LOCUS       sequence-462350-        7169 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RpH-ILV probe strongly colocalises with lysosomal markers with FRB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7169)
  AUTHORS   Cheetham-Wilkinson IJ, Sivalingam B, Flitton C, Flottmann F, Vehling
            L, Drechsler M, Stojchevska M, Raimondi A, Paululat A, Frohlich F, 
            Swan LE, Stagi M
  TITLE     RpH-ILV: Probe for lysosomal pH and acute LLOMe-induced membrane 
            permeabilization in cell lines and Drosophila.
  JOURNAL   Sci Adv. 2025 Jan 3;11(1):eadr7325. doi: 10.1126/sciadv.adr7325. 
            Epub 2025 Jan 3.
  PUBMED    39752501
REFERENCE   2  (bases 1 to 7169)
  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: "Sci Adv."; 
            date: "2025-01-3"; volume: "11(1):eadr7325. doi"; pages: " 
            10.1126/sciadv.adr7325. Epub 2025 Jan 3"
COMMENT     Sequence Label: RpH-ILV-FRB
FEATURES             Location/Qualifiers
     source          1..7169
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    828..962
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(852..871)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     906..925
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        complement(968..1163)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      982..1117
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     protein_bind    1222..1243
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1258..1263,1264..1281,1282..1288)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1296..1312
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     1301..1323
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1320..1336
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1320..1336
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1679..1698
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     polyA_signal    1697..1752
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     1798..1817
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     CDS             complement(1910..2188)
                     /codon_start=1
                     /product="FKBP-rapamycin binding domain of human FRAP"
                     /label=FRB
                     /translation="ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL
                     KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK"
     CDS             complement(2195..2218)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(2324..3031)
                     /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(2599..2618)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2868..2886
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(3305..4018)
                     /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(3345..3365)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     3960..3988
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(4301..4320)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          4328..5344
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(4382..4404)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(5345..5622)
                     /label=chicken beta-actin promoter
     enhancer        5624..6003
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6034..6138
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6139..6207,6208..6999)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6357..6376)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagcggat ccagacatga
      841 taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta
      901 tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag
      961 ttagcttttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     1021 cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg
     1081 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
     1141 tggttgctga ctaattgaga tgcggatccg ctggcacgac aggtttcccg actggaaagc
     1201 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     1261 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     1321 aggaaacagc tatgaccatg attacgccaa gcttgggctg caggtcgagg gatctccata
     1381 agagaagagg gacagctatg actgggagta gtcaggagag gaggaaaaat ctggctagta
     1441 aaacatgtaa ggaaaatttt agggatgtta aagaaaaaaa taacacaaaa caaaatataa
     1501 aaaaaatcta acctcaagtc aaggcttttc tatggaataa ggaatggaca gcagggggct
     1561 gtttcatata ctgatgacct ctttatagcc aacctttgtt catggcagcc agcatatggg
     1621 catatgttgc caaactctaa accaaatact cattctgatg ttttaaatga tttgccctcc
     1681 catatgtcct tccgagtgag agacacaaaa aattccaaca cactattgca atgaaaataa
     1741 atttccttta ttagccagaa gtcagatgct caaggggctt catgatgtcc ccataatttt
     1801 tggcagaggg aaaaagatct cagtggtatt tgtgagccag ggcattggcc acaccagcca
     1861 ccaccttctg ataggcagcc tgcacctgag gagtgaattc ttagatgggc tttgagattc
     1921 gtcggaacac atgataatag aggtcccagg cttgggtgag gtccttgaca ttccctgatt
     1981 tcatgtactt cctgcaccac tcttgggcct ccattaaatc tcgaccatag gcctgattaa
     2041 aggatgtttc cttcagagtc tgggggcccc gttccatcat agcatgcaag ggctccagca
     2101 cctcaaacat gcctttcacg ttcctttccc caaagtacaa acgagatgcc tcttccaggc
     2161 cttcatgcca catctcatgc cagaggatcc catcaggccc ttgaaacaga acctccaatc
     2221 tccttatttt gaccgtatat atcaacagac aaatcagtga gcatataagt tccagagcaa
     2281 gtattgccag aatgaggtcc agatacagcc gtccgctagc tcccttgtac agctcgtcca
     2341 tgccgccggt ggagtggcgg ccctcggcgc gttcgtactg ttccacgatg gtgtagtcct
     2401 cgttgtggga ggtgatgtcc aacttgatgt tgacgttgta ggcgccgggc agctgcacgg
     2461 gcttcttggc cttgtaggtg gtcttgacct cagcgtcgta gtggccgccg tccttcagct
     2521 tcagcctctg cttgatctcg cccttcaggg cgccgtcctc ggggtacatc cgctcggagg
     2581 aggcctccca gcccatggtc ttcttctgca ttacggggcc gtcggagggg aagttggtgc
     2641 cgcgcagctt caccttgtag atgaactcgc cgtcctgcag ggaggagtcc tgggtcacgg
     2701 tcaccacgcc gccgtcctcg aagttcatca cgcgctccca cttgaagccc tcggggaagg
     2761 acagcttcaa gtagtcgggg atgtcggcgg ggtgcttcac gtaggccttg gagccgtaca
     2821 tgaactgagg ggacaggatg tcccaggcga agggcagggg gccacccttg gtcaccttca
     2881 gcttggcggt ctgggtgccc tcgtaggggc ggccctcgcc ctcgccctcg atctcgaact
     2941 cgtggccgtt cacggagccc tccatgtgca ccttgaagcg catgaactcc ttgatgatgg
     3001 ccatgttatc ctcctcgccc ttgctcacca taccgctagc accgcctggt tccgggaaag
     3061 aatgttgcat acacaagatc aacaagagta cagtatttcc agaggactga atcatcaacg
     3121 caagtctctt cagatgccgc gtggatcgat atataagatt gtatccgcga tttctgattt
     3181 ttgaatgatg gtattgtata tagcactcca ggaggagatg cagtatccag agtataactt
     3241 tacccagaat gatcactgtt tgcactttga gaggattggt gtagttccca ggacatttcc
     3301 cgggtttgta tagttcatcc atgccatgtg taatcccagc agctgttaca aactcaagaa
     3361 ggaccatgtg gtctctcttt tcgttgggat ctttcgaaag agtagaagtt gtaaacaggt
     3421 aatggttgtc tggtaaaagg acgggcccat cgccaattgg agtattttgt tgataatggt
     3481 ctgctagttg aacgcctcca tcttcaatgt tgtgtctaat tttgaagtta gctttgattc
     3541 cattcttttg tttgtctgcc atgatgtaca cctggtgatc gttatagttg tattccaatt
     3601 tgtgtccaag aatgtttcca tcttctttaa aatcaatacc ttttaactcg attctattaa
     3661 caagggtatc accttcaaac ttgacttcag cacgtgtctt gtagttcccg tcatctttga
     3721 aaaatatagt tctttcctgt acataacctt cgggcatggc actcttgaaa aagtcatgcc
     3781 gtttcatatg atctgggtat cttgaaaagc attgaacgcc ataagttaaa gtagtgacaa
     3841 gtgttggcca aggaacaggt agttttccag tagtgcaaat aaatttaagg gtaagttttc
     3901 cgtatgttgc atcaccttca ccctctccac tgacagaaaa tttgtgccca ttaacatcac
     3961 catctaattc aacaagaatt gggacaactc cagtgaaaag ttcttctcct ttactcatcc
     4021 cgggcccacc gtcctcgttg gggttagggt aactgcaaag aacaccagta agaaaggcaa
     4081 gaactacaaa aacgagatgt ataaaccaca ggaggttcac tatgataact gttggcagag
     4141 gatgaaagcg cgggcgaaaa tgcgcttgca aactatgatg tggcaaaagc tgagcgtcga
     4201 gcaaggggtc gtcctctatg gattgagtaa tgtcaagaga accatcctcc attcgacctc
     4261 cagcagccat ggtgaattct ttgccaaaat gatgagacag cacaataacc agcacgttgc
     4321 ccaggagctg taggaaaaag aagaaggcat gaacatggtt agcagaggct ctagagccgc
     4381 cggtcacacg ccagaagccg aaccccgccc tgccccgtcc cccccgaagg cagccgtccc
     4441 cccgcggaca gccccgaggc tggagaggga gaaggggacg gcggcgcggc gacgcacgaa
     4501 ggccctcccc gcccatttcc ttcctgccgg cgccgcaccg cttcgccccg cgcccgctag
     4561 agggggtgcg gcggcgcctc ccagatttcg gctccgcaca gatttgggac aaaggaagtc
     4621 cctgcgccct ctcgcacgat taccataaaa ggcaatggct gcggctcgcc gcgcctcgac
     4681 agccgccggc gctccggggg ccgccgcgcc cctcccccga gccctccccg gcccgaggcg
     4741 gccccgcccc gcccggcacc cccacctgcc gccacccccc gcccggcacg gcgagccccg
     4801 cgccacgccc cgtacggagc cccgcacccg aagccgggcc gtgctcagca actcggggag
     4861 gggggtgcag ggggggttgc agcccgaccg acgcgcccac accccctgct caccccccca
     4921 cgcacacacc ccgcacgcag cctttgttcc cctcgcagcc ccccccgcac cgcggggcac
     4981 cgcccccggc cgcgctcccc tcgcgcacac tgcggagcgc acaaagcccc gcgccgcgcc
     5041 cgcagcgctc acagccgccg ggcagcgcgg agccgcacgc ggcgctcccc acgcacacac
     5101 acacgcacgc accccccgag ccgctccccc cgcacaaagg gccctcccgg agcccctcaa
     5161 ggctttcacg cagccacaga aaagaaacaa gccgtcatta aaccaagcgc taattacagc
     5221 ccggaggaga agggccgtcc cgcccgctca cctgtgggag taacgcggtc agtcagagcc
     5281 ggggcgggcg gcgcgaggcg gcggcggagc ggggcacggg gcgaaggcag cgcgcagcga
     5341 ctcccgcccg ccgcgcgctt cgctttttat agggccgccg ccgccgccgc ctcgccataa
     5401 aaggaaactt tcggagcgcg ccgctctgat tggctgccgc cgcacctctc cgcctcgccc
     5461 cgccccgccc ctcgccccgc cccgccccgc ctggcgcgcg cccccccccc cccccgcccc
     5521 catcgctgca caaaataatt aaaaaataaa taaatacaaa attgggggtg gggagggggg
     5581 gggagatggg gagagtgaag cagaacgtgg ggctcacctc gaccatggta atagcgatga
     5641 ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat
     5701 gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca
     5761 cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga
     5821 aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt
     5881 cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat
     5941 atgggctatg aactaatgac cccgtaattg attactatta ataactagtc aataatcaat
     6001 gtcgacccag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     6061 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6121 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6181 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6241 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6301 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6361 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6421 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6481 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6541 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6601 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6661 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6721 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6781 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6841 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6901 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6961 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     7021 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     7081 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7141 gaccccgtag aaaagatcaa aggatcttc
//
