LOCUS       sequence-319738-        7724 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NFAT-driven ZsGreen-1 reporter gene with human CD4 gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7724)
  AUTHORS   Landry LG, Anderson AM, Russ HA, Yu L, Kent SC, Atkinson MA, Mathews
            CE, Michels AW, Nakayama M
  TITLE     Proinsulin-Reactive CD4 T Cells in the Islets of Type 1 Diabetes 
            Organ Donors.
  JOURNAL   Front Endocrinol (Lausanne). 2021 Mar 25;12:622647. doi: 
            10.3389/fendo.2021.622647. eCollection 2021.
  PUBMED    33841327
REFERENCE   2  (bases 1 to 7724)
  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 
            Endocrinol (Lausanne)."; date: "2021-03-25"; volume: "12:622647. 
            doi"; pages: " 10.3389/fendo.2021.622647. eCollection 2021"
COMMENT     Sequence Label: 8xNFAT-ZsG-hCD4
FEATURES             Location/Qualifiers
     source          1..7724
                     /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"
     CDS             join(3235..3309,3310..4611)
                     /codon_start=1
                     /product="human CD4 cell surface glycoprotein"
                     /label=CD4
                     /translation="MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQ
                     KKSIQFHWKNSNQIKILGNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDS
                     DTYICEVEDQKEEVQLLVFGLTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNI
                     QGGKTLSVSQLELQDSGTWTCTVLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFS
                     FPLAFTVEKLTGSGELWWQAERASSSKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLH
                     LTLPQALPQYAGSGNLTLALEAKTGKLHQEVNLVVMRATQLQKNLTCEVWGPTSPKLML
                     SLKLENKEAKVSKREKAVWVLNPEAGMWQCLLSDSGQVLLESNIKVLPTWSTPVQPMAL
                     IVLGGVAGLLLFIGLGIFFCVRCRHRRRQAERMSQIKRLLSEKKTCQCPHRFQKTCSPI
                     "
     LTR             4692..5117
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     rep_origin      complement(5287..5875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5367..5386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6046..6837,6838..6906))
                     /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     6669..6688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6907..7011)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7079..7097
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7135..7157)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7257..7276
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7470..7492
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7620..7639
                     /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 caccatgaac
     3241 cggggagtcc cttttaggca cttgcttctg gtgctgcaac tggcgctcct cccagcagcc
     3301 actcagggaa agaaagtggt gctgggcaaa aaaggggata cagtggaact gacctgtaca
     3361 gcttcccaga agaagagcat acaattccac tggaaaaact ccaaccagat aaagattctg
     3421 ggaaatcagg gctccttctt aactaaaggt ccatccaagc tgaatgatcg cgctgactca
     3481 agaagaagcc tttgggacca aggaaacttt cccctgatca tcaagaatct taagatagaa
     3541 gactcagata cttacatctg tgaagtggag gaccagaagg aggaggtgca attgctagtg
     3601 ttcggattga ctgccaactc tgacacccac ctgcttcagg ggcagagcct gaccctgacc
     3661 ttggagagcc cccctggtag tagcccctca gtgcaatgta ggagtccaag gggtaaaaac
     3721 atacaggggg ggaagaccct ctccgtgtct cagctggagc tccaggatag tggcacctgg
     3781 acatgcactg tcttgcagaa ccagaagaag gtggagttca aaatagacat cgtggtgcta
     3841 gctttccaga aggcctccag catagtctat aagaaagagg gggaacaggt ggagttctcc
     3901 ttcccactcg cctttacagt tgaaaagctg acgggcagtg gcgagctgtg gtggcaggcg
     3961 gagagggctt cctcctccaa gtcttggatc acctttgacc tgaagaacaa ggaagtgtct
     4021 gtaaaacggg ttacccagga ccctaagctc cagatgggca agaagctccc gctccacctc
     4081 accctgcccc aggccttgcc tcagtatgct ggctctggaa acctcaccct ggcccttgaa
     4141 gcgaaaacag gaaagttgca tcaggaagtg aacctggtgg tgatgagagc cactcagctc
     4201 cagaaaaatt tgacctgtga ggtgtgggga cccacctccc ctaagctgat gctgagcttg
     4261 aaactggaga acaaggaggc aaaggtctcg aagcgggaga aggcggtgtg ggtgctgaac
     4321 cctgaggcgg ggatgtggca gtgtctgctg agtgactcgg gacaggtcct gctggaatcc
     4381 aacatcaagg ttctgcccac atggtccacc ccggtgcagc caatggccct gattgtgctg
     4441 gggggcgtcg ccggcctcct gcttttcatt gggctaggca tcttcttctg tgtcaggtgc
     4501 cggcaccgaa ggcgccaagc agagcggatg tctcagatca agagactcct cagtgagaag
     4561 aagacctgcc agtgccctca ccggtttcag aagacatgta gccccatttg aaagcttcac
     4621 cagctgaagc ctatagagta cgagccatag ataaaataaa agattttatt tagtctccag
     4681 aaaaaggggg gaatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca
     4741 ttttgcaagg catggaaaaa tacataactg agaatagaga agttcagatc aaggtcagga
     4801 acagatggaa cagggtcgac cctagagaac catcagatgt ttccagggtg ccccaaggac
     4861 ctgaaatgac cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg
     4921 cgcgcttctg ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt
     4981 cctccgattg actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca
     5041 tccgacttgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc
     5101 agcgggggtc tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac
     5161 cgacccacca ccgggaggta agctggctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     5221 ctctgacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5281 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5341 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5401 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5461 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5521 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5581 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5641 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5701 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5761 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5821 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5881 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5941 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     6001 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6061 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6121 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6181 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6241 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6301 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6361 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6421 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6481 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6541 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6601 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6661 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6721 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6781 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6841 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6901 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6961 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     7021 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     7081 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     7141 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     7201 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     7261 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     7321 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
     7381 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     7441 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggcgca
     7501 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     7561 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     7621 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     7681 atgcgtccgg cgtagaggcg attagtccaa tttgttaaag acag
//
