct
      721 cctctgagtg attgactacc cacgacgggg gtctttcatt tgggggctcg tccgggattt
      781 ggagacccct gcccagggac caccgaccca ccaccgggag gtaagctggc cagcaactta
      841 tctgtgtctg tccgattgtc tagtgtctat gtttgatgtt atgcgcctgc gtctgtacta
      901 gttagctaac tagctctgta tctggcggac ccgtggtgga actgacgagt tctgaacacc
      961 cggccgcaac cctgggagac gtcccaggga ctttgggggc cgtttttgtg gcccgacctg
     1021 aggaagggag tcgatgtgga atccgacccc gtcaggatat gtggttctgg taggagacga
     1081 gaacctaaaa cagttcccgc ctccgtctga atttttgctt tcggtttgga accgaagccg
     1141 cgcgtcttgt ctgctgcagc gctgcagcat cgttctgtgt tgtctctgtc tgactgtgtt
     1201 tctgtatttg tctgaaaatt agggccagac tgttaccact cccttaagtt tgaccttagg
     1261 tcactggaaa gatgtcgagc ggatcgctca caaccagtcg gtagatgtca agaagagacg
     1321 ttgggttacc ttctgctctg cagaatggcc aacctttaac gtcggatggc cgcgagacgg
     1381 cacctttaac cgagacctca tcacccaggt taagatcaag gtcttttcac ctggcccgca
     1441 tggacaccca gaccaggtcc cctacatcgt gacctgggaa gccttggctt ttgacccccc
     1501 tccctgggtc aagccctttg tacaccctaa gcctccgcct cctcttcctc catccgcccc
     1561 gtctctcccc cttgaacctc ctcgttcgac cccgcctcga tcctcccttt atccagccct
     1621 cactccttct ctaggcgccg gaattgaaga tcatcatgtc tggatccaag ctggaggaaa
     1681 aactgtttca tacagaaggc gtggaggaaa aactgtttca tacagaaggc gtggaggaaa
     1741 aactgtttca tacagaaggc gtggaggaaa aactgtttca tacagaaggc gtggaggaaa
     1801 aactgtttca tacagaaggc gtggaggaaa aactgtttca tacagaaggc gtggaggaaa
     1861 aactgtttca tacagaaggc gtggaggaaa aactgtttca tacagaaggc gtcgcggaga
     1921 ctctagaggg tatataatgg aagctcgaat tccagtcccc gagatctcgc caccatggcc
     1981 cagtccaagc acggcctgac caaggagatg accatgaagt accgcatgga gggctgcgtg
     2041 gacggccaca agttcgtgat caccggcgag ggcatcggct accccttcaa gggcaagcag
     2101 gccatcaacc tgtgcgtggt ggagggcggc cccttgccct tcgccgagga catcttgtcc
     2161 gccgccttca tgtacggcaa ccgcgtgttc accgagtacc cccaggacat cgtcgactac
     2221 ttcaagaact cctgccccgc cggctacacc tgggaccgct ccttcctgtt cgaggacggc
     2281 gccgtgtgca tctgcaacgc cgacatcacc gtgagcgtgg aggagaactg catgtaccac
     2341 gagtccaagt tctacggcgt gaacttcccc gccgacggcc ccgtgatgaa gaagatgacc
     2401 gacaactggg agccctcctg cgagaagatc atccccgtgc ccaagcaggg catcttgaag
     2461 ggcgacgtga gcatgtacct gctgctgaag gacggtggcc gcttgcgctg ccagttcgac
     2521 accgtgtaca aggccaagtc cgtgccccgc aagatgcccg actggcactt catccagcac
     2581 aagctgaccc gcgaggaccg cagcgacgcc aagaaccaga agtggcacct gaccgagcac
     2641 gccatcgcct ccggctccgc cttgccctga ctcgaggtta acgaattcta ccgggtaggg
     2701 gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct gggcacttgg
     2761 cgctacacaa gtggcctctg gcctcgcaca cattccacat ccaccggtag gcgccaaccg
     2821 gctccgttct ttggtggccc cttcgcgcca ccttctactc ctcccctagt caggaagttc
     2881 ccccccgccc cgcagctcgc gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact
     2941 agtctcgtgc agatggacag caccgctgag caatggaagc gggtaggcct ttggggcagc
     3001 ggccaatagc agctttgctc cttcgctttc tgggctcaga ggctgggaag gggtgggtcc
     3061 gggggcgggc tcaggggcgg gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc
     3121 ccggcattct gcacgcttca aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct
     3181 ccgggccttt cgacctgcat cccgccacca tggccttacc agtgaccgcc ttgctcctgc
     3241 cgctggcctt gctgctccac gccgccaggc cgagccagtt ccgggtgtcg ccgctggatc
     3301 ggacctggaa cctgggcgag acagtggagc tgaagtgcca ggtgctgctg tccaacccga
     3361 cgtcgggctg ctcgtggctc ttccagccgc gcggcgccgc cgccagtccc accttcctcc
     3421 tatacctctc ccaaaacaag cccaaggcgg ccgaggggct ggacacccag cggttctcgg
     3481 gcaagaggtt gggggacacc ttcgtcctca ccctgagcga cttccgccga gagaacgagg
     3541 gctactattt ctgctcggcc ctgagcaact ccatcatgta cttcagccac ttcgtgccgg
     3601 tcttcctgcc agcgaagccc accacgacgc cagcgccgcg accaccaaca ccggcgccca
     3661 ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg
     3721 cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg cccttggccg
     3781 ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaac cacaggaacc
     3841 gaagacgtgt ttgcaaatgt ccccggcctg tggtcaaatc gggagacaag cccagccttt
     3901 cggcgagata cgtcggctcc ggagccacga acttctctct gttaaagcaa gcaggagacg
     3961 tggaagaaaa ccccggtccc atgcggccgc ggctgtggct cctcctggcc gcgcagctga
     4021 cagttctcca tggcaactca gtcctccagc agacccctgc atacataaag gtgcaaacca
     4081 acaagatggt gatgctgtcc tgcgaagcta aaatctccct cagtaacatg cgcatctact
     4141 ggctgagaca gcgccaggca ccgagcagtg acagtcacca cgagttcctg gccctctggg
     4201 attccgcaaa agggactatc cacggtgaag aggtggaaca ggagaagata gctgtgtttc
     4261 gggatgcaag ccggttcatt ctcaatctca caagcgtgaa gccggaagac agtggcatct
     4321 acttctgcat gatcgtcggg agccccgagc tgaccttcgg gaagggaact cagctgagtg
     4381 tggttgattt ccttcccacc actgcccagc ccaccaagaa gtccaccctc aagaagagag
     4441 tgtgccggtt acccaggcca gagacccaga agggcccact ttgtagcccc atcacccttg
     4501 gcctgctggt ggctggcatc ctggttctgc tggtttccct gggagtggcc atccacctgt
     4561 gctgccggcg gaggagagcc cggcttcgtt tcatgaaaca gcctcaaggg gaaggtatat
     4621 caggaacctt tgtcccccaa tgcctgcatg gatactacag caatactaca acctcacaga
     4681 agctgcttaa cccatggatc ctgaaaacat agaagcttca ccagctgaag cctatagagt
     4741 acgagccata gataaaataa aagattttat ttagtctcca gaaaaagggg ggaatgaaag
     4801 accccacctg taggtttggc aagctagctt aagtaacgcc attttgcaag gcatggaaaa
     4861 atacataact gagaatagag aagttcagat caaggtcagg aacagatgga acagggtcga
     4921 ccctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga ccctgtgcct
     4981 tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct gctccccgag
     5041 ctcaataaaa gagcccacaa cccctcactc ggggcgccag tcctccgatt gactgagtcg
     5101 cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg tggtctcgct
     5161 gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt ctttcatttg
     5221 ggggctcgtc cgggatcggg agacccctgc ccagggacca ccgacccacc accgggaggt
     5281 aagctggctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacat gtgagcaaaa
     5341 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5401 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5461 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5521 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5581 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5641 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5701 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5761 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5821 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5881 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     5941 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6001 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6061 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6121 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6181 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6241 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6301 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6361 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6421 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6481 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6541 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6601 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6661 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6721 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6781 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6841 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6901 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     6961 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7021 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7081 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7141 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     7201 tttcgtctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
     7261 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
     7321 gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca gattgtactg
     7381 agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     7441 aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     7501 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     7561 ccagggtttt cccagtcacg acgttgtaaa acgacggcgc aaggaatggt gcatgcaagg
     7621 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     7681 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     7741 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagaggc
     7801 gattagtcca atttgttaaa gacaggatat cccaacatta ccgccatgtt
//
