LOCUS       sequence-494655-        7513 bp    DNA     circular SYN 04-DEC-2025
DEFINITION  Lysosomal channel with a low-affinity GECI fused to the C-terminus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7513)
  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 7513)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 4, 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-G-GECO1.2 (F90L)
FEATURES             Location/Qualifiers
     source          1..7513
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(2266..2469)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2295..2315)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2470..2773
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(2948..3536)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3028..3047)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    3865..3912
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     3937..3956
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4144..4938)
                     /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             complement(4144..4938)
                     /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(4255..4274)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4865..4884
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4973..5330)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4987..5122
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        5139..5330
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     5287..5307
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(5332..5436)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5463..5918
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5550..5569)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5760..5781
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5925..6046
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5936..5955)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5990..6009
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(6168..6611)
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREALRVFDKDGNGYIGAAEL
                     RHVMTNLGEKLTDEEVDEMIRVADIDGDGQVNYEEFVQMMTAK"
     primer_bind     complement(7119..7140)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(7353..7409)
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
ORIGIN
        1 cctcgggctc ctccagaata tccctgaaca ggagctccac agtcatctgg taggtggcct
       61 ctggggtccc agcaaggggc tgcaggtggc tgcgggggtc ccacttgtga aggaagttct
      121 ccagaatcag ggccagaaac aggttgaccc agatgacaga cgacaccagc caccacaata
      181 caaaatagat cttggaccac gggcctgagt agcgccgata tgcatccaga aacacctgcc
      241 agttgttcac caccatcaag ttccacagag tgaccagggc agccgcaaag tcatcgaagt
      301 tgttggccca gtactccagc tgctcgaagc tcccacaggg cgccgagcca ttggcagggg
      361 ccaggctgct gtttccagga agagccacaa tgacgcctct aaacaagttg atcccaatga
      421 tggcaaatac gtagtagacc accaccagga tcccgccaaa agcacgcatg ttctgcacca
      481 ggcccaggac ggtactggcc accacggcca tcggcttcat gctggggatg atacgcagga
      541 agcggaacac gatgagcatg ttcagcatgc gggtcatgtc ccacagcgac agcaggccca
      601 ccatctccgg cctccagcct gggtgtggca atcggtacac agccagagtt gagatctcca
      661 aaaccagcag gacaacggtg aggagcccgt caaacacgtt gctggggtag gacaggtacc
      721 ctcgcaggcc cagggcaaag accttgagca gcatctccaa caggtagtac acaatgaaga
      781 cgcagttgag aatccccagg atgaagtcat cacgctcagc aggcagcaca tctgcatcca
      841 gcaccaggaa cacgcaaatg gacaccaggt ttgccagggc gatgaggttc cccaggtagt
      901 caaagtagta gtggccgaag aggaactggg cgctctgcag aaacggagac tggtactcgg
      961 gcctcggcgg gtgctcttta accacacttc tgtcaagctc gttgaagagc ttctgaaact
     1021 cctcagctga cagcagaaca ctgccgtagg aacgcacctt ctccatcatg gcctgtttgt
     1081 gggagctgtc cagctggacc ttctgaagca cctgcagcaa gttctggggc ttcaccccaa
     1141 ctgcctgagg gaaggctcct ccctccccca ccatggagga taggacttca aaggcagccc
     1201 gggttcccag ccgcctccga aacagcgagg tctggagaga tttcatcagg tagccccgga
     1261 actgactgta gatgatggct gtcagcaggt tcatcagaaa caggcttcct atcacagtga
     1321 agactatgaa gaagatggca taggcccggt tcttggaata cgcaggaatc atcacatcgg
     1381 ggttgttggc cgtggtcagc agcaccagga gggaagtcag agactcaggc aggttctgga
     1441 agtaggtcag cctctccctg tcctgcccat catcctgctt cccaccagcg aacagcagca
     1501 ttccgaacat ggtgaagagg cacaggtgga tggccagcag cagcccgacg ctggccattt
     1561 ccggcagcga ccagcggatg catttcaagg tcttcttcat catagaggag ttctgcagca
     1621 ggaagaaggg acggagaagc cggcggatcc gcaggggctc atgacacacg agactcaggg
     1681 acacggtcca gtccaccaga gacaccacca gcaccacgag gtagcccagc agccaaaggt
     1741 ttttctggaa atgggcccac ccgaacaggt aacccttcac agagaggtcg gccgcaaaga
     1801 ccagcaggca gagcacctcg acactctcgg tcaggccgca gggcggctcc cagggggcag
     1861 cgcggtagcg cacgtccgcc gtgctggtga gtgaggatgg ggtctcgata aaagccaaaa
     1921 acaggatcaa gaagatggtg aagctcaaag tccgttggca tacgttcgag taataccgtc
     1981 ggtaaagcca catcgagctg gcatccaccc ggtggttgat ggagcggtac tgaatagcat
     2041 cttcgatgaa gaccacagcc tgatcaatgc agaggtccca cctggccgcg gcaccagggc
     2101 cgacttggat gctgcggtaa gtggtcagcc ccgccggcca gtcgccgcca ccgccgcggg
     2161 ccccgcccag caggggctcc gactccgcct ggggttccgc catggtaagc ttgagctcga
     2221 gatctgagtc cggtagcgct agcggatctg acggttcact aaaccagctc tgcttatata
     2281 gacctcccac cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca
     2341 ttttggaaag tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg
     2401 gagacttgga aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa
     2461 ccgcatcacc atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc
     2521 ccataaggtc atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat
     2581 agggggcgta cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa
     2641 atactccacc cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt
     2701 cattattgac gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt
     2761 aagttatgta acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta
     2821 ctattaataa ctaatgcatg gcggtaatac ggttatccac agaatcaggg gataacgcag
     2881 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2941 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     3001 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     3061 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     3121 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     3181 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     3241 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     3301 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     3361 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     3421 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     3481 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     3541 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     3601 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     3661 gttttaaatc aatctaaagt atatatgagt aacctgaggc tatggcaggg cctgccgccc
     3721 cgacgttggc tgcgagccct gggccttcac ccgaacttgg ggggtggggt ggggaaaagg
     3781 aagaaacgcg ggcgtattgg ccccaatggg gtctcggtgg ggtatcgaca gagtgccagc
     3841 cctgggaccg aaccccgcgt ttatgaacaa acgacccaac accgtgcgtt ttattctgtc
     3901 tttttattgc cgtcatagcg cgggttcctt ccggtattgt ctccttccgt gtttcagtta
     3961 gcctccccct agggtgggcg aagaactcca gcatgagatc cccgcgctgg aggatcatcc
     4021 agccggcgtc ccggaaaacg attccgaagc ccaacctttc atagaaggcg gcggtggaat
     4081 cgaaatctcg tgatggcagg ttgggcgtcg cttggtcggt catttcgaac cccagagtcc
     4141 cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc
     4201 gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc
     4261 acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat
     4321 gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt
     4381 cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg
     4441 cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg
     4501 agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc
     4561 aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag
     4621 gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc
     4681 ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag
     4741 ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag
     4801 aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg
     4861 ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa
     4921 tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcga tctttgcaaa
     4981 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg
     5041 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     5101 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     5161 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg
     5221 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     5281 ttccacaccc taactgacac acattccaca gctggttctt tccgcctcag gactcttcct
     5341 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     5401 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     5461 tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     5521 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     5581 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     5641 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     5701 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     5761 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     5821 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     5881 taacgcgaat tttaacaaaa tattaacgct tacaatttac gccttaagat acattgatga
     5941 gtttggacaa accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga
     6001 tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg
     6061 cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa
     6121 cctctacaaa tgtggtatgg ctgattatga tctagagtcg cggccgctta cttcgctgtc
     6181 atcatttgta caaactcttc gtagtttacc tgaccatccc catcgatgtc tgctaccctg
     6241 atcatttcat caacctcttc atctgttaac ttctctccaa ggtttgtcat cacgtggcga
     6301 agctctgctg cgccgatgta gccattgccg tccttatcaa acacacgtaa cgcttctcta
     6361 atttcttctt cactgtctgt gtctttcatt tttcttgcca tcattgtcag gaactcaggg
     6421 aagtcgattg tgccgtcacc gtcggcatct acttcattga tcatgtcctg cagctctgct
     6481 tctgtggggt tctgccccag agaccgcatc accgtcccca gctccttggt tgttatcgtc
     6541 ccatccccgt ccttgtcaaa tagggagaaa gcctctttaa attctgcgat ctgctcttca
     6601 gtcagttggt cacgcgtgtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg
     6661 aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg
     6721 gcgcgggtct tgtagttgcc gtcgcccttg aagaagatgg tgcgctcctg gatgtagcct
     6781 tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag
     6841 cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg
     6901 gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc accctcgccg
     6961 gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgacctggat gggcaccacc
     7021 ccggtgaaca gctcctcgcc cttgctcacc atggattcgc tccctccggt accgcccttg
     7081 tacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag caggaccatg
     7141 tgatcgcgct tctcgttggg gtctttcgaa agcatggact gcacgctcag gtagtggttg
     7201 tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtaggcgagc
     7261 tgcacgccgc cgccctcgat gttgtggcgg atcttgaagt acgccttgat gccgttcttc
     7321 tgctcgtcgg ccttgatata gacgttctcg agtgagctca gccgacctat agctctgact
     7381 gcgtgacctg tcttattcca cttacgacgt gatgagtcga ccatgaccgg tggatcccgg
     7441 gcccgcggta ccgtcgactg cctgcacagc cacaggtgcg ggtgctggct cagcctctct
     7501 gtgagctcat cct
//
