LOCUS       sequence-274438-        6528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  genetically encoded pH sensor made from mutagenesis of the red 
            fluorescent protein mKeima.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6528)
  AUTHORS   Tantama M, Hung YP, Yellen G
  TITLE     Imaging Intracellular pH in Live Cells with a Genetically Encoded 
            Red Fluorescent Protein Sensor.
  JOURNAL   J Am Chem Soc. 2011 Jul 6. 133(26):10034-7.
  PUBMED    21631110
REFERENCE   2  (bases 1 to 6528)
  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: "J Am Chem 
            Soc. 2011 Jul 6. 133(26):10034-7."
COMMENT     Sequence Label: GW1-pHRed
FEATURES             Location/Qualifiers
     source          1..6528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(168..756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(248..267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(927..1718,1719..1787))
                     /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     1550..1569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(1844..2357)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2039..2060)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2251..2270
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2529..2547
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2585..2607)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2707..2726
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2958..2980
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2972..2989
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2973..2989
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        3555..3934
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3935..4138
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4089..4109
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4135..4159
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          4274..5100
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
     CDS             join(5306..5308,5309..5311,5312..5971)
                     /codon_start=1
                     /product="red fluorescent protein that serves as a 
                     ratiometric pH sensor (Tantama et al., 2011)"
                     /label=pHRed
                     /note="contains an A213S mutation relative to the parental 
                     mKeima protein"
                     /translation="MVSVIAKQMTYKVYMSGTVNGHYFEVEGDGKGKPYEGEQTVKLTV
                     TKGGPLPFAWDILSPQLQYGSIPFTKYPEDIPDYFKQSFPEGYTWERSMNFEDGAVCTV
                     SNDSSIQGNCFIYNVKISGENFPPNGPVMQKKTQGWEPSTERLFARDGMLIGNDYMALK
                     LEGGGHYLCEFKSTYKAKKPVRMPGRHEIDRKLDVTSHNRDYTSVEQCEISIARHSLLG
                     "
     polyA_signal    6138..6272
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6175..6194)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6229..6248
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6295..6311)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6295..6311)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6308..6330)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6319..6335
                     /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)."
     promoter        complement(join(6343..6349,6350..6367,6368..6373))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(join(6525..6528,1..14))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
       61 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      121 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      181 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      241 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      301 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      361 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      421 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      481 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      541 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      601 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      661 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      721 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      781 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
      841 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
      901 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
      961 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1021 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     1081 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1141 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1201 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1261 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     1321 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     1381 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     1441 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     1501 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     1561 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     1621 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     1681 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     1741 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     1801 caataagcgg ccgcggccat gccggccact agtctcgagg gccaccccgg ttgataatca
     1861 gaaaagcccc aaaaacagga agattgtata agcaaatatt taaattgtaa gcgttaatat
     1921 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     1981 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     2041 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     2101 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     2161 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     2221 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     2281 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     2341 gccgctacag ggcgcgtggg gatccctcga gttattattg aagcatttat cagggttatt
     2401 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     2461 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     2521 cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg
     2581 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     2641 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta
     2701 actatgcggc atcagagcag attgtactga gagtgcacca tatgaagacg tcgcctcctc
     2761 actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa ataaaaaaaa
     2821 ttagtcaggg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg
     2881 ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg
     2941 ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg cccgtgtcaa
     3001 ggacggtgac tgcagtgaat aataaaatgt gtgtttgtcc gaaatacgcg ttttgagatt
     3061 tctgtcgccg actaaattca tgtcgcgcga tagtggtgtt tatcgccgat agagatggcg
     3121 atattggaaa aatcgatatt tgaaaatatg gcatattgaa aatgtcgccg atgtgagttt
     3181 ctgtgtaact gatatcgcca tttttccaaa agtgattttt gggcatacgc gatatctggc
     3241 gatagcgctt atatcgttta cgggggatgg cgatagacga ctttggtgac ttgggcgatt
     3301 ctgtgtgtcg caaatatcgc agtttcgata taggtgacag acgatatgag gctatatcgc
     3361 cgatagaggc gacatcaagc tggcacatgg ccaatgcata tcgatctata cattgaatca
     3421 atattggcca ttagccatat tattcattgg ttatatagca taaatcaata ttggctattg
     3481 gccattgcat acgttgtatc catatcataa tatgtacatt tatattggct catgtccaac
     3541 attaccgcca tgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
     3601 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
     3661 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
     3721 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
     3781 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
     3841 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
     3901 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
     3961 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
     4021 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     4081 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctcg
     4141 tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag
     4201 acaccgggac cgatccagcc tccgcggccg ggaacggtgc attggaacgc ggattccccg
     4261 tgccaagagt gacgtaagta ccgcctatag agtctatagg cccaccccct tggcttctta
     4321 tgcatgctat actgtttttg gcttggggtc tatacacccc cgcttcctca tgttataggt
     4381 gatggtatag cttagcctat aggtgtgggt tattgaccat tattgaccac tcccctattg
     4441 gtgacgatac tttccattac taatccataa catggctctt tgccacaact ctctttattg
     4501 gctatatgcc aatacactgt ccttcagaga ctgacacgga ctctgtattt ttacaggatg
     4561 gggtctcatt tattatttac aaattcacat atacaacacc accgtcccca gtgcccgcag
     4621 tttttattaa acataacgtg ggatctccac gcgaatctcg ggtacgtgtt ccggacatgg
     4681 gctcttctcc ggtagcggcg gagcttctac atccgagccc tgctcccatg cctccagcga
     4741 ctcatggtcg ctcggcagct ccttgctcct aacagtggag gccagactta ggcacagcac
     4801 gatgcccacc accaccagtg tgccgcacaa ggccgtggcg gtagggtatg tgtctgaaaa
     4861 tgagctcggg gagcgggctt gcaccgctga cgcatttgga agacttaagg cagcggcaga
     4921 agaagatgca ggcagctgag ttgttgtgtt ctgataagag tcagaggtaa ctcccgttgc
     4981 ggtgctgtta acggtggagg gcagtgtagt ctgagcagta ctcgttgctg ccgcgcgcgc
     5041 caccagacat aatagctgac agactaacag actgttcctt tccatgggtc ttttctgcag
     5101 tcaccgtcct tgacacgaag cttcccggcc acagcaatcg atccatgaca caaccagtga
     5161 caagcagtcc cccagtgaaa ccgcatccgc atccgagtcc gataccgata aagattctga
     5221 atcggagtga gtgccgcgtc cgagagcgtt ccctgtccac gtccaccatc ggcggagcag
     5281 gtgtgcctga ggcctctaga ccaccatggt ttccgttatc gcaaagcaaa tgacctataa
     5341 agtttatatg tctggcaccg ttaacggcca ctacttcgag gttgaaggtg acggcaaggg
     5401 caagccttac gagggcgagc agaccgttaa gctgaccgtt accaaaggcg gcccactgcc
     5461 tttcgcctgg gatatcctgt cccctcagct ccaatacggc tccatccctt tcaccaaata
     5521 ccccgaggac atccctgact actttaagca gagcttccca gagggctaca cctgggagcg
     5581 ctctatgaac ttcgaggacg gcgcagtttg taccgtttcc aacgacagct ccatccaggg
     5641 taactgcttc atctacaacg ttaagatttc cggcgagaac ttcccaccta acggcccagt
     5701 tatgcagaag aaaacccaag gctgggagcc cagcaccgaa cgcctgtttg cccgcgacgg
     5761 tatgctgatt ggtaacgatt atatggccct gaaactggaa ggcggcggtc actacctgtg
     5821 cgagttcaag agcacttaca aggctaaaaa gcctgttcgc atgcctggcc gccacgaaat
     5881 cgaccgcaag ctggatgtta cctctcacaa ccgcgactac accagcgttg agcagtgtga
     5941 aatctctatc gcccgccact ctctgctggg gacaggcaac tccgccgacg gtggtggctc
     6001 tggtggtgct agctgagtcg acgaattcat tgatcataat cagccatacc acatttgtag
     6061 aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa cataaaatga
     6121 atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     6181 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     6241 aactcatcaa tgtatcttat catgtctgga tccgtaccga gctcgcgtaa tcatgtcata
     6301 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     6361 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctacattaat tgcgttgcgc
     6421 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     6481 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcg
//
