LOCUS       sequence-311981-        6276 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gateway donor vector encoding ratiometric sensor of lysosomal 
            lumenal pH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6276)
  AUTHORS   Ponsford AH, Ryan TA, Raimondi A, Cocucci E, Wycislo SA, Frohlich F,
            Swan LE, Stagi M
  TITLE     Live imaging of intra-lysosome pH in cell lines and primary neuronal
            culture using a novel genetically encoded biosensor.
  JOURNAL   Autophagy. 2020 Jun 9:1-19. doi: 10.1080/15548627.2020.1771858.
  PUBMED    32515674
REFERENCE   2  (bases 1 to 6276)
  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"; journalName: "Autophagy. 
            2020 Jun 9:1-19. doi: 10.1080/15548627.2020.1771858."
COMMENT     Sequence Label: pDONOR223  R2pH-LAMP1-3xFLAG
FEATURES             Location/Qualifiers
     source          1..6276
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(1..28)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(120..206)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     255..277
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     269..286
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     270..286
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    302..401
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      411..420
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             494..1201
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             1214..1921
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     CDS             1943..2656
                     /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(1973..2001)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2596..2616
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             3812..3877
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(3888..3987)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(4004..4023)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4005..4023)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4028..4044)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4028..4044)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4475..5266
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      5359..5947
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5848..5867
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6101..6118
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaaggccat ccgtcaggat ggccttctgc ttagtttgat gcctggcagt ttatggcggg
       61 cgtcctgccc gccaccctcc gggccgttgc ttcacaacgt tcaaatccgc tcccggcgga
      121 tttgtcctac tcaggagagc gttcaccgac aaacaacaga taaaacgaaa ggcccagtct
      181 tccgactgag cctttcgttt tatttgatgc ctggcagttc cctactctcg cgttaacgct
      241 agcatggatg ttttcccagt cacgacgttg taaaacgacg gccagtctta agctcgggcc
      301 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca
      361 atgctttttt ataatgccaa ctttgtacaa aaaagcaggc tgggaattca ccatggcggc
      421 ccccggcgcc cggcggccgc tgctcctgct gctgctggca ggccttgcac atggcgcctc
      481 agcagctagc ggtatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
      541 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
      601 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
      661 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
      721 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
      781 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
      841 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
      901 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
      961 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     1021 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     1081 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     1141 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     1201 gggagctagc ggtatggtga gcaagggcga ggaggataac atggccatca tcaaggagtt
     1261 catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg
     1321 cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg
     1381 tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc
     1441 ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc ccgagggctt
     1501 caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc
     1561 ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca acttcccctc
     1621 cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg agcggatgta
     1681 ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg
     1741 ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg
     1801 cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact acaccatcgt
     1861 ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa
     1921 gggagctagc ggggggcccg ggatgagtaa aggagaagaa cttttcactg gagttgtccc
     1981 aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg
     2041 tgaaggtgat gcaacatacg gaaaacttac ccttaaattt atttgcacta ctggaaaact
     2101 acctgttcct tggccaacac ttgtcactac tttaacttat ggcgttcaat gcttttcaag
     2161 atacccagat catatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt
     2221 acaggaaaga actatatttt tcaaagatga cgggaactac aagacacgtg ctgaagtcaa
     2281 gtttgaaggt gatacccttg ttaatagaat cgagttaaaa ggtattgatt ttaaagaaga
     2341 tggaaacatt cttggacaca aattggaata caactataac gatcaccagg tgtacatcat
     2401 ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa attagacaca acattgaaga
     2461 tggaggcgtt caactagcag accattatca acaaaatact ccaattggcg atgggcccgt
     2521 ccttttacca gacaaccatt acctgtttac aacttctact ctttcgaaag atcccaacga
     2581 aaagagagac cacatggtcc ttcttgagtt tgtaacagct gctgggatta cacatggcat
     2641 ggatgaacta tacaaacccg gggggctctt tgaggtgaaa aacaatggca caacgtgtat
     2701 aatggccagc ttctctgcct cctttctgac cacctacgag actgcgaatg gttctcagat
     2761 cgtgaacatt tccctgccag cctctgcaga agtactgaaa aatggcagtt cttgtggtaa
     2821 agaaaatgtt tctgacccca gcctcacaat tacttttgga agaggatatt tactgacact
     2881 caacttcaca aaaaatacaa cacgttacag tgtccagcat atgtatttta catataactt
     2941 gtcagataca gaacattttc ccaatgccat cagcaaagag atctacacca tggattccac
     3001 aactgacatc aaggcagaca tcaacaaagc ataccggtgt gtcagtgata tccgggtcta
     3061 catgaagaat gtgaccgttg tgctccggga tgccactatc caggcctacc tgtcgagtgg
     3121 caacttcagc aaggaagaga cacactgcac acaggatgga ccttccccaa ccactgggcc
     3181 acccagcccc tcaccaccac ttgtgcccac aaaccccact gtatccaagt acaatgttac
     3241 tggtaacaac ggaacctgcc tgctggcctc tatggcactg caactgaata tcacctacct
     3301 gaaaaaggac aacaagacgg tgaccagagc gttcaacatc agcccaaatg acacatctag
     3361 tgggagttgc ggtatcaact tggtgaccct gaaagtggag aacaagaaca gagccctgga
     3421 attgcagttt gggatgaatg ccagctctag cctgtttttc ctgcaaggag tgcgcttgaa
     3481 tatgactctt cctgatgccc tagtgcccac attcagcatc tccaaccatt cactgaaagc
     3541 tcttcaggcc actgtgggaa actcatacaa gtgcaacact gaggaacaca tctttgtcag
     3601 caagatgctc tccctcaatg tcttcagtgt gcaggtccag gctttcaagg tggacagtga
     3661 caggtttggg tctgtggaag agtgtgttca ggatggtaac aacatgttga tccccattgc
     3721 tgtgggcggt gccctggcag ggctggtcct catcgtcctc attgcctacc tcattggcag
     3781 gaagaggagt cacgccggct atcagaccat cgactacaaa gaccatgacg gtgattataa
     3841 agatcatgac atcgactaca aggatgacga tgacaagtag gaattccacc cagctttctt
     3901 gtacaaagtt ggcattataa gaaagcattg cttatcaatt tgttgcaacg aacaggtcac
     3961 tatcagtcaa aataaaatca ttatttgcca tccagctgat atcccctata gtgagtcgta
     4021 ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc tctgatgtta
     4081 cattgcacaa gataaaaata tatcatcatg cctcctctag accagccagg acagaaatgc
     4141 ctcgacttcg ctgctaccca aggttgccgg gtgacgcaca ccgtggaaac ggatgaaggc
     4201 acgaacccag tggacataag cctgttcggt tcgtaagctg taatgcaagt agcgtatgcg
     4261 ctcacgcaac tggtccagaa ccttgaccga acgcagcggt ggtaacggcg cagtggcggt
     4321 tttcatggct tgttatgact gtttttttgg ggtacagtct atgcctcggg catccaagca
     4381 gcaagcgcgt tacgccgtgg gtcgatgttt gatgttatgg agcagcaacg atgttacgca
     4441 gcagggcagt cgccctaaaa caaagttaaa cattatgagg gaagcggtga tcgccgaagt
     4501 atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac cgacgttgct
     4561 ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca gtgatattga
     4621 tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt tgatcaacga
     4681 ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg tagaagtcac
     4741 cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg aactgcaatt
     4801 tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca cgatcgacat
     4861 tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg taggtccagc
     4921 ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc taaatgaaac
     4981 cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg tagtgcttac
     5041 gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg atgtcgctgc
     5101 cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg aagctagaca
     5161 ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt tggaagaatt
     5221 tgtccactac gtgaaaggcg agatcaccaa ggtagtcggc aaataaccct cgagccaccc
     5281 atgaccaaaa tcccttaacg tgagttacgc gtcgttccac tgagcgtcag accccgtaga
     5341 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     5401 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     5461 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
     5521 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     5581 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
     5641 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
     5701 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
     5761 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
     5821 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
     5881 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
     5941 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
     6001 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
     6061 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
     6121 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     6181 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatac
     6241 gcgtaccgct agccaggaag agtttgtaga aacgca
//
