LOCUS       sequence-218681-        5627 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ORAI1 channel with C-terminal YFP, carrying the double mutation 
            L273D-L276D in the STIM1 binding domain, leading to a channel gating
            deficiency..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5627)
  AUTHORS   Bulla M, Gyimesi G, Kim JH, Bhardwaj R, Hediger MA, Frieden M, 
            Demaurex N
  TITLE     ORAI1 channel gating and selectivity is differentially altered by 
            natural mutations in the first or third transmembrane domain.
  JOURNAL   J Physiol. 2018 Nov 1. doi: 10.1113/JP277079.
  PUBMED    30382595
REFERENCE   2  (bases 1 to 5627)
  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: "J Physiol. 
            2018 Nov 1. doi: 10.1113/JP277079."
COMMENT     Sequence Label: ORAI1-YFP L273D-L276D
FEATURES             Location/Qualifiers
     source          1..5627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        54..357
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        358..561
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     512..532
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      910..919
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      1560..1569
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1566..1568,1569..1571,1572..2285)
                     /codon_start=1
                     /product="enhanced YFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1611..1632)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1872..1891)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2219..2240
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2408..2529
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2445..2464)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2499..2518
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2536..2991)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2673..2694)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2885..2904
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3018..3122
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3124..3481
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3124..3315
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3147..3167)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3332..3467
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3516..4310
                     /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
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQLIAFYRLLDEFF"
     primer_bind     complement(3570..3589)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4180..4199
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4498..4517)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4542..4589
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4918..5506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5407..5426
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
       61 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      121 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      181 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      241 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      301 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      361 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      421 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      481 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      541 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact
      601 cagatctcga gatgcatccg gagcccgccc cgcccccgag ccgtagcagt cccgagcttc
      661 ccccaagcgg cggcagcacc accagcggca gccgccggag ccgccgccgc agcggggacg
      721 gggagccccc gggggccccg ccaccgccgc cgtccgccgt cacctacccg gactggatcg
      781 gccagagtta ctccgaggtg atgagcctca acgagcactc catgcaggcg ctgtcctggc
      841 gcaagctcta cttgagccgc gccaagctta aagcctccag ccggacctcg gctctgctct
      901 ccggcttcgc catggtggca atggtggagg tgcagctgga cgctgaccac gactacccac
      961 cggggctgct catcgccttc agtgcctgca ccacagtgct ggtggctgtg cacctgtttg
     1021 cgctcatgat cagcacctgc atcctgccca acatcgaggc ggtgagcaac gtgcacaatc
     1081 tcaactcggt caaggagtcc ccccatgagc gcatgcaccg ccacatcgag ctggcctggg
     1141 ccttctccac cgtcattggc acgctgctct tcctagctga ggtggtgctg ctctgctggg
     1201 tcaagttctt gcccctcaag aagcagccag gccagccaag gcccaccagc aagccccccg
     1261 ccagtggcgc agcagccaac gtcagcacca gcggcatcac cccgggccag gcagctgcca
     1321 tcgcctcgac caccatcatg gtgcccttcg gcctgatctt tatcgtcttc gccgtccact
     1381 tctaccgctc actggttagc cataagaccg accgacagtt ccaggaggac aacgaggacg
     1441 cggagtttgc ccgcttacag gaccagctgg accacagagg ggaccacccc ctgacgcccg
     1501 gcagccacta tgcccgaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggtcg
     1561 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1621 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1681 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1741 ccaccctcgt gaccaccttc ggctacggcc tgcagtgctt cgcccgctac cccgaccaca
     1801 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1861 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1921 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1981 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     2041 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     2101 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     2161 accactacct gagctaccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     2221 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     2281 agtaaagcgg ccgcgactct agatcataat cagccatacc acatttgtag aggttttact
     2341 tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt
     2401 tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     2461 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     2521 tgtatcttaa ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     2581 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     2641 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2701 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2761 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2821 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2881 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2941 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca
     3001 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3061 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3121 gtcctgaggc ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     3181 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     3241 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     3301 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     3361 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     3421 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     3481 agatcgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg
     3541 caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa
     3601 tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg
     3661 tcaagaccga cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt
     3721 ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa
     3781 gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc
     3841 ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg
     3901 ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg
     3961 aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg
     4021 aactgttcgc caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg
     4081 gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact
     4141 gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg
     4201 ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc
     4261 ccgattcgca gctcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct
     4321 ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac
     4381 cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat
     4441 cctccagcgc ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga
     4501 aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa
     4561 taaaacgcac ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca
     4621 ctctgtcgat accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc
     4681 cccaccccac cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc
     4741 aggccctgcc atagcctcag gttactcata tatactttag attgatttaa aacttcattt
     4801 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     4861 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     4921 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     4981 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5041 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5101 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5161 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5221 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5281 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     5341 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     5401 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     5461 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     5521 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     5581 atcccctgat tctgtggata accgtattac cgccatgcat tagttat
//
