LOCUS       sequence-413480-        6263 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lysosome-targeted rapalog dimerizer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6263)
  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 6263)
  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: LAMP1-mCherry-FRB*
FEATURES             Location/Qualifiers
     source          1..6263
                     /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(641..724,725..1891)
                     /codon_start=1
                     /product="human lysosome-associated membrane glycoprotein 
                     1"
                     /label=LAMP1
                     /translation="MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMAN
                     FSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNF
                     TRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHM
                     NNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKY
                     NVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSE
                     GTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNA
                     EEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIV
                     LIAYLVGRKRSHAGYQTI"
     CDS             1931..2635
                     /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(2073..2091)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2341..2360
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2648..2926
                     /codon_start=1
                     /product="T2098L mutant of FKBP-rapamycin binding domain of
                     human FRAP"
                     /label=FRB*
                     /note="binds synthetic ligands such as AP21967"
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK"
     polyA_signal    3051..3172
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3088..3107)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3142..3161
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3179..3634)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3316..3337)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3528..3547
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3661..3765
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3767..4124
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3767..3958
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3790..3810)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3975..4110
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4159..4953
                     /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             4159..4953
                     /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(4213..4232)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4823..4842
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5141..5160)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5185..5232
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5561..6149
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6050..6069
                     /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 caagcttcga attcagggac atggcggccc ccggcagcgc
      661 ccggcgaccc ctgctgctgc tactgctgtt gctgctgctc ggcctcatgc attgtgcgtc
      721 agcagcaatg tttatggtga aaaatggcaa cgggaccgcg tgcataatgg ccaacttctc
      781 tgctgccttc tcagtgaact acgacaccaa gagtggccct aagaacatga cctttgacct
      841 gccatcagat gccacagtgg tgctcaaccg cagctcctgt ggaaaagaga acacttctga
      901 ccccagtctc gtgattgctt ttggaagagg acatacactc actctcaatt tcacgagaaa
      961 tgcaacacgt tacagcgtcc agctcatgag ttttgtttat aacttgtcag acacacacct
     1021 tttccccaat gcgagctcca aagaaatcaa gactgtggaa tctataactg acatcagggc
     1081 agatatagat aaaaaataca gatgtgttag tggcacccag gtccacatga acaacgtgac
     1141 cgtaacgctc catgatgcca ccatccaggc gtacctttcc aacagcagct tcagcagggg
     1201 agagacacgc tgtgaacaag acaggccttc cccaaccaca gcgccccctg cgccacccag
     1261 cccctcgccc tcacccgtgc ccaagagccc ctctgtggac aagtacaacg tgagcggcac
     1321 caacgggacc tgcctgctgg ccagcatggg gctgcagctg aacctcacct atgagaggaa
     1381 ggacaacacg acggtgacaa ggcttctcaa catcaacccc aacaagacct cggccagcgg
     1441 gagctgcggc gcccacctgg tgactctgga gctgcacagc gagggcacca ccgtcctgct
     1501 cttccagttc gggatgaatg caagttctag ccggtttttc ctacaaggaa tccagttgaa
     1561 tacaattctt cctgacgcca gagaccccgc ctttaaagct gccaacggct ccctgcgagc
     1621 gctgcaggcc acagtcggca attcctacaa gtgcaacgcg gaggagcacg tccgtgtcac
     1681 gaaggcgttt tcagtcaata tattcaaagt gtgggtccag gctttcaagg tggaaggtgg
     1741 ccagtttggc tctgtggagg agtgtctgct ggacgagaac agcatgctga tccccatcgc
     1801 tgtgggtggt gccctggcgg ggctggtcct catcgtcctc atcgcctacc tcgtcggcag
     1861 gaagaggagt cacgcaggct accagactat cggctccacc ggctccacag gctccaccgg
     1921 cgcggtcgac gtgagcaagg gcgaggagga taacttagcc atcatcaagg agttcatgcg
     1981 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     2041 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
     2101 cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt
     2161 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg
     2221 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     2281 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
     2341 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga
     2401 ggacggcgcc ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta
     2461 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta
     2521 caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     2581 gtacgaacgc gccgagggcc gccactccac cggcggcatg gacgagctgt acaagggagc
     2641 aagtggaatc ctctggcatg agatgtggca tgaaggcctg gaagaggcat ctcgtttgta
     2701 ctttggggaa aggaacgtga aaggcatgtt tgaggtgctg gagcccttgc atgctatgat
     2761 ggaacggggc ccccagactc tgaaggaaac atcctttaat caggcctatg gtcgagattt
     2821 aatggaggcc caagagtggt gcaggaagta catgaaatca gggaatgtca aggacctcct
     2881 ccaagcctgg gacctctatt atcatgtgtt ccgacgaatc tcaaagtagg cggccgcgac
     2941 tctagatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     3001 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     3061 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     3121 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taaggcgtaa
     3181 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     3241 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     3301 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     3361 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     3421 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     3481 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     3541 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     3601 cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg
     3661 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3721 caataaccct gataaatgct tcaataatat tgaaaaagga agagtcctga ggcggaaaga
     3781 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     3841 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     3901 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     3961 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     4021 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
     4081 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaagatcga tcaagagaca
     4141 ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct
     4201 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     4261 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     4321 ggtgccctga atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc
     4381 gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
     4441 ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     4501 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     4561 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     4621 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     4681 aaggcgagca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     4741 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     4801 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     4861 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     4921 gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
     4981 accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa
     5041 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     5101 tcatgctgga gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa
     5161 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacggtgttg
     5221 ggtcgtttgt tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac
     5281 cgagacccca ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
     5341 gttcgggtga aggcccaggg ctcgcagcca acgtcggggc ggcaggccct gccatagcct
     5401 caggttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     5461 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     5521 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     5581 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     5641 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     5701 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     5761 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     5821 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     5881 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     5941 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     6001 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     6061 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     6121 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     6181 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     6241 ataaccgtat taccgccatg cat
//
