LOCUS       sequence-487628-        5464 bp    DNA     circular SYN 01-OCT-2025
DEFINITION  Lysosomal purification of lysosome marker LAMP1-3xFlag-twin-strep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5464)
  TITLE     Martin lab plasmids
REFERENCE   2  (bases 1 to 5464)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 1, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: LAMP1 3xFlag twin strep
FEATURES             Location/Qualifiers
     source          1..5464
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             join(585..668,669..1835)
                     /codon_start=1
                     /product="human lysosome-associated membrane glycoprotein 
                     1"
                     /label=LAMP1
                     /translation="MAAPGSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMAN
                     FSAAFSVNYDTKSGPKNMTFDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNF
                     TRNATRYSVQLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHM
                     NNVTVTLHDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKY
                     NVSGTNGTCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSE
                     GTTVLLFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNA
                     EEHVRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSMLIPIAVGGALAGLVLIV
                     LIAYLVGRKRSHAGYQTI"
     CDS             1860..1925
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1944..1964
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(1971..1976,1977..2000,2001..2036,2037..2060)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     polyA_signal    2192..2313
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2229..2248)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2283..2302
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2320..2775)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2457..2478)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2669..2688
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2802..2906
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2908..3265
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2908..3099
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2931..2951)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3116..3251
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3300..4094
                     /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             3300..4094
                     /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(3354..3373)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3964..3983
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4282..4301)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4326..4373
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4702..5290
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5191..5210
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      541 ccggactcag atctcgagct caagcttcga attctgcagt cgacatggcg gcccccggca
      601 gcgcccggcg acccctgctg ctgctactgc tgttgctgct gctcggcctc atgcattgtg
      661 cgtcagcagc aatgtttatg gtgaaaaatg gcaacgggac cgcgtgcata atggccaact
      721 tctctgctgc cttctcagtg aactacgaca ccaagagtgg ccctaagaac atgacctttg
      781 acctgccatc agatgccaca gtggtgctca accgcagctc ctgtggaaaa gagaacactt
      841 ctgaccccag tctcgtgatt gcttttggaa gaggacatac actcactctc aatttcacga
      901 gaaatgcaac acgttacagc gtccagctca tgagttttgt ttataacttg tcagacacac
      961 accttttccc caatgcgagc tccaaagaaa tcaagactgt ggaatctata actgacatca
     1021 gggcagatat agataaaaaa tacagatgtg ttagtggcac ccaggtccac atgaacaacg
     1081 tgaccgtaac gctccatgat gccaccatcc aggcgtacct ttccaacagc agcttcagca
     1141 ggggagagac acgctgtgaa caagacaggc cttccccaac cacagcgccc cctgcgccac
     1201 ccagcccctc gccctcaccc gtgcccaaga gcccctctgt ggacaagtac aacgtgagcg
     1261 gcaccaacgg gacctgcctg ctggccagca tggggctgca gctgaacctc acctatgaga
     1321 ggaaggacaa cacgacggtg acaaggcttc tcaacatcaa ccccaacaag acctcggcca
     1381 gcgggagctg cggcgcccac ctggtgactc tggagctgca cagcgagggc accaccgtcc
     1441 tgctcttcca gttcgggatg aatgcaagtt ctagccggtt tttcctacaa ggaatccagt
     1501 tgaatacaat tcttcctgac gccagagacc ccgcctttaa agctgccaac ggctccctgc
     1561 gagcgctgca ggccacagtc ggcaattcct acaagtgcaa cgcggaggag cacgtccgtg
     1621 tcacgaaggc gttttcagtc aatatattca aagtgtgggt ccaggctttc aaggtggaag
     1681 gtggccagtt tggctctgtg gaggagtgtc tgctggacga gaacagcatg ctgatcccca
     1741 tcgctgtggg tggtgccctg gcggggctgg tcctcatcgt cctcatcgcc tacctcgtcg
     1801 gcaggaagag gagtcacgca ggctaccaga ctatcaagga tccaggctcg ggctccggag
     1861 actacaagga ccatgacggt gactataagg accacgacat cgactacaaa gatgatgatg
     1921 ataagggcaa gggaggttct ggtgagaatc tctacttcca atctggtgct agcgcatgga
     1981 gtcatcctca attcgagaaa ggtggaggtt ctggcggtgg atcgggaggt tcagcgtgga
     2041 gccacccgca gttcgaaaaa taaaagtaaa gcggccgcga ctctagatca taatcagcca
     2101 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     2161 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     2221 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2281 ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc gttaatattt
     2341 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     2401 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     2461 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     2521 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     2581 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2641 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2701 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2761 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2821 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2881 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     2941 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     3001 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     3061 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     3121 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     3181 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     3241 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     3301 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     3361 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3421 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3481 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3541 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3601 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     3661 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     3721 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     3781 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     3841 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3901 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3961 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     4021 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     4081 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     4141 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     4201 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     4261 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     4321 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     4381 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     4441 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     4501 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     4561 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4621 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4681 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4741 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4801 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4861 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4921 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4981 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     5041 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     5101 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     5161 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     5221 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     5281 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     5341 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
     5401 gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
     5461 tccg
//
