LOCUS       sequence-355811-        5972 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lysosomal expression of ratiometric pHLuorin2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5972)
  AUTHORS   Linders PTA, Ioannidis M, Ter Beest M, van den Bogaart G
  TITLE     Fluorescence Lifetime Imaging of pH along the Secretory Pathway.
  JOURNAL   ACS Chem Biol. 2022 Jan 21;17(1):240-251. doi: 
            10.1021/acschembio.1c00907. Epub 2022 Jan 10.
  PUBMED    35000377
REFERENCE   2  (bases 1 to 5972)
  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: 
            "10.1021/acschembio.1c00907"; journalName: "ACS Chem Biol"; date: 
            "2022-01-21- 21"; volume: "17"; issue: "1"; pages: "240-251"
COMMENT     Sequence Label: LAMP1-RpHLuorin2
FEATURES             Location/Qualifiers
     source          1..5972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        75..378
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        379..582
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     533..553
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     complement(1060..1079)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2774..2895
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2811..2830)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2865..2884
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2902..3357)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3039..3060)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3251..3270
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3384..3488
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3490..3847
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3490..3681
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3513..3533)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3698..3833
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3882..4676
                     /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             3882..4676
                     /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(3936..3955)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4546..4565
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4864..4883)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4908..4955
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5284..5872
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5773..5792
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttaccgccat gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
       61 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      121 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      181 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      241 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      301 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      361 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      421 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      481 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      541 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
      601 atccgctagc gctaccggac tcagatctcg agctcaagct tcgaattcag ggacatggcg
      661 gcccccggca gcgcccggcg acccctgctg ctgctactgc tgttgctgct gctcggcctc
      721 atgcattgtg cgtcagcagc aacaccggtc gccaccatga gcaagggcga ggagctgttc
      781 accggcgtgg tgcccatcct ggtggagctg gacggcgacg tgaacggcca caagttcagc
      841 gtgagcggcg agggcgaggg cgacgccacc tacggcaagc tgaccctgaa gttcatctgc
      901 accaccggca agctgcccgt gccctggccc accctggtga ccaccctgag ctacggcgtg
      961 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gagcgccatg
     1021 cccgagggct acgtgcagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1081 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1141 gacttcaagg aggacggcaa catcctgggc cacaagctgg agtacaacta caacgagcac
     1201 ctggtctaca tcatggccga caagcagaag aacggcacca aggccatctt ccaggtgcac
     1261 cacaacatcg aggacggcgg cgtgcagctg gccgaccact accagcagaa cacccccatc
     1321 ggcgacggcc ccgtgctgct gcccgacaac cactacctgc acacccagag cgccctgagc
     1381 aaggacccca acgagaagcg cgaccacatg gtgctgctgg agttcgtgac cgccgccggc
     1441 atcacccacg gcatggacga gctgtacaaa ggctcaggct cagcaatgtt tatggtgaaa
     1501 aatggcaacg ggaccgcgtg cataatggcc aacttctctg ctgccttctc agtgaactac
     1561 gacaccaaga gtggccctaa gaacatgacc tttgacctgc catcagatgc cacagtggtg
     1621 ctcaaccgca gctcctgtgg aaaagagaac acttctgacc ccagtctcgt gattgctttt
     1681 ggaagaggac atacactcac tctcaatttc acgagaaatg caacacgtta cagcgtccag
     1741 ctcatgagtt ttgtttataa cttgtcagac acacaccttt tccccaatgc gagctccaaa
     1801 gaaatcaaga ctgtggaatc tataactgac atcagggcag atatagataa aaaatacaga
     1861 tgtgttagtg gcacccaggt ccacatgaac aacgtgaccg taacgctcca tgatgccacc
     1921 atccaggcgt acctttccaa cagcagcttc agcaggggag agacacgctg tgaacaagac
     1981 aggccttccc caaccacagc gccccctgcg ccacccagcc cctcgccctc acccgtgccc
     2041 aagagcccct ctgtggacaa gtacaacgtg agcggcacca acgggacctg cctgctggcc
     2101 agcatggggc tgcagctgaa cctcacctat gagaggaagg acaacacgac ggtgacaagg
     2161 cttctcaaca tcaaccccaa caagacctcg gccagcggga gctgcggcgc ccacctggtg
     2221 actctggagc tgcacagcga gggcaccacc gtcctgctct tccagttcgg gatgaatgca
     2281 agttctagcc ggtttttcct acaaggaatc cagttgaata caattcttcc tgacgccaga
     2341 gaccccgcct ttaaagctgc caacggctcc ctgcgagcgc tgcaggccac agtcggcaat
     2401 tcctacaagt gcaacgcgga ggagcacgtc cgtgtcacga aggcgttttc agtcaatata
     2461 ttcaaagtgt gggtccaggc tttcaaggtg gaaggtggcc agtttggctc tgtggaggag
     2521 tgtctgctgg acgagaacag catgctgatc cccatcgctg tgggtggtgc cctggcgggg
     2581 ctggtcctca tcgtcctcat cgcctacctc gtcggcagga agaggagtca cgcaggctac
     2641 cagactatct aagcggccgc gactctagat cataatcagc cataccacat ttgtagaggt
     2701 tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc
     2761 aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     2821 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     2881 catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     2941 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     3001 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     3061 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     3121 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     3181 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     3241 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     3301 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag
     3361 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3421 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3481 ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa
     3541 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     3601 caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3661 ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag
     3721 ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc
     3781 cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt
     3841 ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa caagatggat
     3901 tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac tgggcacaac
     3961 agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg cgcccggttc
     4021 tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag gcagcgcggc
     4081 tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag
     4141 cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg tcatctcacc
     4201 ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg catacgcttg
     4261 atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga gcacgtactc
     4321 ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag gggctcgcgc
     4381 cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga
     4441 cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt tctggattca
     4501 tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg gctacccgtg
     4561 atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg
     4621 ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc ttctgagcgg
     4681 gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga
     4741 ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg
     4801 gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta gggggaggct
     4861 aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag
     4921 acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg
     4981 ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc gcgtttcttc
     5041 cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag ccaacgtcgg
     5101 ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg atttaaaact
     5161 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     5221 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     5281 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     5341 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     5401 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     5461 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     5521 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     5581 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     5641 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     5701 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     5761 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     5821 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     5881 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     5941 tgcgttatcc cctgattctg tggataaccg ta
//
