LOCUS       sequence-320947-        7796 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NFAT-driven ZsGreen-1 reporter gene with mouse CD8 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7796)
  AUTHORS   Landry LG, Mann SE, Anderson AM, Nakayama M
  TITLE     Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation 
            Reporter-based Detection System.
  JOURNAL   Bio Protoc. 2021 Jan 20;11(2):e3883. doi: 10.21769/BioProtoc.3883. 
            eCollection 2021 Jan 20.
  PUBMED    33732772
REFERENCE   2  (bases 1 to 7796)
  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: "Bio 
            Protoc."; date: "2021-01-20"; volume: "11(2):e3883. doi"; pages: " 
            10.21769/BioProtoc.3883. eCollection 2021 Jan 20"
COMMENT     Sequence Label: 8xNFAT-ZsG-mCD8
FEATURES             Location/Qualifiers
     source          1..7796
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        26..405
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        406..609
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     560..580
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             610..785
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    848..1047
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1248..1664
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1594..1616
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1632..1648
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     protein_bind    1698..1727
                     /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    1728..1757
                     /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    1758..1787
                     /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    1788..1817
                     /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    1818..1847
                     /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    1848..1877
                     /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    1878..1907
                     /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    1908..1937
                     /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        1949..1980
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      1994..2003
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2000..2695
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     promoter        2718..3217
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2753..2776)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     3150..3169
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      3229..3238
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3985..4041
                     /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             4764..5189
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     rep_origin      complement(5359..5947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5439..5458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6118..6909,6910..6978))
                     /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     6741..6760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6979..7083)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7151..7169
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7207..7229)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7329..7348
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7542..7564
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7692..7711
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatatcccaa cattaccgcc atgttgacat tgattattga ctagttatta atagtaatca
       61 attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta
      121 aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat
      181 gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg
      241 taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac
      301 gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt
      361 cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg
      421 cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc
      481 attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt
      541 aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      601 agcagagctc aataaaagag cccacaaccc ctcactcggc gcgccagtct tccgatagac
      661 tgcgtcgccc gggtacccgt attcccaata aagcctcttg ctgtttgcat ccgaatcgtg
      721 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccacga cgggggtctt
      781 tcatttgggg gctcgtccgg gatttggaga cccctgccca gggaccaccg acccaccacc
      841 gggaggtaag ctggccagca acttatctgt gtctgtccga ttgtctagtg tctatgtttg
      901 atgttatgcg cctgcgtctg tactagttag ctaactagct ctgtatctgg cggacccgtg
      961 gtggaactga cgagttctga acacccggcc gcaaccctgg gagacgtccc agggactttg
     1021 ggggccgttt ttgtggcccg acctgaggaa gggagtcgat gtggaatccg accccgtcag
     1081 gatatgtggt tctggtagga gacgagaacc taaaacagtt cccgcctccg tctgaatttt
     1141 tgctttcggt ttggaaccga agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc
     1201 tgtgttgtct ctgtctgact gtgtttctgt atttgtctga aaattagggc cagactgtta
     1261 ccactccctt aagtttgacc ttaggtcact ggaaagatgt cgagcggatc gctcacaacc
     1321 agtcggtaga tgtcaagaag agacgttggg ttaccttctg ctctgcagaa tggccaacct
     1381 ttaacgtcgg atggccgcga gacggcacct ttaaccgaga cctcatcacc caggttaaga
     1441 tcaaggtctt ttcacctggc ccgcatggac acccagacca ggtcccctac atcgtgacct
     1501 gggaagcctt ggcttttgac ccccctccct gggtcaagcc ctttgtacac cctaagcctc
     1561 cgcctcctct tcctccatcc gccccgtctc tcccccttga acctcctcgt tcgaccccgc
     1621 ctcgatcctc cctttatcca gccctcactc cttctctagg cgccggaatt gaagatcatc
     1681 atgtctggat ccaagctgga ggaaaaactg tttcatacag aaggcgtgga ggaaaaactg
     1741 tttcatacag aaggcgtgga ggaaaaactg tttcatacag aaggcgtgga ggaaaaactg
     1801 tttcatacag aaggcgtgga ggaaaaactg tttcatacag aaggcgtgga ggaaaaactg
     1861 tttcatacag aaggcgtgga ggaaaaactg tttcatacag aaggcgtgga ggaaaaactg
     1921 tttcatacag aaggcgtcgc ggagactcta gagggtatat aatggaagct cgaattccag
     1981 tccccgagat ctcgccacca tggcccagtc caagcacggc ctgaccaagg agatgaccat
     2041 gaagtaccgc atggagggct gcgtggacgg ccacaagttc gtgatcaccg gcgagggcat
     2101 cggctacccc ttcaagggca agcaggccat caacctgtgc gtggtggagg gcggcccctt
     2161 gcccttcgcc gaggacatct tgtccgccgc cttcatgtac ggcaaccgcg tgttcaccga
     2221 gtacccccag gacatcgtcg actacttcaa gaactcctgc cccgccggct acacctggga
     2281 ccgctccttc ctgttcgagg acggcgccgt gtgcatctgc aacgccgaca tcaccgtgag
     2341 cgtggaggag aactgcatgt accacgagtc caagttctac ggcgtgaact tccccgccga
     2401 cggccccgtg atgaagaaga tgaccgacaa ctgggagccc tcctgcgaga agatcatccc
     2461 cgtgcccaag cagggcatct tgaagggcga cgtgagcatg tacctgctgc tgaaggacgg
     2521 tggccgcttg cgctgccagt tcgacaccgt gtacaaggcc aagtccgtgc cccgcaagat
     2581 gcccgactgg cacttcatcc agcacaagct gacccgcgag gaccgcagcg acgccaagaa
     2641 ccagaagtgg cacctgaccg agcacgccat cgcctccggc tccgccttgc cctgactcga
     2701 ggttaacgaa ttctaccggg taggggaggc gcttttccca aggcagtctg gagcatgcgc
     2761 tttagcagcc ccgctgggca cttggcgcta cacaagtggc ctctggcctc gcacacattc
     2821 cacatccacc ggtaggcgcc aaccggctcc gttctttggt ggccccttcg cgccaccttc
     2881 tactcctccc ctagtcagga agttcccccc cgccccgcag ctcgcgtcgt gcaggacgtg
     2941 acaaatggaa gtagcacgtc tcactagtct cgtgcagatg gacagcaccg ctgagcaatg
     3001 gaagcgggta ggcctttggg gcagcggcca atagcagctt tgctccttcg ctttctgggc
     3061 tcagaggctg ggaaggggtg ggtccggggg cgggctcagg ggcgggctca ggggcggggc
     3121 gggcgcccga aggtcctccg gaggcccggc attctgcacg cttcaaaagc gcacgtctgc
     3181 cgcgctgttc tcctcttcct catctccggg cctttcgacc tgcatcccgc caccatggcc
     3241 tcaccgttga cccgctttct gtcgctgaac ctgctgctgc tgggtgagtc gattatcctg
     3301 gggagtggag aagctaagcc acaggcaccc gaactccgaa tctttccaaa gaaaatggac
     3361 gccgaacttg gccagaaggt ggacctggta tgtgaagtgt tggggtccgt ttcgcaagga
     3421 tgctcttggc tcttccagaa ctccagctcc aaactccccc agcccacctt cgttgtctat
     3481 atggcttcat cccacaacaa gataacgtgg gacgagaagc tgaattcgtc gaaactgttt
     3541 tctgccatga gggacacgaa taataagtac gttctcaccc tgaacaagtt cagcaaggaa
     3601 aacgaaggct actatttctg ctcagtcatc agcaactcgg tgatgtactt cagttctgtc
     3661 gtgccagtcc ttcagaaagt gaactctact actaccaagc cagtgctgcg aactccctca
     3721 cctgtgcacc ctaccgggac atctcagccc cagagaccag aagattgtcg gccccgtggc
     3781 tcagtgaagg ggaccggatt ggacttcgcc tgtgatattt acatctgggc acccttggcc
     3841 ggaatctgcg tggcccttct gctgtccttg atcatcactc tcatctgcta ccacaggagc
     3901 cgaaagcgtg tttgcaaatg tcccaggccg ctagtcagac aggaaggcaa gcccagacct
     3961 tcagagaaaa ttgtgggctc cggagccacg aacttctctc tgttaaagca agcaggagac
     4021 gtggaagaaa accccggtcc catgcagcca tggctctggc tggtcttcag tatgaagctg
     4081 gcagctctct ggagcagctc tgccctcatt cagacccctt cgtccctgct ggttcaaacc
     4141 aaccatacgg caaagatgtc ctgtgaggtt aaaagcatct ctaagttaac aagcatctac
     4201 tggctgcggg agcgccagga ccccaaggac aagtactttg agttcctggc ctcctggagt
     4261 tcttccaaag gagttttgta tggtgaaagt gtggacaaga aaagaaatat aattcttgag
     4321 tcttcagact caagccggcc ctttctcagt atcatgaatg tgaagccaga ggacagtgac
     4381 ttctacttct gcgcgacggt tgggagcccc aagatggtct ttgggacagg gacgaagctg
     4441 actgtggttg atgtccttcc tacaactgcc ccaaccaaga agactaccct gaagatgaag
     4501 aagaagaagc aatgcccgtt cccccaccca gagacccaga agggcctgac atgcagcctt
     4561 accaccctca gcctgctggt agtctgcatc ctgcttctgc tggcattcct cggagtggcc
     4621 gtctactttt actgtgtgcg gaggagagcc cgaattcact tcatgaaaca gtttcacaaa
     4681 tgaaagcttc accagctgaa gcctatagag tacgagccat agataaaata aaagatttta
     4741 tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagct
     4801 taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga gaagttcaga
     4861 tcaaggtcag gaacagatgg aacagggtcg accctagaga accatcagat gtttccaggg
     4921 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct
     4981 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact
     5041 cggggcgcca gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc aataaaccct
     5101 cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
     5161 tgactacccg tcagcggggg tctttcattt gggggctcgt ccgggatcgg gagacccctg
     5221 cccagggacc accgacccac caccgggagg taagctggct gcctcgcgcg tttcggtgat
     5281 gacggtgaaa acctctgaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5341 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5401 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5461 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5521 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5581 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5641 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5701 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5761 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5821 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5881 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5941 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6001 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6061 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6121 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6181 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6241 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6301 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6361 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6421 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6481 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6541 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6601 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6661 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6721 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6781 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6841 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6901 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6961 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7021 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7081 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     7141 aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg
     7201 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     7261 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct
     7321 taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc
     7381 gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa
     7441 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg
     7501 atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa
     7561 aacgacggcg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     7621 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     7681 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     7741 atgccggcca cgatgcgtcc ggcgtagagg cgattagtcc aatttgttaa agacag
//
