LOCUS       sequence-356184-        7981 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The ORAI1N223A mutant containing-TEV-pHluorin-TEV sequence in the 
            extracellular loop 2 .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7981)
  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 7981)
  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: MO91-ORAI1N223A-TEV-pHluorin (ORAI1-EC-pHluorin)
FEATURES             Location/Qualifiers
     source          1..7981
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     307..329
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     345..361
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      682..691
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1008..1028
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1041..1757
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MGSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1074..1102)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1697..1717
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1770..1790
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2106..2129
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2153..2179
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2180..2206
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        2304..2803
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2339..2362)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2736..2755
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2824..3423
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2824..2843)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3320..3340
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3523..4037
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4199..4215
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4223..4229,4230..4247,4248..4253))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4268..4289
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4406..4423)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4577..5165)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4657..4676)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5336..6127,6128..6196))
                     /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     5959..5978
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6197..6301)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6369..6387
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6425..6447)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6547..6566
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6711..7090
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        7091..7294
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     7245..7265
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    7534..7875
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             join(7942..7981,1..377)
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
ORIGIN
        1 actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt
       61 gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca
      121 cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg
      181 gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc
      241 cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt
      301 ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca
      361 ctccttctct aggcgccgga attagatcta tgcatccgga gcccgccccg agccgtagca
      421 gtcccgagct tcccccaagc ggcggcagca ccaccagcgg cagccgccgg agccgccgcc
      481 gcagcgggga cggggagccc ccgggggccc cgccaccgcc gccgtccgcc gtcacctacc
      541 cggactggat cggccagagt tactccgagg tgatgagcct caacgagcac tccatgcagg
      601 cgctgtcctg gcgcaagctc tacttgagcc gcgccaagct taaagcctcc agccggacct
      661 cggctctgct ctccggcttc gccatggtgg caatggtgga ggtgcagctg gacgctgacc
      721 acgactaccc accggggctg ctcatcgcct tcagtgcctg caccacagtg ctggtggctg
      781 tgcacctgtt tgcgctcatg atcagcacct gcatcctgcc caacatcgag gcggtgagca
      841 acgtgcacaa tctcaactcg gtcaaggagt ccccccatga gcgcatgcac cgccacatcg
      901 agctggcctg ggccttctcc accgtcattg gcacgctgct cttcctagct gaggtggtgc
      961 tgctctgctg ggtcaagttc ttgcccctca agaagcagcc agtcgacgaa aacctgtatt
     1021 ttcagggcgg agctggagct atgggaagta aaggagaaga acttttcact ggagttgtcc
     1081 caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg
     1141 gtgaaggtga tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac
     1201 tacctgttcc ttggccaaca cttgtcacta ctttaactta tggtgttcaa tgcttttcaa
     1261 gatacccaga tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg
     1321 tacaggaaag aactatattt ttcaaagatg acgggaacta caagacacgt gctgaagtca
     1381 agtttgaagg tgataccctt gttaatagaa tcgagttaaa aggtattgat tttaaagaag
     1441 atggaaacat tcttggacac aaattggaat acaactataa cgatcaccag gtgtacatca
     1501 tggcagacaa acaaaagaat ggaatcaaag ctaacttcaa aattagacac aacattgaag
     1561 atggaggcgt tcaactagca gaccattatc aacaaaatac tccaattggc gatgggcccg
     1621 tccttttacc agacaaccat tacctgttta caacttctac tctttcgaaa gatcccaacg
     1681 aaaagagaga ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca
     1741 tggatgaact atacaaagga gctggagctg aaaacctgta ttttcagggc acgcgtggcc
     1801 agccaaggcc caccagcaag ccccccgcca gtggcgcagc agccgccgtc agcaccagcg
     1861 gcatcacccc gggccaggca gctgccatcg cctcgaccac catcatggtg cccttcggcc
     1921 tgatctttat cgtcttcgcc gtccacttct accgctcact ggttagccat aagaccgacc
     1981 gacagttcca ggagctcaac gagctggcgg agtttgcccg cttacaggac cagctggacc
     2041 acagagggga ccaccccctg acgcccggca gccactatgc cctcgagcac gtacgagcgg
     2101 ccgctgacta caaggacgac gatgacaagt gagaattgtc gaacgggccg cttacccata
     2161 cgatgttccg gattacgctt acccatatga tgttccggat tacgcttaac tagagtgagg
     2221 gtccccacct gggacccttg agagtatcag gtctcccacg tgggagacaa gaaatccctg
     2281 tttaatattt aacgaattct accgggtagg ggaggcgctt ttcccaaggc agtctggagc
     2341 atgcgcttta gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac
     2401 acattccaca tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc
     2461 accttctact cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag
     2521 gacgtgacaa atggaagtag cacgtctcac tagtctcgtg cagatggaca gcaccgctga
     2581 gcaatggaag cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt
     2641 ctgggctcag aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg
     2701 cggggcgggc gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac
     2761 gtctgccgcg ctgttctcct cttcctcatc tccgggcctt tcgacctgca gcccaagctt
     2821 accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     2881 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     2941 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     3001 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     3061 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     3121 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     3181 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     3241 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     3301 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     3361 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     3421 tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc ccatgcatcg
     3481 ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt
     3541 aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaat acataactga
     3601 gaatagagaa gttcagatca aggttaggaa cagagagaca gcagaatatg ggccaaacag
     3661 gatatctgtg gtaagcagtt cctgccccgg ctcagggcca agaacagatg gtccccagat
     3721 gcggtcccgc cctcagcagt ttctagagaa ccatcagatg tttccagggt gccccaagga
     3781 cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc
     3841 gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc ggcgcgccag
     3901 tcctccgata gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc
     3961 atccgacttg tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt
     4021 cagcgggggt ctttcatggg taacagtttc ttgaagttgg agaacaacat tctgagggta
     4081 ggagtcgaat attaagtaat cctgactcaa ttagccactg ttttgaatcc acatactcca
     4141 atactcctga aatagttcat tatggacagc gcagaaagag ctggggagaa ttgtgaaatt
     4201 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4261 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4321 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4381 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4441 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4501 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4561 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4621 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4681 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4741 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4801 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     4861 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     4921 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     4981 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5041 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5101 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5161 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5221 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5281 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5341 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5401 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5461 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5521 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5581 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     5641 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     5701 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     5761 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     5821 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     5881 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     5941 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6001 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6061 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6121 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6181 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6241 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6301 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     6361 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     6421 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     6481 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     6541 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     6601 acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact
     6661 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagcgcgtt gacattgatt
     6721 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     6781 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     6841 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     6901 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     6961 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     7021 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     7081 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
     7141 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
     7201 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
     7261 gcgtgtacgg tgggaggtct atataagcag agctcaataa aagagcccac aacccctcac
     7321 tcggcgcgcc agtcctccga tagactgcgt cgcccgggta cccgtattcc caataaagcc
     7381 tcttgctgtt tgcatccgaa tcgtggactc gctgatcctt gggagggtct cctcagattg
     7441 attgactgcc cacctcgggg gtctttcatt tggaggttcc accgagattt ggagacccct
     7501 gcccagggac caccgacccc cccgccggga ggtaagctgg ccagcggtcg tttcgtgtct
     7561 gtctctgtct ttgtgcgtgt ttgtgccggc atctaatgtt tgcgcctgcg tctgtactag
     7621 ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt ctgaacaccc
     7681 ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg cccgacctga
     7741 ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt aggagacgag
     7801 aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa ccgaagccgc
     7861 gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct gactgtgttt
     7921 ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt gaccttaggt
     7981 c
//
