LOCUS       sequence-349874-        5683 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes hORAI1 with HA-tag in its extracellular loop 2 and an 
            N-terminal GFP fusion (GFP-ORAI1-EC-HA)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5683)
  AUTHORS   Wu B, Woo JS, Vila P, Jew M, Leung J, Sun Z, Srikanth S, Gwack Y
  TITLE     NKD2 mediates stimulation-dependent ORAI1 trafficking to augment 
            Ca(2+) entry in T cells.
  JOURNAL   Cell Rep. 2021 Aug 24;36(8):109603. doi: 
            10.1016/j.celrep.2021.109603.
  PUBMED    34433025
REFERENCE   2  (bases 1 to 5683)
  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: "Cell Rep.";
            date: "2021-08-24"; volume: "36(8):109603. doi"; pages: " 
            10.1016/j.celrep.2021.109603"
COMMENT     Sequence Label: pEGFPC1-ORAI1-EC-HA
FEATURES             Location/Qualifiers
     source          1..5683
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(613..615,616..618,619..1329)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(658..679)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(919..938)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1266..1287
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     regulatory      1640..1649
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1978..2004
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    2471..2592
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2508..2527)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2562..2581
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2599..3054)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2736..2757)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2948..2967
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3081..3185
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3187..3544
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3187..3378
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3210..3230)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3395..3530
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3579..4373
                     /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             3579..4373
                     /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(3633..3652)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4243..4262
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4561..4580)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4605..4652
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4981..5569
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5470..5489
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
      661 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
      721 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
      781 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
      841 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
      901 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
      961 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     1021 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     1081 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     1141 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     1201 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     1261 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     1321 ctgtacaagt ccggactcag atctcatccg gagcccgccc cgcccccgag ccgtagcagt
     1381 cccgagcttc ccccaagcgg cggcagcacc accagcggca gccgccggag ccgccgccgc
     1441 agcggggacg gggagccccc gggggccccg ccaccgccgc cgtccgccgt cacctacccg
     1501 gactggatcg gccagagtta ctccgaggtg atgagcctca acgagcactc catgcaggcg
     1561 ctgtcctggc gcaagctcta cttgagccgc gccaagctta aagcctccag ccggacctcg
     1621 gctctgctct ccggcttcgc catggtggca atggtggagg tgcagctgga cgctgaccac
     1681 gactacccac cggggctgct catcgccttc agtgcctgca ccacagtgct ggtggctgtg
     1741 cacctgtttg cgctcatgat cagcacctgc atcctgccca acatcgaggc ggtgagcaac
     1801 gtgcacaatc tcaactcggt caaggagtcc ccccatgagc gcatgcaccg ccacatcgag
     1861 ctggcctggg ccttctccac cgtcattggc acgctgctct tcctagctga ggtggtgctg
     1921 ctctgctggg tcaagttctt gcccctcaag aagcagccag tcgacggcag cggcagctac
     1981 ccatacgatg ttccagatta cgctggcagc ggcagcacgc gtggccagcc aaggcccacc
     2041 agcaagcccc ccgccagtgg cgcagcagcc aacgtcagca ccagcggcat caccccgggc
     2101 caggcagctg ccatcgcctc gaccaccatc atggtgccct tcggcctgat ctttatcgtc
     2161 ttcgccgtcc acttctaccg ctcactggtt agccataaga ccgaccgaca gttccaggag
     2221 ctcaacgagc tggcggagtt tgcccgctta caggaccagc tggaccacag aggggaccac
     2281 cccctgacgc ccggcagcca ctatgcctag gaattctgca gtcgacggta ccgcgggccc
     2341 gggatccacc ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     2401 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     2461 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     2521 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     2581 caatgtatct taacgcgtaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     2641 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     2701 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     2761 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     2821 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     2881 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     2941 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     3001 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg
     3061 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3121 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3181 agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3241 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3301 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3361 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3421 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3481 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3541 caaagatcga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3601 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3661 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3721 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca agacgaggca gcgcggctat
     3781 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3841 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3901 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3961 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     4021 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     4081 ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc
     4141 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     4201 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     4261 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     4321 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     4381 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     4441 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4501 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccctaggg ggaggctaac
     4561 tgaaacacgg aaggagacaa taccggaagg aacccgcgct atgacggcaa taaaaagaca
     4621 gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc ggggttcggt cccagggctg
     4681 gcactctgtc gataccccac cgagacccca ttggggccaa tacgcccgcg tttcttcctt
     4741 ttccccaccc caccccccaa gttcgggtga aggcccaggg ctcgcagcca acgtcggggc
     4801 ggcaggccct gccatagcct caggttactc atatatactt tagattgatt taaaacttca
     4861 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4921 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4981 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     5041 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5101 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     5161 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5221 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5281 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5341 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5401 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5461 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5521 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5581 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5641 gttatcccct gattctgtgg ataaccgtat taccgccatg cat
//
