LOCUS       sequence-413485-        6987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fusion of Ca2+ reporter and lysosomal Ca2+-permeable channel, with 
            ER-export signal.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6987)
  AUTHORS   Davis LC, Morgan AJ, Galione A
  TITLE     NAADP-regulated two-pore channels drive phagocytosis through 
            endo-lysosomal Ca(2+) nanodomains, calcineurin and dynamin.
  JOURNAL   EMBO J. 2020 Jul 15;39(14):e104058. doi: 10.15252/embj.2019104058. 
            Epub 2020 Jun 8.
  PUBMED    32510172
REFERENCE   2  (bases 1 to 6987)
  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: "EMBO J."; 
            date: "2020-07-15"; volume: "39(14):e104058. doi"; pages: " 
            10.15252/embj.2019104058. Epub 2020 Jun 8"
COMMENT     Sequence Label: TRPML1-G-GECO1.2-ERES
FEATURES             Location/Qualifiers
     source          1..6987
                     /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"
     CDS             2391..2447
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     2660..2681
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3189..3629
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYIGAAEL
                     RHVMTNLGEKLTDEEVDEMIRVADIDGDGQVNYEEFVQMMTAK"
     polyA_signal    3775..3896
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3812..3831)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3866..3885
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3903..4358)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4040..4061)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4252..4271
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4385..4489
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4491..4848
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4491..4682
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4514..4534)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4699..4834
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4883..5677
                     /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             4883..5677
                     /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(4937..4956)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5547..5566
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5865..5884)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5909..5956
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6285..6873
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6774..6793
                     /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 gctagcacca
      601 tgacagcccc ggcgggtccg cgcggctcag agaccgagcg gcttctgacc cccaaccccg
      661 ggtatgggac ccaggcgggg ccttcaccgg cccctccgac acccccagaa gaggaagacc
      721 ttcgccgtcg tctcaaatac tttttcatga gtccctgcga caagtttcga gccaagggcc
      781 gcaagccctg caagctgatg ctgcaagtgg tcaagatcct ggtggtcacg gtgcagctca
      841 tcctgtttgg gctcagtaat cagctggctg tgacattccg ggaagagaac accatcgcct
      901 tccgacacct cttcctgctg ggctactcgg acggagcgga tgacaccttc gcagcctaca
      961 cgcgggagca gctgtaccag gccatcttcc atgctgtgga ccagtacctg gcgttgcctg
     1021 acgtgtcact gggccggtat gcgtatgtcc gtggtggggg tgacccttgg accaatggct
     1081 cagggcttgc tctctgccag cggtactacc accgaggcca cgtggacccg gccaacgaca
     1141 catttgacat tgatccgatg gtggttactg actgcatcca ggtggatccc cccgagcggc
     1201 cccctccgcc ccccagcgac gatctcaccc tcttggaaag cagctccagt tacaagaacc
     1261 tcacgctcaa attccacaag ctggtcaatg tcaccatcca cttccggctg aagaccatta
     1321 acctccagag cctcatcaat aatgagatcc cggactgcta taccttcagc gtcctgatca
     1381 cgtttgacaa caaagcacac agtgggcgga tccccatcag cctggagacc caggcccaca
     1441 tccaggagtg taagcacccc agtgtcttcc agcacggaga caacagcttc cggctcctgt
     1501 ttgacgtggt ggtcatcctc acctgctccc tgtccttcct cctctgcgcc cgctcactcc
     1561 ttcgaggctt cctgctgcag aatgagtttg tggggttcat gtggcggcag cggggacggg
     1621 tcatcagcct gtgggagcgg ctggaatttg tcaatggctg gtacatcctg ctcgtcacca
     1681 gcgatgtgct caccatctcg ggcaccatca tgaagatcgg catcgaggcc aagaacttgg
     1741 cgagctacga cgtctgcagc atcctcctgg gcacctcgac gctgctggtg tgggtgggcg
     1801 tgatccgcta cctgaccttc ttccacaact acaatatcct catcgccaca ctgcgggtgg
     1861 ccctgcccag cgtcatgcgc ttctgctgct gcgtggctgt catctacctg ggctactgct
     1921 tctgtggctg gatcgtgctg gggccctatc atgtgaagtt ccgctcactc tccatggtgt
     1981 ctgagtgcct gttctcgctc atcaatgggg acgacatgtt tgtgacgttc gccgccatgc
     2041 aggcgcagca gggccgcagc agcctggtgt ggctcttctc ccagctctac ctttactcct
     2101 tcatcagcct cttcatctac atggtgctca gcctcttcat cgcgctcatc accggcgcct
     2161 acgacaccat caagcatccc ggcggcgcag gcgcagagga gagcgagctg caggcctaca
     2221 tcgcacagtg ccaggacagc cccacctccg gcaagttccg ccgcgggagc ggctcggcct
     2281 gcagccttct ctgctgctgc ggaagggacc cctcggagga gcattcgctg ctggtgaatc
     2341 agtcgacggt accgcgggcc cgggatccac cggtcatggt cgactcatca cgtcgtaagt
     2401 ggaataagac aggtcacgca gtcagagcta taggtcggct gagctcactc gagaacgtct
     2461 atatcaaggc cgacgagcag aagaacggca tcaaggcgta cttcaagatc cgccacaaca
     2521 tcgagggcgg cggcgtgcag ctcgcctacc actaccagca gaacaccccc atcggcgacg
     2581 gccccgtgct gctgcccgac aaccactacc tgagcgtgca gtccatgctt tcgaaagacc
     2641 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2701 tcggcatgga cgagctgtac aagggcggta ccggagggag cgaatccatg gtgagcaagg
     2761 gcgaggagct gttcaccggg gtggtgccca tccaggtcga gctggacggc gacgtaaacg
     2821 gccacaagtt cagcgtgtcc ggcgagggtg agggcgatgc cacctacggc aagctgaccc
     2881 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     2941 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     3001 tcaagtccgc catgcccgaa ggctacatcc aggagcgcac catcttcttc aagggcgacg
     3061 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     3121 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     3181 acacgcgtga ccaactgact gaagagcaga tcgcagaatt taaagaggct ttctccctat
     3241 ttgacaagga cggggatggg acgataacaa ccaaggagct ggggacggtg atgcggtctc
     3301 tggggcagaa ccccacagaa gcagagctgc aggacatgat caatgaagta gatgccgacg
     3361 gtgacggcac aatcgacttc cctgagttcc tgacaatgat ggcaagaaaa atgaaagaca
     3421 cagacagtga agaagaaatt agagaagcgt tccgtgtgtt tgataaggac ggcaatggct
     3481 acatcggcgc agcagagctt cgccacgtga tgacaaacct tggagagaag ttaacagatg
     3541 aagaggttga tgaaatgatc agggtagcag acatcgatgg ggatggtcag gtaaactacg
     3601 aagagtttgt acaaatgatg acagcgaagt tctgctacga gaacgaggtg taagcggccg
     3661 cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc tttaaaaaac
     3721 ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt tgttaacttg
     3781 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3841 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttaaggc
     3901 gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3961 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     4021 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     4081 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     4141 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     4201 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     4261 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     4321 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa
     4381 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     4441 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc ctgaggcgga
     4501 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4561 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4621 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4681 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4741 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4801 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga tcgatcaaga
     4861 gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc
     4921 cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga
     4981 tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct
     5041 gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac
     5101 gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct
     5161 attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt
     5221 atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt
     5281 cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt
     5341 cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag
     5401 gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt
     5461 gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg
     5521 tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg
     5581 cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg
     5641 catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgaaatg
     5701 accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc cgccttctat
     5761 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     5821 gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac acggaaggag
     5881 acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa aacgcacggt
     5941 gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc
     6001 ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc
     6061 ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata
     6121 gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6181 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6241 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6301 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6361 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6421 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6481 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6541 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6601 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6661 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6721 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6781 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6841 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6901 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     6961 gtggataacc gtattaccgc catgcat
//
