LOCUS       sequence-325846-        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        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"
     regulatory      637..646
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      641..650
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             715..1428
                     /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(745..773)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1368..1388
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     regulatory      2593..2602
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2599..3273
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             2599..3273
                     /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(2729..2747)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2997..3016
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3191..3214
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     polyA_signal    3408..3529
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3445..3464)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3499..3518
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3536..3991)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3673..3694)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3885..3904
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4018..4122
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4124..4481
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4124..4315
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4147..4167)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4332..4467
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4516..5310
                     /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             4516..5310
                     /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(4570..4589)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5180..5199
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5498..5517)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5542..5589
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5918..6506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6407..6426
                     /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 attctcgcca ccatggcggc cccgggcgcc
      661 cggcggccgc tgctcctgtt gctgctggca ggccttgcac acagcgcccc agcaatgagt
      721 aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt
      781 aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata cggaaaactt
      841 acccttaaat ttatttgcac tactggaaaa ctacctgttc cttggccaac acttgtcact
      901 actttaactt atggtgttca atgcttttca agatacccag atcatatgaa acggcatgac
      961 tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt tttcaaagat
     1021 gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct tgttaataga
     1081 atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca caaattggaa
     1141 tacaactata acgatcacca ggtgtacatc atggcagaca aacaaaagaa tggaatcaaa
     1201 gctaacttca aaattagaca caacattgaa gatggaggcg ttcaactagc agaccattat
     1261 caacaaaata ctccaattgg cgatgggccc gtccttttac cagacaacca ttacctgttt
     1321 acaacttcta ctctttcgaa agatcccaac gaaaagagag accacatggt ccttcttgag
     1381 tttgtaacag ctgctgggat tacacatggc atggatgaac tatacaaact gttcgaggtg
     1441 aaagacaaca acggcacagc gtgtataatg gccagcttct ctgcctcctt tctgaccacc
     1501 tatgaggctg gacatgtttc taaggtctcg aatatgaccc tgccagcctc tgcagaagtc
     1561 ctgaagaata gcagctcttg tggtgaaaag aatgcttctg agcccaccct cgcaatcacc
     1621 tttggagaag gatatttact gaaactcacc ttcacaaaaa acacaacacg ttacagtgtc
     1681 cagcacatgt atttcacata taacctgtca gacacacaat tctttcccaa tgccagctcc
     1741 aaagggcccg acactgtgga ttccacaact gacatcaagg cagacatcaa caaaacatac
     1801 cgatgtgtca gcgacatcag ggtctacatg aagaatgtga ccattgtgct ctgggacgct
     1861 actatccagg cctacctgcc gagtagcaac ttcagcaagg aagagacacg ctgcccacag
     1921 gatcaacctt ccccaactac tgggccaccc agcccctcac caccactcgt gcccacaaac
     1981 cccagtgtgt ccaagtacaa tgtgactggt gacaatggaa cctgcctgct ggcctctatg
     2041 gcactgcaac tcaacatcac ctacatgaag aaggacaaca cgactgtgac cagagcattc
     2101 aacatcaacc caagtgacaa atatagtggg acttgcggtg cccagttggt gaccctgaag
     2161 gtggggaaca agagcagagt cctggagctg cagtttggga tgaatgccac ttctagcctg
     2221 tttttcctgc aaggagttca gttgaacatg actcttcctg atgccataga gcccacgttc
     2281 agcacctcca actattccct gaaagctctt caggccagtg tcggcaactc atacaagtgc
     2341 aacagtgagg agcacatctt tgtcagcaag gcgctcgccc tcaatgtctt cagcgtgcaa
     2401 gtccaggctt tcagggtaga aagtgacagg tttgggtctg tggaagagtg tgtacaggac
     2461 ggtaacaaca tgctgatccc cattgctgtg ggcggggccc tggcagggct ggtcctcatc
     2521 gtcctcatcg cctacctcat cggcaggaag aggagtcacg cgggctatca gaccatccgg
     2581 gatccaccgg tcgccaccat ggcctcctcc gaggacgtca tcaaggagtt catgcgcttc
     2641 aaggtgcgca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag
     2701 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg
     2761 cccttcgcct gggacatcct gtcccctcag ttccagtacg gctccaaggc ctacgtgaag
     2821 caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag
     2881 cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag
     2941 gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc
     3001 gtaatgcaga agaagaccat gggctgggag gcctccaccg agcggatgta ccccgaggac
     3061 ggcgccctga agggcgagat caagatgagg ctgaagctga aggacggcgg ccactacgac
     3121 gccgaggtca agaccaccta catggccaag aagcccgtgc agctgcccgg cgcctacaag
     3181 accgacatca agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     3241 gagcgcgccg agggccgcca ctccaccggc gccttgtaca agtaaagcgg ccgcgactct
     3301 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     3361 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     3421 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3481 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa ggcgtaaatt
     3541 gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3601 aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg
     3661 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3721 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca
     3781 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3841 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3901 ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3961 gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc
     4021 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     4081 taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc ggaaagaacc
     4141 agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa
     4201 gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc
     4261 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc
     4321 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     4381 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     4441 agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca agagacagga
     4501 tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg
     4561 gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc
     4621 gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt
     4681 gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt
     4741 ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc
     4801 gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc
     4861 atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac
     4921 caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag
     4981 gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag
     5041 gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat
     5101 atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg
     5161 gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa
     5221 tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc
     5281 ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgacc
     5341 aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt
     5401 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     5461 tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag gagacaatac
     5521 cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac ggtgttgggt
     5581 cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat accccaccga
     5641 gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac cccccaagtt
     5701 cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc atagcctcag
     5761 gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5821 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5881 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5941 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6001 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6061 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6121 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     6181 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     6241 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     6301 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     6361 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     6421 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     6481 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     6541 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     6601 accgtattac cgccatgcat
//
