LOCUS       sequence-492928-        6964 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  Red-tagged lysosomal channel (pore-dead).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6964)
  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 6964)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 18, 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-D276K-mCherry
FEATURES             Location/Qualifiers
     source          1..6964
                     /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             2923..3630
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNLAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3065..3083)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3333..3352
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3752..3873
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3789..3808)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3843..3862
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3880..4335)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4017..4038)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4229..4248
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4362..4466
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4468..4825
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4468..4659
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4491..4511)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4676..4811
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4860..5654
                     /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             4860..5654
                     /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(4914..4933)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5524..5543
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5842..5861)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5886..5933
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6262..6850
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6751..6770
                     /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 accccaaagt 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 tcgtgagcaa gggcgaggag
     2941 gataacttag ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     3001 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     3061 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     3121 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     3181 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     3241 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     3301 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc
     3361 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     3421 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     3481 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatcacc
     3541 tcccacaacg aggactacac catcgtggaa cagtacgaac gcgccgaggg ccgccactcc
     3601 accggcggca tggacgagct gtacaagtag gcggccgcga ctctagatca taatcagcca
     3661 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     3721 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     3781 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     3841 ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt
     3901 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     3961 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     4021 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     4081 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     4141 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     4201 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     4261 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     4321 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4381 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4441 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     4501 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     4561 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     4621 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     4681 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     4741 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     4801 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     4861 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4921 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4981 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     5041 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     5101 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     5161 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     5221 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     5281 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     5341 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     5401 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     5461 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     5521 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     5581 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     5641 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     5701 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     5761 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     5821 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     5881 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     5941 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     6001 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     6061 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     6121 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     6181 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     6241 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     6301 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     6361 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     6421 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     6481 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     6541 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6601 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6661 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6721 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6781 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6841 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6901 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
     6961 gcat
//
