LOCUS       sequence-488287-        6085 bp    DNA     circular SYN 02-OCT-2025
DEFINITION  Lysosomal purification of lysosome marker LAMP1-mScarlet-twin-strep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6085)
  TITLE     Martin lab plasmids
REFERENCE   2  (bases 1 to 6085)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 2, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: LAMP1 mScarlet twin strep
FEATURES             Location/Qualifiers
     source          1..6085
                     /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"
     regulatory      1851..1860
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1857..2552
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(1990..2008)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2258..2277
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2565..2585
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(2592..2597,2598..2621,2622..2657,2658..2681)
                     /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    2813..2934
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2850..2869)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2904..2923
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2941..3396)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3078..3099)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3290..3309
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3423..3527
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3529..3886
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3529..3720
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3552..3572)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3737..3872
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3921..4715
                     /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             3921..4715
                     /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(3975..3994)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4585..4604
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4903..4922)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4947..4994
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5323..5911
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5812..5831
                     /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 tccaccggtc gccaccatgg
     1861 tgagcaaggg cgaggcagtg atcaaggagt tcatgcggtt caaggtgcac atggagggct
     1921 ccatgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
     1981 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttctcc tgggacatcc
     2041 tgtcccctca gttcatgtac ggctccaggg ccttcatcaa gcaccccgcc gacatccccg
     2101 actactataa gcagtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
     2161 acggcggcgc cgtgaccgtg acccaggaca cctccctgga ggacggcacc ctgatctaca
     2221 aggtgaagct ccgcggcacc aacttccctc ctgacggccc cgtaatgcag aagaagacaa
     2281 tgggctggga agcgtccacc gagcggttgt accccgagga cggcgtgctg aagggcgaca
     2341 ttaagatggc cctgcgcctg aaggacggcg gccgctacct ggcggacttc aagaccacct
     2401 acaaggccaa gaagcccgtg cagatgcccg gcgcctacaa cgtcgaccgc aagttggaca
     2461 tcacctccca caacgaggac tacaccgtgg tggaacagta cgaacgctcc gagggccgcc
     2521 actccaccgg cggcatggac gagctgtaca agggaggttc tggtgagaat ctctacttcc
     2581 aatctggtgc tagcgcatgg agtcatcctc aattcgagaa aggtggaggt tctggcggtg
     2641 gatcgggagg ttcagcgtgg agccacccgc agttcgaaaa ataaaagtaa agcggccgcg
     2701 actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt taaaaaacct
     2761 cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     2821 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     2881 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttaaggcgt
     2941 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     3001 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     3061 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     3121 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     3181 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     3241 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     3301 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     3361 cacccgccgc gcttaatgcg ccgctacagg gcgcgtcagg tggcactttt cggggaaatg
     3421 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     3481 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtcct gaggcggaaa
     3541 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3601 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3661 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     3721 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3781 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     3841 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaagatc gatcaagaga
     3901 caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg
     3961 cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg
     4021 ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt
     4081 ccggtgccct gaatgaactg caagacgagg cagcgcggct atcgtggctg gccacgacgg
     4141 gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat
     4201 tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat
     4261 ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg
     4321 accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg
     4381 atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc
     4441 tcaaggcgag catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc
     4501 cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg
     4561 tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg
     4621 gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca
     4681 tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac
     4741 cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga
     4801 aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
     4861 tctcatgctg gagttcttcg cccaccctag ggggaggcta actgaaacac ggaaggagac
     4921 aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacggtgt
     4981 tgggtcgttt gttcataaac gcggggttcg gtcccagggc tggcactctg tcgatacccc
     5041 accgagaccc cattggggcc aatacgcccg cgtttcttcc ttttccccac cccacccccc
     5101 aagttcgggt gaaggcccag ggctcgcagc caacgtcggg gcggcaggcc ctgccatagc
     5161 ctcaggttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     5221 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     5281 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5341 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5401 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5461 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5521 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5581 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5641 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5701 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5761 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5821 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5881 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5941 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6001 ggataaccgt attaccgcca tgcattagtt attaatagta atcaattacg gggtcattag
     6061 ttcatagccc atatatggag ttccg
//
