LOCUS       sequence-133094-        6196 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mouse NFAT1 (amino acids 4-460) with HA and GFP tags in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6196)
  AUTHORS   Aramburu J, Yaffe MB, Lopez-Rodriguez C, Cantley LC, Hogan PG, Rao A
  TITLE     Affinity-driven peptide selection of an NFAT inhibitor more 
            selective than cyclosporin A.
  JOURNAL   Science. 1999 Sep 24. 285(5436):2129-33.
  PUBMED    10497131
REFERENCE   2  (bases 1 to 6196)
  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: "Science. 
            1999 Sep 24. 285(5436):2129-33."
COMMENT     Sequence Label: HA-NFAT1(4-460)-GFP
FEATURES             Location/Qualifiers
     source          1..6196
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        68..371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        372..575
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     526..546
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             649..738
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAGYPYDVPDYAGSYPYDVPDYA"
     regulatory      2143..2152
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2149..2151,2152..2154,2155..2868)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2149..2151,2152..2154,2155..2865)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2194..2215)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2455..2474)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2802..2823
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2991..3112
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3028..3047)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3082..3101
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3119..3574)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3256..3277)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3468..3487
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3601..3705
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3707..4064
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3707..3898
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3730..3750)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3915..4050
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4099..4893
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4099..4893
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4153..4172)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4763..4782
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5081..5100)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5125..5172
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5501..6089
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5990..6009
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
       61 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      121 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      181 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      241 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      301 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      361 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      421 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      481 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      541 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      601 agcgctaccg gactcagatc tcgagctcaa gcttccacca tgatctttta cccatacgat
      661 gttcctgact atgcgggcta tccctatgac gtcccggact atgcaggatc ctatccatat
      721 gacgttccag attacgctgc tcagtgcggc cgctctagcc cggagccgca gcccgacccc
      781 gatggcgggg acggccccgg ccacgagccc gggggcagtc cccaagacga gctggacttt
      841 tccatcctct tcgattatga ctatctgaac cctatcgaag aagaaccgat cgcacataag
      901 gccatcagct caccctccgg actcgcatac ccggatgatg tcctggacta tggcctcaag
      961 ccatgcaacc cccttgccag tccctctggc gagccccctg gccggttcgg agagccggat
     1021 agtatagggt tccagaactt tctgagcccg gtcaagccag caggggcttc gggcccgagc
     1081 cctcggatcg agatcactcc atcccacgaa ctgatgcagg cagggggggc cctccgtggg
     1141 agagacgccg gcctgtcccc cgagcagccg gccctggccc tggccggcgt ggccgccagc
     1201 ccgaggttca cactgcccgt gcccggctac gagggctacc gcgagccgct ttgcttgagc
     1261 cccgctagca gcggctcctc tgccagcttc atttctgaca ccttctcccc ctacacctcg
     1321 ccctgcgtct cacccaataa cgccgggccc gacgacctgt gtccccagtt tcaaaacatc
     1381 cctgctcatt attcccccag aacctctcca ataatgtcac ctcgaaccag cctcgccgag
     1441 gacagctgcc tgggccgaca ctcgcccgtg ccccgtccgg catcccgctc ctcctcaccc
     1501 ggtgccaagc ggaggcattc gtgcgcagag gctttggttg ctcctctgcc cgcagcctca
     1561 ccccagcgct cccggagccc ctcgccacag ccctcgcctc acgtggcacc gcaggacgac
     1621 agcatccccg ctgggtaccc ccccacggcc ggctctgctg ttctcatgga tgccctcaac
     1681 accctggcca ccgactcgcc ctgcgggatc ccctccaaga tatggaagac cagtcctgac
     1741 ccgacgcctg tgtccaccgc tccgtccaag gctggcctgg cccgccacat ctaccctact
     1801 gtggagttcc tggggccatg tgagcaggag gagaggagga attccgctcc agagtccatc
     1861 ctgctggtac cacctacttg gcccaagcag ttggtgccgg ccattcccat ctgcagcatc
     1921 cctgtgactg catccctccc accactcgag tggccactct ccaatcagtc gggctcctat
     1981 gagctacgga ttgaggtcca acccaagccc catcaccggg cccactatga gacggagggc
     2041 agccgtggcg ctgtcaaagc cccaacagga ggacaccctg tggtgcagct ccacggctac
     2101 atggagaaca agcctctggg gcttcagatc gatccaccgg tcgccaccat ggtgagcaag
     2161 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2221 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     2281 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2341 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
     2401 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
     2461 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     2521 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
     2581 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
     2641 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
     2701 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
     2761 cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2821 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag cggccgcgac
     2881 tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     2941 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     3001 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3061 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa
     3121 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     3181 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     3241 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     3301 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     3361 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     3421 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     3481 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     3541 cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg
     3601 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3661 caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga
     3721 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     3781 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3841 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3901 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     3961 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     4021 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca
     4081 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4141 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     4201 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4261 ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     4321 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4381 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4441 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4501 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4561 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4621 aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4681 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4741 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4801 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4861 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4921 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     4981 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5041 tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa
     5101 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg
     5161 ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac
     5221 cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
     5281 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct
     5341 caggttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5401 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5461 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5521 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5581 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5641 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5701 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5761 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5821 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5881 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     5941 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6001 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6061 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6121 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6181 ataaccgtat taccgc
//
