LOCUS       sequence-492862-        6724 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bioluminescent endolysosomal Ca2+ indicator lower Ca2+ affinity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6724)
  AUTHORS   Calvo B, Torres-Vidal P, Delrio-Lorenzo A, Rodriguez C, Aulestia FJ,
            Rojo-Ruiz J, Callejo B, McVeigh BM, Keller M, Grimm C, Oorschot V, 
            Moiseenkova-Bell V, Yule DI, Garcia-Sancho J, Patel S, Alonso MT
  TITLE     Direct measurements of luminal Ca2+ with endo-lysosomal GFP-aequorin
            reveal functional IP3 receptors.
  JOURNAL   J Cell Biol. 2025 Dec 1;224(12):e202410094. doi: 
            10.1083/jcb.202410094. Epub 2025 Nov 7.
  PUBMED    41201441
REFERENCE   2  (bases 1 to 6724)
  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 Cell 
            Biol."; date: "2025-12-1"; volume: "224(12):e202410094. doi"; pages:
            " 10.1083/jcb.202410094. Epub 2025 Nov 7"
COMMENT     Sequence Label: pHSV-ELGA1
FEATURES             Location/Qualifiers
     source          1..6724
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     154..171
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    186..326
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(252..271)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     412..434
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     regulatory      1506..1515
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2196..2198,2199..2909)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="ASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKA
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(2226..2254)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2849..2869
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     polyA_signal    3854..3988
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3891..3910)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3945..3964
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4914..4932)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5000..5104
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5105..5173,5174..5965)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5323..5342)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6136..6724
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6625..6644
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
       61 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      121 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      181 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
      241 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat
      301 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct
      361 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
      421 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagcat gaattattcc
      481 attatgcacg accccgcccc gacgccggca cgccgggggc ccgtggccgc ggcccgttgg
      541 tcgaaccccc ggccccgccc atccgcgcca tctgccatgg gcggggcgcg agggcgggtg
      601 ggtccgcgcc ccgccccgca tggcatctca ttaccgcccg atccggcggt ttccgcttcc
      661 gttccgcatg ctaacgagga acaggcaggg ggcggggccc gggccccgac ttcccggttc
      721 ggcggtaatg agatacgagc cccgcgcgcc cgttggccgt ccccgggccc cccggtcccg
      781 cccgccggac gccgggacca acgggacggc gggcggccca agggccgccc gccttgccgc
      841 ccccccattg gccggcgggc gggaccgccc caagggggcg gggccgccgg gtaaaagaag
      901 tgagaacgcg aagcgttcgc acttcgtccc aatatatata tattattagg gcgaagtgcg
      961 agcactggcg ccgtgcccga ctccgcgccg gccccggggg cgggcccggg cggcgggggg
     1021 cgggtctctc cggcgcacat aaaggcccgg cgcgaccgac gcccgcagac ggcgccggcc
     1081 acgaacgacg ggagcggctg cggagcacgc ggaccgggag cgggagtcgc agagggccgt
     1141 cggagcggac ggcgtcggca tcgcgacgcc ccggctcggg atcgggatcg catcggaaag
     1201 ggacacgcgg acgcgggggg gaaagacccg cccaccccac ccacgaaaca caggggacgc
     1261 accccggggg cctccgacga cagaaaccca ccggtccgcc ttttttgcac gggtaagcac
     1321 cttgggtggg cggaggaggg ggggacgcgg gggcggagga ggggggacgc gggggcggag
     1381 gaggggggac gcgggggcgg aggagggggg acgcgggggc ggaggagggg ggacgcgggg
     1441 gcggaggagg gggctcaccc gcgttcgtgc cttcccgcag gaggaacgtc ctcgtcgata
     1501 agcttgccac catggcgatt ctttttgctg ttgttgccag ggggaccact atccttgcca
     1561 aacatgcttg gtgtggagga aacttcctgg aggtgacaga gcagattctg gctaagatac
     1621 cttctgaaaa taacaaacta acgtactcac atggcaatta tttgtttcat tacatctgcc
     1681 aagacaggat tgtatatctt tgtatcactg atgatgattt tgaacgttcc cgagccttta
     1741 attttctgaa tgagataaag aagaggttcc agactactta cggttcaaga gcacagacag
     1801 cacttccata tgccatgaat agcgagttct caagtgtctt agctgcacag ctgaagcatc
     1861 actctgagaa taagggccta gacaaagtga tggagactca agcccaagtg gatgaactga
     1921 aaggaatcat ggtcagaaac atagatctgg tagctcagcg aggagaaaga ttggaattat
     1981 tgattgacaa aacagaaaat cttgtggatt cttctgtcac cttcaaagct accagcagaa
     2041 atcttgctcg agccatgtgt atgaagaacc tcaagctcac tattatcatc atcatcgtat
     2101 caattgtgtt catctatatc attgtttcac ctctctgtgg tggatttaca tggccaagct
     2161 gtgtgaagaa aaagctttat gatgttcctg attatgctag caaaggagaa gaacttttca
     2221 ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg
     2281 tcagtggaga gggtgaaggt gatgctacat acggaaaact tacccttaaa tttatttgca
     2341 ctactggaaa actacctgtt ccatggccaa cacttgtcac tactttctct tatggtgttc
     2401 aatgcttttc ccgttatccg gatcatatga aacggcatga ctttttcaag agtgccatgc
     2461 ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga tgacgggaac tacaagacgc
     2521 gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg tatcgagtta aaaggtattg
     2581 attttaaaga agatggaaac attctcggac acaaactcga gtacaactat aactcacaca
     2641 atgtatacat cacggcagac aaacaaaaga atggaatcaa agctaacttc aaaattcgcc
     2701 acaacattga agatggatcc gttcaactag cagaccatta tcaacaaaat actccaattg
     2761 gcgatggccc tgtcctttta ccagacaacc attacctgtc gacacaatct gccctttcga
     2821 aagatcccaa cgaaaagcgt gaccacatgg tccttcttga gtttgtaact gctgctggga
     2881 ttacacatgg catggatgag ctctacaaac cgggtacagc gaccccagca accacgccga
     2941 ccactgcacc gaccgcgggt accggtaaac ttacatcaga cttcgacaac ccaagatgga
     3001 ttggacgaca caagcatatg ttcaatttcc ttgatgtcaa ccacaatgga aaaatctctc
     3061 ttgacgagat ggtctacaag gcatctgata ttgtcatcaa taaccttgga gcaacacctg
     3121 agcaagccaa acgacacaaa gatgctgtag aagccttctt cggaggagct ggaatgaaat
     3181 atggtgtgga aactgattgg cctgcatata ttgaaggatg gaaaaaattg gctactgatg
     3241 aattggagaa atacgccaaa aacgaaccaa cgctcatccg tatatggggt gatgctttgt
     3301 ttgacatcgt tgccaaagcc caaaatggag ccattacact ggatgaatgg aaagcataca
     3361 ccaaagctgc tggtatcatc caatcatcag aagattgcga ggaaacattc agagtgtgcg
     3421 atattgatga aagtggacaa ctcgatgttg ccgagatgac aagacaacat ttaggatttt
     3481 ggtacaccat ggatcctgct tgcgaaaagc tctacggtgg agctgtcccc taagaattcc
     3541 agctgagcgc cggtcgctac cattaccagt tggtctggtg tcaaaaataa taataaccgg
     3601 gcaggccatg tctgcccgta tttcgcgtaa ggaaatccat tatgtactat ttaaaaaaca
     3661 caaacttttg gatgttcggt ttattctttt tcttttactt ttttatcatg ggagcctact
     3721 tcccgttttt cccgatttgg ctacatgaca tcaaccatat cagcaaaagt gatacgggta
     3781 ttatttttgc cgctatttct ctgttctcgc tattattcca accgctgttt ggtctgcttt
     3841 ctgacaaact cggaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     3901 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     3961 catcaatgta tcttatcatg tctggatctc tgacctgaga ttggcggcac tgaggtagag
     4021 atgcccgaac ccccccgagg gggggggggg gggggggggg gggggggggg gggggggggg
     4081 gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
     4141 gggggggggg gggggggggg gggggggcgg gggaggggcg gtggtgtgta gcaggagcgg
     4201 tgtgttgcgc cggggtacgt ctggaggagc gggaggtgcg cggtgacgtg tggatgagga
     4261 acaggagttg ttgcgcggtg agttgtcgct gtgagttgtg ttgttgggca ggtgtggtgg
     4321 atgacgtgac gtgtgacgtg cggagtgcgc cgtgctctgt tggtttcacc tgtggcagcc
     4381 cgggcccccc gcgggcgcgc gcgcgcgcaa aaaaggcggg cggcggtccg ggcggcgtgc
     4441 gcgcgcgcgg cgggcgtggg gggcggggcc gcgggagcgg ggggaggagc ggggggagga
     4501 gcggggggag gagcgggggg aggagcgggg ggaggagcgg ggggaggagc ggggggagga
     4561 gcggggggag gagcgggggg aggagcgggg ggaggagcgg ccagaccccg aaaacgggcc
     4621 ccccccaaaa cacacccccc gggggtcgcg cgcggccctt taaagcggcg gcggcgggca
     4681 gcccgggccc cccgcggccg agactagcga gttagacagg caagcactac tcgcctctgc
     4741 acgcacatgc ttgcctgtca aactctacca ccccggcacg ctctctgtct ccatggcccg
     4801 ccgccgccgc catcgcggcc cccgccgccc ccggccgccc gggcccacgg gcgccgtccc
     4861 aaccgcacag tcccaggtaa cgactctagg aattaattct tgaagacgaa agggcctcgt
     4921 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     4981 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     5041 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     5101 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     5161 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     5221 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     5281 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     5341 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     5401 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     5461 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     5521 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     5581 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     5641 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     5701 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     5761 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     5821 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     5881 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     5941 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     6001 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     6061 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     6121 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     6181 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     6241 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     6301 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     6361 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     6421 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     6481 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     6541 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     6601 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     6661 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     6721 gaaa
//
