LOCUS       sequence-318959-        5556 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 5556)
  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 5556)
  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 RpH-LAMP1-3xFLAG
FEATURES             Location/Qualifiers
     source          1..5556
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    33..132
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     regulatory      142..151
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             225..932
                     /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"
     primer_bind     complement(370..388)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     638..657
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             954..1667
                     /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(984..1012)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1607..1627
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             2823..2888
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     protein_bind    complement(2899..2998)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3015..3034)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3016..3034)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3039..3055)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3039..3055)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3486..4277
                     /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      4370..4958
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4859..4878
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5112..5129
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(5288..5315)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(5407..5493)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(5542..5556,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(5556,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc cccaaataat gattttattt tgactgatag
       61 tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca actttgtaca
      121 aaaaagcagg ctgggaattc accatggcgg cccccggcgc ccggcggccg ctgctcctgc
      181 tgctgctggc aggccttgca catggcgcct cagcagctag cggtatggtg agcaagggcg
      241 aggaggataa catggccatc atcaaggagt tcatgcgctt caaggtgcac atggagggct
      301 ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga gggccgcccc tacgagggca
      361 cccagaccgc caagctgaag gtgaccaagg gtggccccct gcccttcgcc tgggacatcc
      421 tgtcccctca gttcatgtac ggctccaagg cctacgtgaa gcaccccgcc gacatccccg
      481 actacttgaa gctgtccttc cccgagggct tcaagtggga gcgcgtgatg aacttcgagg
      541 acggcggcgt ggtgaccgtg acccaggact cctccctgca ggacggcgag ttcatctaca
      601 aggtgaagct gcgcggcacc aacttcccct ccgacggccc cgtaatgcag aagaagacca
      661 tgggctggga ggcctcctcc gagcggatgt accccgagga cggcgccctg aagggcgaga
      721 tcaagcagag gctgaagctg aaggacggcg gccactacga cgctgaggtc aagaccacct
      781 acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa cgtcaacatc aagttggaca
      841 tcacctccca caacgaggac tacaccatcg tggaacagta cgaacgcgcc gagggccgcc
      901 actccaccgg cggcatggac gagctgtaca agggagctag cggggggccc gggatgagta
      961 aaggagaaga acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta
     1021 atgggcacaa attttctgtc agtggagagg gtgaaggtga tgcaacatac ggaaaactta
     1081 cccttaaatt tatttgcact actggaaaac tacctgttcc ttggccaaca cttgtcacta
     1141 ctttaactta tggcgttcaa tgcttttcaa gatacccaga tcatatgaaa cggcatgact
     1201 ttttcaagag tgccatgccc gaaggttatg tacaggaaag aactatattt ttcaaagatg
     1261 acgggaacta caagacacgt gctgaagtca agtttgaagg tgataccctt gttaatagaa
     1321 tcgagttaaa aggtattgat tttaaagaag atggaaacat tcttggacac aaattggaat
     1381 acaactataa cgatcaccag gtgtacatca tggcagacaa acaaaagaat ggaatcaaag
     1441 ctaacttcaa aattagacac aacattgaag atggaggcgt tcaactagca gaccattatc
     1501 aacaaaatac tccaattggc gatgggcccg tccttttacc agacaaccat tacctgttta
     1561 caacttctac tctttcgaaa gatcccaacg aaaagagaga ccacatggtc cttcttgagt
     1621 ttgtaacagc tgctgggatt acacatggca tggatgaact atacaaaccc ggggggctct
     1681 ttgaggtgaa aaacaatggc acaacgtgta taatggccag cttctctgcc tcctttctga
     1741 ccacctacga gactgcgaat ggttctcaga tcgtgaacat ttccctgcca gcctctgcag
     1801 aagtactgaa aaatggcagt tcttgtggta aagaaaatgt ttctgacccc agcctcacaa
     1861 ttacttttgg aagaggatat ttactgacac tcaacttcac aaaaaataca acacgttaca
     1921 gtgtccagca tatgtatttt acatataact tgtcagatac agaacatttt cccaatgcca
     1981 tcagcaaaga gatctacacc atggattcca caactgacat caaggcagac atcaacaaag
     2041 cataccggtg tgtcagtgat atccgggtct acatgaagaa tgtgaccgtt gtgctccggg
     2101 atgccactat ccaggcctac ctgtcgagtg gcaacttcag caaggaagag acacactgca
     2161 cacaggatgg accttcccca accactgggc cacccagccc ctcaccacca cttgtgccca
     2221 caaaccccac tgtatccaag tacaatgtta ctggtaacaa cggaacctgc ctgctggcct
     2281 ctatggcact gcaactgaat atcacctacc tgaaaaagga caacaagacg gtgaccagag
     2341 cgttcaacat cagcccaaat gacacatcta gtgggagttg cggtatcaac ttggtgaccc
     2401 tgaaagtgga gaacaagaac agagccctgg aattgcagtt tgggatgaat gccagctcta
     2461 gcctgttttt cctgcaagga gtgcgcttga atatgactct tcctgatgcc ctagtgccca
     2521 cattcagcat ctccaaccat tcactgaaag ctcttcaggc cactgtggga aactcataca
     2581 agtgcaacac tgaggaacac atctttgtca gcaagatgct ctccctcaat gtcttcagtg
     2641 tgcaggtcca ggctttcaag gtggacagtg acaggtttgg gtctgtggaa gagtgtgttc
     2701 aggatggtaa caacatgttg atccccattg ctgtgggcgg tgccctggca gggctggtcc
     2761 tcatcgtcct cattgcctac ctcattggca ggaagaggag tcacgccggc tatcagacca
     2821 tcgactacaa agaccatgac ggtgattata aagatcatga catcgactac aaggatgacg
     2881 atgacaagta ggaattccac ccagctttct tgtacaaagt tggcattata agaaagcatt
     2941 gcttatcaat ttgttgcaac gaacaggtca ctatcagtca aaataaaatc attatttgcc
     3001 atccagctga tatcccctat agtgagtcgt attacatggt catagctgtt tcctggcagc
     3061 tctggcccgt gtctcaaaat ctctgatgtt acattgcaca agataaaaat atatcatcat
     3121 gcctcctcta gaccagccag gacagaaatg cctcgacttc gctgctaccc aaggttgccg
     3181 ggtgacgcac accgtggaaa cggatgaagg cacgaaccca gtggacataa gcctgttcgg
     3241 ttcgtaagct gtaatgcaag tagcgtatgc gctcacgcaa ctggtccaga accttgaccg
     3301 aacgcagcgg tggtaacggc gcagtggcgg ttttcatggc ttgttatgac tgtttttttg
     3361 gggtacagtc tatgcctcgg gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt
     3421 tgatgttatg gagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttaa
     3481 acattatgag ggaagcggtg atcgccgaag tatcgactca actatcagag gtagttggcg
     3541 tcatcgagcg ccatctcgaa ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg
     3601 atggcggcct gaagccacac agtgatattg atttgctggt tacggtgacc gtaaggcttg
     3661 atgaaacaac gcggcgagct ttgatcaacg accttttgga aacttcggct tcccctggag
     3721 agagcgagat tctccgcgct gtagaagtca ccattgttgt gcacgacgac atcattccgt
     3781 ggcgttatcc agctaagcgc gaactgcaat ttggagaatg gcagcgcaat gacattcttg
     3841 caggtatctt cgagccagcc acgatcgaca ttgatctggc tatcttgctg acaaaagcaa
     3901 gagaacatag cgttgccttg gtaggtccag cggcggagga actctttgat ccggttcctg
     3961 aacaggatct atttgaggcg ctaaatgaaa ccttaacgct atggaactcg ccgcccgact
     4021 gggctggcga tgagcgaaat gtagtgctta cgttgtcccg catttggtac agcgcagtaa
     4081 ccggcaaaat cgcgccgaag gatgtcgctg ccgactgggc aatggagcgc ctgccggccc
     4141 agtatcagcc cgtcatactt gaagctagac aggcttatct tggacaagaa gaagatcgct
     4201 tggcctcgcg cgcagatcag ttggaagaat ttgtccacta cgtgaaaggc gagatcacca
     4261 aggtagtcgg caaataaccc tcgagccacc catgaccaaa atcccttaac gtgagttacg
     4321 cgtcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4381 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4441 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4501 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4561 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4621 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4681 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4741 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4801 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4861 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4921 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4981 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5041 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5101 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     5161 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5221 aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc tagccaggaa gagtttgtag
     5281 aaacgcaaaa aggccatccg tcaggatggc cttctgctta gtttgatgcc tggcagttta
     5341 tggcgggcgt cctgcccgcc accctccggg ccgttgcttc acaacgttca aatccgctcc
     5401 cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc
     5461 ccagtcttcc gactgagcct ttcgttttat ttgatgcctg gcagttccct actctcgcgt
     5521 taacgctagc atggatgttt tcccagtcac gacgtt
//
