LOCUS       sequence-489543-        7612 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  Lysosomal channel with a high-affinity GECI fused to the C-terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7612)
  AUTHORS   Davis LC, Morgan AJ, Galione A
  TITLE     Optical profiling of autonomous Ca(2+) nanodomains generated by 
            lysosomal TPC2 and TRPML1.
  JOURNAL   Cell Calcium. 2023 Dec;116:102801. doi: 10.1016/j.ceca.2023.102801. 
            Epub 2023 Sep 18.
  PUBMED    37742482
REFERENCE   2  (bases 1 to 7612)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell 
            Calcium."; date: "2023-12"; volume: "116:102801. doi"; pages: " 
            10.1016/j.ceca.2023.102801. Epub 2023 Sep 18"
COMMENT     Sequence Label: TPC2-GCaMP6s
FEATURES             Location/Qualifiers
     source          1..7612
                     /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             join(2926..2934,2935..2952,2953..2955,2956..2988,2989..2991,
                     2992..3015,3016..3042,3043..3099,3100..3105,3106..3375,
                     3376..3393,3394..3828,3829..3834,3835..4278)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFHIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      3022..3031
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     3312..3333
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3439..3460)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3700..3719)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    4400..4521
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4437..4456)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4491..4510
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(4528..4983)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4665..4686)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4877..4896
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5010..5114
                     /gene="bla"
                     /label=AmpR promoter
     promoter        5116..5473
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        5116..5307
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(5139..5159)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      5324..5459
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5508..6302
                     /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             5508..6302
                     /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(5562..5581)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6172..6191
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(6490..6509)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    6534..6581
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6910..7498
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7399..7418
                     /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 ccggactcag atctcgagct caagcttacc atggcggaac cccaggcgga gtcggagccc
      661 ctgctgggcg gggcccgcgg cggtggcggc gactggccgg cggggctgac cacttaccgc
      721 agcatccaag tcggccctgg tgccgcggcc aggtgggacc tctgcattga tcaggctgtg
      781 gtcttcatcg aagatgctat tcagtaccgc tccatcaacc accgggtgga tgccagctcg
      841 atgtggcttt accgacggta ttactcgaac gtatgccaac ggactttgag cttcaccatc
      901 ttcttgatcc tgtttttggc ttttatcgag accccatcct cactcaccag cacggcggac
      961 gtgcgctacc gcgctgcccc ctgggagccg ccctgcggcc tgaccgagag tgtcgaggtg
     1021 ctctgcctgc tggtctttgc ggccgacctc tctgtgaagg gttacctgtt cgggtgggcc
     1081 catttccaga aaaacctttg gctgctgggc tacctcgtgg tgctggtggt gtctctggtg
     1141 gactggaccg tgtccctgag tctcgtgtgt catgagcccc tgcggatccg ccggcttctc
     1201 cgtcccttct tcctgctgca gaactcctct atgatgaaga agaccttgaa atgcatccgc
     1261 tggtcgctgc cggaaatggc cagcgtcggg ctgctgctgg ccatccacct gtgcctcttc
     1321 accatgttcg gaatgctgct gttcgctggt gggaagcagg atgatgggca ggacagggag
     1381 aggctgacct acttccagaa cctgcctgag tctctgactt ccctcctggt gctgctgacc
     1441 acggccaaca accccgatgt gatgattcct gcgtattcca agaaccgggc ctatgccatc
     1501 ttcttcatag tcttcactgt gataggaagc ctgtttctga tgaacctgct gacagccatc
     1561 atctacagtc agttccgggg ctacctgatg aaatctctcc agacctcgct gtttcggagg
     1621 cggctgggaa cccgggctgc ctttgaagtc ctatcctcca tggtggggga gggaggagcc
     1681 ttccctcagg cagttggggt gaagccccag aacttgctgc aggtgcttca gaaggtccag
     1741 ctggacagct cccacaaaca ggccatgatg gagaaggtgc gttcctacgg cagtgttctg
     1801 ctgtcagctg aggagtttca gaagctcttc aacgagcttg acagaagtgt ggttaaagag
     1861 cacccgccga ggcccgagta ccagtctccg tttctgcaga gcgcccagtt cctcttcggc
     1921 cactactact ttgactacct ggggaacctc atcgccctgg caaacctggt gtccatttgc
     1981 gtgttcctgg tgctggatgc agatgtgctg cctgctgagc gtgatgactt catcctgggg
     2041 attctcaact gcgtcttcat tgtgtactac ctgttggaga tgctgctcaa ggtctttgcc
     2101 ctgggcctgc gagggtacct gtcctacccc agcaacgtgt ttgacgggct cctcaccgtt
     2161 gtcctgctgg ttttggagat ctcaactctg gctgtgtacc gattgccaca cccaggctgg
     2221 aggccggaga tggtgggcct gctgtcgctg tgggacatga cccgcatgct gaacatgctc
     2281 atcgtgttcc gcttcctgcg tatcatcccc agcatgaagc cgatggccgt ggtggccagt
     2341 accgtcctgg gcctggtgca gaacatgcgt gcttttggcg ggatcctggt ggtggtctac
     2401 tacgtatttg ccatcattgg gatcaacttg tttagaggcg tcattgtggc tcttcctgga
     2461 aacagcagcc tggcccctgc caatggctcg gcgccctgtg ggagcttcga gcagctggag
     2521 tactgggcca acaacttcga tgactttgcg gctgccctgg tcactctgtg gaacttgatg
     2581 gtggtgaaca actggcaggt gtttctggat gcatatcggc gctactcagg cccgtggtcc
     2641 aagatctatt ttgtattgtg gtggctggtg tcgtctgtca tctgggtcaa cctgtttctg
     2701 gccctgattc tggagaactt ccttcacaag tgggaccccc gcagccacct gcagcccctt
     2761 gctgggaccc cagaggccac ctaccagatg actgtggagc tcctgttcag ggatattctg
     2821 gaggagcccg aggaggatga gctcacagag aggctgagcc agcacccgca cctgtggctg
     2881 tgcaggcagt cgacggtacc gcgggcccgg gatccaccgg tcaccatggg ttctcatcat
     2941 catcatcatc atggtatggc tagcatgact ggtggacagc aaatgggtcg ggatctgtac
     3001 gacgatgacg ataaggatct cgccaccatg gtcgactcat cacgtcgtaa gtggaataag
     3061 acaggtcacg cagtcagagc tataggtcgg ctgagctcac tcgagaacgt ctatatcaag
     3121 gccgacaagc agaagaacgg catcaaggcg aacttccaca tccgccacaa catcgaggac
     3181 ggcggcgtgc agctcgccta ccactaccag cagaacaccc ccatcggcga cggccccgtg
     3241 ctgctgcccg acaaccacta cctgagcgtg cagtccaaac tttcgaaaga ccccaacgag
     3301 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
     3361 gacgagctgt acaagggcgg taccggaggg agcatggtga gcaagggcga ggagctgttc
     3421 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     3481 gtgtccggcg agggtgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     3541 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     3601 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     3661 cccgaaggct acatccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     3721 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     3781 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacct gccggaccaa
     3841 ctgactgaag agcagatcgc agaatttaaa gaggctttct ccctatttga caaggacggg
     3901 gatgggacaa taacaaccaa ggagctgggg acggtgatgc ggtctctggg gcagaacccc
     3961 acagaagcag agctgcagga catgatcaat gaagtagatg ccgacggtga cggcacaatc
     4021 gacttccctg agttcctgac aatgatggca agaaaaatga aatacaggga cacggaagaa
     4081 gaaattagag aagcgttcgg tgtgtttgat aaggatggca atggctacat cagtgcagca
     4141 gagcttcgcc acgtgatgac aaaccttgga gagaagttaa cagatgaaga ggttgatgaa
     4201 atgatcaggg aagcagacat cgatggggat ggtcaggtaa actacgaaga gtttgtacaa
     4261 atgatgacag cgaagtgagc ggccgcgact ctagatcata atcagccata ccacatttgt
     4321 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     4381 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     4441 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4501 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg
     4561 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     4621 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     4681 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     4741 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     4801 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     4861 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     4921 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     4981 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5041 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5101 gaaaaaggaa gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg
     5161 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     5221 agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     5281 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     5341 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     5401 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     5461 aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag
     5521 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     5581 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     5641 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     5701 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     5761 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     5821 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     5881 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     5941 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     6001 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     6061 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     6121 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     6181 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     6241 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     6301 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     6361 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     6421 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     6481 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     6541 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     6601 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     6661 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     6721 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     6781 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6841 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6901 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6961 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     7021 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     7081 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     7141 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     7201 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     7261 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     7321 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     7381 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     7441 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     7501 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     7561 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc at
//
