LOCUS       sequence-368435-        6282 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mCherry-FRB at the outer surface of the lysosome using
            LAMP1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6282)
  AUTHORS   Kuey C, Sittewelle M, Larocque G, Hernandez-Gonzalez M, Royle SJ
  TITLE     Recruitment of clathrin to intracellular membranes is sufficient for
            vesicle formation.
  JOURNAL   Elife. 2022 Jul 19;11. pii: 78929. doi: 10.7554/eLife.78929.
  PUBMED    35852853
REFERENCE   2  (bases 1 to 6282)
  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: "Elife."; 
            date: "2022-07-19"; volume: "11. pii: 78929. doi"; pages: " 
            10.7554/eLife.78929"
COMMENT     Sequence Label: Lamp1-mCherry-FRB
FEATURES             Location/Qualifiers
     source          1..6282
                     /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"
     regulatory      1907..1916
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1913..2617
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELY"
     primer_bind     complement(2058..2076)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2326..2345
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2621..2647
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2666..2944
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             2678..2947
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS
                     FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKQ"
     polyA_signal    3070..3191
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3107..3126)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3161..3180
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3198..3653)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3335..3356)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3547..3566
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3680..3784
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3786..4143
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3786..3977
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3809..3829)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3994..4129
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4178..4972
                     /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             4178..4972
                     /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(4232..4251)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4842..4861
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5160..5179)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5204..5251
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5580..6168
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6069..6088
                     /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 caaggatcca ccggtcgcca ccatggtgag
     1921 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat
     1981 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta
     2041 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg
     2101 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga
     2161 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa
     2221 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt
     2281 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa
     2341 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa
     2401 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa
     2461 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa
     2521 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga
     2581 gggccgccac tccaccggcg gcatggacga gctgtacact tacccctacg acgtgcccga
     2641 ctacgcaggc tccgcacgag tggccatcct ctggcatgag atgtggcatg aaggcctgga
     2701 agaggcatct cgtttgtact ttggggaaag gaacgtgaaa ggcatgtttg aggtgctgga
     2761 gcccttgcat gctatgatgg aacggggccc ccagactctg aaggaaacat cctttaatca
     2821 ggcctatggt cgagatttaa tggaggccca agagtggtgc aggaagtaca tgaaatcagg
     2881 gaatgtcaag gacctcaccc aagcctggga cctctattat catgtgttcc gacgaatctc
     2941 aaagcaggcc ggctgataat ctagatcata atcagccata ccacatttgt agaggtttta
     3001 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
     3061 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3121 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3181 aatgtatctt aaggcgtaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     3241 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     3301 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     3361 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     3421 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     3481 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     3541 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     3601 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg
     3661 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     3721 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     3781 gagtcctgag gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3841 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3901 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3961 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4021 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4081 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4141 aaagatcgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4201 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4261 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4321 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc
     4381 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4441 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4501 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4561 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4621 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4681 cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg tcgtgaccca
     4741 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4801 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4861 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4921 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4981 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     5041 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     5101 atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg gaggctaact
     5161 gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat aaaaagacag
     5221 aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     5281 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     5341 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     5401 gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt aaaacttcat
     5461 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     5521 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     5581 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     5641 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     5701 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     5761 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     5821 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     5881 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     5941 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     6001 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     6061 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     6121 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     6181 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     6241 ttatcccctg attctgtgga taaccgtatt accgccatgc at
//
