LOCUS       sequence-462349-        7047 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  RpH-ILV probe strongly colocalises with lysosomal markers..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7047)
  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 7047)
  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-3xALFA
FEATURES             Location/Qualifiers
     source          1..7047
                     /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..1948)
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             complement(1964..2002)
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             complement(2018..2056)
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             complement(2072..2095)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(2201..2908)
                     /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(2476..2495)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2745..2763
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(3182..3895)
                     /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(3222..3242)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     3837..3865
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(4178..4197)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          4205..5221
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(4259..4281)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(5222..5500)
                     /label=chicken beta-actin promoter
     enhancer        5502..5881
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5912..6016
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6017..6085,6086..6877)
                     /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(6235..6254)
                     /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 ttagatatct tccgtgaggc
     1921 gtcggcgaag ttcttcttca agccgagaac cccctccgat atcctcagtc aaccttctcc
     1981 tgagctcctc ttccagtcgg gatccaccac cgatatcctc cgtaagccgt cgcctcaact
     2041 cttcttcgag tcttgaacct cccccgatat caggcccttg aaacagaacc tccaatctcc
     2101 ttattttgac cgtatatatc aacagacaaa tcagtgagca tataagttcc agagcaagta
     2161 ttgccagaat gaggtccaga tacagccgtc cgctagctcc cttgtacagc tcgtccatgc
     2221 cgccggtgga gtggcggccc tcggcgcgtt cgtactgttc cacgatggtg tagtcctcgt
     2281 tgtgggaggt gatgtccaac ttgatgttga cgttgtaggc gccgggcagc tgcacgggct
     2341 tcttggcctt gtaggtggtc ttgacctcag cgtcgtagtg gccgccgtcc ttcagcttca
     2401 gcctctgctt gatctcgccc ttcagggcgc cgtcctcggg gtacatccgc tcggaggagg
     2461 cctcccagcc catggtcttc ttctgcatta cggggccgtc ggaggggaag ttggtgccgc
     2521 gcagcttcac cttgtagatg aactcgccgt cctgcaggga ggagtcctgg gtcacggtca
     2581 ccacgccgcc gtcctcgaag ttcatcacgc gctcccactt gaagccctcg gggaaggaca
     2641 gcttcaagta gtcggggatg tcggcggggt gcttcacgta ggccttggag ccgtacatga
     2701 actgagggga caggatgtcc caggcgaagg gcagggggcc acccttggtc accttcagct
     2761 tggcggtctg ggtgccctcg taggggcggc cctcgccctc gccctcgatc tcgaactcgt
     2821 ggccgttcac ggagccctcc atgtgcacct tgaagcgcat gaactccttg atgatggcca
     2881 tgttatcctc ctcgcccttg ctcaccatac cgctagcacc gcctggttcc gggaaagaat
     2941 gttgcataca caagatcaac aagagtacag tatttccaga ggactgaatc atcaacgcaa
     3001 gtctcttcag atgccgcgtg gatcgatata taagattgta tccgcgattt ctgatttttg
     3061 aatgatggta ttgtatatag cactccagga ggagatgcag tatccagagt ataactttac
     3121 ccagaatgat cactgtttgc actttgagag gattggtgta gttcccagga catttcccgg
     3181 gtttgtatag ttcatccatg ccatgtgtaa tcccagcagc tgttacaaac tcaagaagga
     3241 ccatgtggtc tctcttttcg ttgggatctt tcgaaagagt agaagttgta aacaggtaat
     3301 ggttgtctgg taaaaggacg ggcccatcgc caattggagt attttgttga taatggtctg
     3361 ctagttgaac gcctccatct tcaatgttgt gtctaatttt gaagttagct ttgattccat
     3421 tcttttgttt gtctgccatg atgtacacct ggtgatcgtt atagttgtat tccaatttgt
     3481 gtccaagaat gtttccatct tctttaaaat caataccttt taactcgatt ctattaacaa
     3541 gggtatcacc ttcaaacttg acttcagcac gtgtcttgta gttcccgtca tctttgaaaa
     3601 atatagttct ttcctgtaca taaccttcgg gcatggcact cttgaaaaag tcatgccgtt
     3661 tcatatgatc tgggtatctt gaaaagcatt gaacgccata agttaaagta gtgacaagtg
     3721 ttggccaagg aacaggtagt tttccagtag tgcaaataaa tttaagggta agttttccgt
     3781 atgttgcatc accttcaccc tctccactga cagaaaattt gtgcccatta acatcaccat
     3841 ctaattcaac aagaattggg acaactccag tgaaaagttc ttctccttta ctcatcccgg
     3901 gcccaccgtc ctcgttgggg ttagggtaac tgcaaagaac accagtaaga aaggcaagaa
     3961 ctacaaaaac gagatgtata aaccacagga ggttcactat gataactgtt ggcagaggat
     4021 gaaagcgcgg gcgaaaatgc gcttgcaaac tatgatgtgg caaaagctga gcgtcgagca
     4081 aggggtcgtc ctctatggat tgagtaatgt caagagaacc atcctccatt cgacctccag
     4141 cagccatggt gaattctttg ccaaaatgat gagacagcac aataaccagc acgttgccca
     4201 ggagctgtag gaaaaagaag aaggcatgaa catggttagc agaggctcta gagccgccgg
     4261 tcacacgcca gaagccgaac cccgccctgc cccgtccccc ccgaaggcag ccgtcccccc
     4321 gcggacagcc ccgaggctgg agagggagaa ggggacggcg gcgcggcgac gcacgaaggc
     4381 cctccccgcc catttccttc ctgccggcgc cgcaccgctt cgccccgcgc ccgctagagg
     4441 gggtgcggcg gcgcctccca gatttcggct ccgcacagat ttgggacaaa ggaagtccct
     4501 gcgccctctc gcacgattac cataaaaggc aatggctgcg gctcgccgcg cctcgacagc
     4561 cgccggcgct ccgggggccg ccgcgcccct cccccgagcc ctccccggcc cgaggcggcc
     4621 ccgccccgcc cggcaccccc acctgccgcc accccccgcc cggcacggcg agccccgcgc
     4681 cacgccccgt acggagcccc gcacccgaag ccgggccgtg ctcagcaact cggggagggg
     4741 ggtgcagggg gggttgcagc ccgaccgacg cgcccacacc ccctgctcac ccccccacgc
     4801 acacaccccg cacgcagcct ttgttcccct cgcagccccc cccgcaccgc ggggcaccgc
     4861 ccccggccgc gctcccctcg cgcacactgc ggagcgcaca aagccccgcg ccgcgcccgc
     4921 agcgctcaca gccgccgggc agcgcggagc cgcacgcggc gctccccacg cacacacaca
     4981 cgcacgcacc ccccgagccg ctccccccgc acaaagggcc ctcccggagc ccctcaaggc
     5041 tttcacgcag ccacagaaaa gaaacaagcc gtcattaaac caagcgctaa ttacagcccg
     5101 gaggagaagg gccgtcccgc ccgctcacct gtgggagtaa cgcggtcagt cagagccggg
     5161 gcgggcggcg cgaggcggcg gcggagcggg gcacggggcg aaggcagcgc gcagcgactc
     5221 ccgcccgccg cgcgcttcgc tttttatagg gccgccgccg ccgccgcctc gccataaaag
     5281 gaaactttcg gagcgcgccg ctctgattgg ctgccgccgc acctctccgc ctcgccccgc
     5341 cccgcccctc gccccgcccc gccccgcctg gcgcgcgccc cccccccccc cccgccccca
     5401 tcgctgcaca aaataattaa aaaataaata aatacaaaat tgggggtggg gagggggggg
     5461 gagatgggga gagtgaagca gaacgtgggg ctcacctcga ccatggtaat agcgatgact
     5521 aatacgtaga tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc
     5581 caggcgggcc atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact
     5641 tgatgtactg ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa
     5701 gtccctattg gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg
     5761 ttgggcggtc agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat
     5821 gggctatgaa ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt
     5881 cgacccaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     5941 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     6001 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     6061 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     6121 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     6181 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     6241 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     6301 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     6361 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     6421 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     6481 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     6541 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     6601 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     6661 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     6721 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     6781 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     6841 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     6901 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     6961 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     7021 ccccgtagaa aagatcaaag gatcttc
//
