LOCUS       sequence-489542-        6508 bp    DNA     circular SYN 16-OCT-2025
DEFINITION  Mid-affinity GECI targeted to cytosolic face of the lysosome.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6508)
  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 6508)
  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: LAMP1-G-GECO1.2
FEATURES             Location/Qualifiers
     source          1..6508
                     /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"
     regulatory      629..638
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(631..714,715..1881)
                     /codon_start=1
                     /product="human lysosome-associated membrane glycoprotein 
                     1"
                     /label=LAMP1
                     /translation="MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMAN
                     FSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNF
                     TRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHM
                     NNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKY
                     NVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSE
                     GTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNA
                     EEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIV
                     LIAYLVGRKRSHAGYQTI"
     CDS             1933..1989
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     2202..2223
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2731..3174
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYIGAAEL
                     RHVMTNLGEKLTDEEVDEMIRVADIDGDGQVNYEEFVQMMTAK"
     polyA_signal    3296..3417
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3333..3352)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3387..3406
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3424..3879)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3561..3582)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3773..3792
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3906..4010
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4012..4369
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4012..4203
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4035..4055)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4220..4355
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4404..5198
                     /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             4404..5198
                     /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(4458..4477)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5068..5087
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5386..5405)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5430..5477
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5806..6394
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6295..6314
                     /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 atggcggccc ccggcagcgc ccggcgaccc
      661 ctgctgctgc tactgctgtt gctgctgctc ggcctcatgc attgtgcgtc agcagcaatg
      721 tttatggtga aaaatggcaa cgggaccgcg tgcataatgg ccaacttctc tgctgccttc
      781 tcagtgaact acgacaccaa gagtggccct aagaacatga cctttgacct gccatcagat
      841 gccacagtgg tgctcaaccg cagctcctgt ggaaaagaga acacttctga ccccagtctc
      901 gtgattgctt ttggaagagg acatacactc actctcaatt tcacgagaaa tgcaacacgt
      961 tacagcgtcc agctcatgag ttttgtttat aacttgtcag acacacacct tttccccaat
     1021 gcgagctcca aagaaatcaa gactgtggaa tctataactg acatcagggc agatatagat
     1081 aaaaaataca gatgtgttag tggcacccag gtccacatga acaacgtgac cgtaacgctc
     1141 catgatgcca ccatccaggc gtacctttcc aacagcagct tcagcagggg agagacacgc
     1201 tgtgaacaag acaggccttc cccaaccaca gcgccccctg cgccacccag cccctcgccc
     1261 tcacccgtgc ccaagagccc ctctgtggac aagtacaacg tgagcggcac caacgggacc
     1321 tgcctgctgg ccagcatggg gctgcagctg aacctcacct atgagaggaa ggacaacacg
     1381 acggtgacaa ggcttctcaa catcaacccc aacaagacct cggccagcgg gagctgcggc
     1441 gcccacctgg tgactctgga gctgcacagc gagggcacca ccgtcctgct cttccagttc
     1501 gggatgaatg caagttctag ccggtttttc ctacaaggaa tccagttgaa tacaattctt
     1561 cctgacgcca gagaccccgc ctttaaagct gccaacggct ccctgcgagc gctgcaggcc
     1621 acagtcggca attcctacaa gtgcaacgcg gaggagcacg tccgtgtcac gaaggcgttt
     1681 tcagtcaata tattcaaagt gtgggtccag gctttcaagg tggaaggtgg ccagtttggc
     1741 tctgtggagg agtgtctgct ggacgagaac agcatgctga tccccatcgc tgtgggtggt
     1801 gccctggcgg ggctggtcct catcgtcctc atcgcctacc tcgtcggcag gaagaggagt
     1861 cacgcaggct accagactat ccagtcgacg gtaccgcggg cccgggatcc accggtcatg
     1921 gtcgactcat cacgtcgtaa gtggaataag acaggtcacg cagtcagagc tataggtcgg
     1981 ctgagctcac tcgagaacgt ctatatcaag gccgacgagc agaagaacgg catcaaggcg
     2041 tacttcaaga tccgccacaa catcgagggc ggcggcgtgc agctcgccta ccactaccag
     2101 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcgtg
     2161 cagtccatgc tttcgaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
     2221 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggcgg taccggaggg
     2281 agcgaatcca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catccaggtc
     2341 gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg tgagggcgat
     2401 gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc
     2461 tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac
     2521 cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacat ccaggagcgc
     2581 accatcttct tcaagggcga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc
     2641 gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc
     2701 ctggggcaca agctggagta caacacgcgt gaccaactga ctgaagagca gatcgcagaa
     2761 tttaaagagg ctttctccct atttgacaag gacggggatg ggacgataac aaccaaggag
     2821 ctggggacgg tgatgcggtc tctggggcag aaccccacag aagcagagct gcaggacatg
     2881 atcaatgaag tagatgccga cggtgacggc acaatcgact tccctgagtt cctgacaatg
     2941 atggcaagaa aaatgaaaga cacagacagt gaagaagaaa ttagagaagc gttccgtgtg
     3001 tttgataagg acggcaatgg ctacatcggc gcagcagagc ttcgccacgt gatgacaaac
     3061 cttggagaga agttaacaga tgaagaggtt gatgaaatga tcagggtagc agacatcgat
     3121 ggggatggtc aggtaaacta cgaagagttt gtacaaatga tgacagcgaa gtaagcggcc
     3181 gcgactctag atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa
     3241 cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt
     3301 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3361 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg
     3421 cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     3481 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     3541 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3601 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     3661 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     3721 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     3781 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3841 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa
     3901 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3961 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg
     4021 aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
     4081 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
     4141 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
     4201 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
     4261 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct
     4321 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag
     4381 agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg
     4441 ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg
     4501 atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc
     4561 tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga
     4621 cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc
     4681 tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag
     4741 tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat
     4801 tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg
     4861 tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca
     4921 ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct
     4981 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     5041 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     5101 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     5161 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat
     5221 gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta
     5281 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     5341 ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga
     5401 gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg
     5461 tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac
     5521 cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc
     5581 cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat
     5641 agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     5701 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     5761 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     5821 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     5881 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     5941 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6001 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6061 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6121 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6181 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6241 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6301 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6361 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6421 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6481 tgtggataac cgtattaccg ccatgcat
//
