LOCUS       sequence-331200-        6620 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the chimeric construct SEP-LAMP1-RFP where superecliptic 
            pHluorin (SEP) is fused to the luminal domain of LAMP1-RFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6620)
  AUTHORS   Farias GG, Guardia CM, De Pace R, Britt DJ, Bonifacino JS
  TITLE     BORC/kinesin-1 ensemble drives polarized transport of lysosomes into
            the axon.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Apr 4;114(14):E2955-E2964. doi: 
            10.1073/pnas.1616363114. Epub 2017 Mar 20.
  PUBMED    28320970
REFERENCE   2  (bases 1 to 6620)
  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"; doi: 
            "10.1073/pnas.1616363114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-04-4- 4"; volume: "114"; issue: "14"; pages: 
            "E2955-E2964"
COMMENT     Sequence Label: pSEP-LAMP1-RFP
FEATURES             Location/Qualifiers
     source          1..6620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        65..368
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        369..572
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     523..543
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      641..650
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      645..654
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             719..1432
                     /codon_start=1
                     /product="pH-sensitive mutant of green fluorescent protein 
                     (Ng et al., 2002)"
                     /label=superecliptic pHluorin
                     /note="excited strongly by blue light at neutral pH but 
                     minimally fluorescent at acidic pH"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKA
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(749..777)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1372..1392
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     regulatory      2597..2606
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2603..3277
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2603..3277
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(2733..2751)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3001..3020
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3195..3218
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3412..3533
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3449..3468)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3503..3522
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3540..3995)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3677..3698)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3889..3908
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4022..4126
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4128..4485
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4128..4319
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4151..4171)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4336..4471
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4520..5314
                     /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             4520..5314
                     /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(4574..4593)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5184..5203
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5502..5521)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5546..5593
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5922..6510
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6411..6430
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
       61 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      121 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      181 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      241 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      301 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      361 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      421 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      481 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      541 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc
      601 gctaccggac tcagatctcg agctcaagct tcgaattctc gccaccatgg cggccccggg
      661 cgcccggcgg ccgctgctcc tgttgctgct ggcaggcctt gcacacagcg ccccagcaat
      721 gagtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat tagatggtga
      781 tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa catacggaaa
      841 acttaccctt aaatttattt gcactactgg aaaactacct gttccttggc caacacttgt
      901 cactacttta acttatggtg ttcaatgctt ttcaagatac ccagatcata tgaaacggca
      961 tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta tatttttcaa
     1021 agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata cccttgttaa
     1081 tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg gacacaaatt
     1141 ggaatacaac tataacgatc accaggtgta catcatggca gacaaacaaa agaatggaat
     1201 caaagctaac ttcaaaatta gacacaacat tgaagatgga ggcgttcaac tagcagacca
     1261 ttatcaacaa aatactccaa ttggcgatgg gcccgtcctt ttaccagaca accattacct
     1321 gtttacaact tctactcttt cgaaagatcc caacgaaaag agagaccaca tggtccttct
     1381 tgagtttgta acagctgctg ggattacaca tggcatggat gaactataca aactgttcga
     1441 ggtgaaagac aacaacggca cagcgtgtat aatggccagc ttctctgcct cctttctgac
     1501 cacctatgag gctggacatg tttctaaggt ctcgaatatg accctgccag cctctgcaga
     1561 agtcctgaag aatagcagct cttgtggtga aaagaatgct tctgagccca ccctcgcaat
     1621 cacctttgga gaaggatatt tactgaaact caccttcaca aaaaacacaa cacgttacag
     1681 tgtccagcac atgtatttca catataacct gtcagacaca caattctttc ccaatgccag
     1741 ctccaaaggg cccgacactg tggattccac aactgacatc aaggcagaca tcaacaaaac
     1801 ataccgatgt gtcagcgaca tcagggtcta catgaagaat gtgaccattg tgctctggga
     1861 cgctactatc caggcctacc tgccgagtag caacttcagc aaggaagaga cacgctgccc
     1921 acaggatcaa ccttccccaa ctactgggcc acccagcccc tcaccaccac tcgtgcccac
     1981 aaaccccagt gtgtccaagt acaatgtgac tggtgacaat ggaacctgcc tgctggcctc
     2041 tatggcactg caactcaaca tcacctacat gaagaaggac aacacgactg tgaccagagc
     2101 attcaacatc aacccaagtg acaaatatag tgggacttgc ggtgcccagt tggtgaccct
     2161 gaaggtgggg aacaagagca gagtcctgga gctgcagttt gggatgaatg ccacttctag
     2221 cctgtttttc ctgcaaggag ttcagttgaa catgactctt cctgatgcca tagagcccac
     2281 gttcagcacc tccaactatt ccctgaaagc tcttcaggcc agtgtcggca actcatacaa
     2341 gtgcaacagt gaggagcaca tctttgtcag caaggcgctc gccctcaatg tcttcagcgt
     2401 gcaagtccag gctttcaggg tagaaagtga caggtttggg tctgtggaag agtgtgtaca
     2461 ggacggtaac aacatgctga tccccattgc tgtgggcggg gccctggcag ggctggtcct
     2521 catcgtcctc atcgcctacc tcatcggcag gaagaggagt cacgcgggct atcagaccat
     2581 ccgggatcca ccggtcgcca ccatggcctc ctccgaggac gtcatcaagg agttcatgcg
     2641 cttcaaggtg cgcatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     2701 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggcggccc
     2761 cctgcccttc gcctgggaca tcctgtcccc tcagttccag tacggctcca aggcctacgt
     2821 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg
     2881 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct
     2941 gcaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
     3001 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc accgagcgga tgtaccccga
     3061 ggacggcgcc ctgaagggcg agatcaagat gaggctgaag ctgaaggacg gcggccacta
     3121 cgacgccgag gtcaagacca cctacatggc caagaagccc gtgcagctgc ccggcgccta
     3181 caagaccgac atcaagctgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     3241 gtacgagcgc gccgagggcc gccactccac cggcgccttg tacaagtaaa gcggccgcga
     3301 ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc
     3361 ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt
     3421 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     3481 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta
     3541 aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt
     3601 ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat
     3661 agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa
     3721 cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta
     3781 atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc
     3841 ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc
     3901 gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac
     3961 acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt
     4021 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4081 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag
     4141 aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     4201 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     4261 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     4321 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4381 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc
     4441 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac
     4501 aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc
     4561 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     4621 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     4681 cggtgccctg aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg
     4741 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     4801 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     4861 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     4921 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     4981 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     5041 caaggcgagc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     5101 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     5161 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     5221 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     5281 cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc
     5341 gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa
     5401 aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat
     5461 ctcatgctgg agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca
     5521 ataccggaag gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt
     5581 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca
     5641 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca
     5701 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc
     5761 tcaggttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5821 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5881 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5941 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6001 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6061 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6121 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6181 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6241 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6301 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     6361 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     6421 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     6481 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     6541 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     6601 gataaccgta ttaccgccat
//
