LOCUS       sequence-223644-        6977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  superfolder pHluorin for intracellular pH measurements in S. 
            cerevisiae (improved performance when targeted to the ER or 
            subsequent compartments).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6977)
  AUTHORS   Reifenrath M, Boles E
  TITLE     A superfolder variant of pH-sensitive pHluorin for in vivo pH 
            measurements in the endoplasmic reticulum.
  JOURNAL   Sci Rep. 2018 Aug 10;8(1):11985. doi: 10.1038/s41598-018-30367-z.
  PUBMED    30097598
REFERENCE   2  (bases 1 to 6977)
  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 Rep."; 
            date: "2018-08-10"; volume: "8(1):11985. doi"; pages: " 
            10.1038/s41598-018-30367-z"
COMMENT     Sequence Label: p426MET25_sfpHluorin (MRV55)
FEATURES             Location/Qualifiers
     source          1..6977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      69..1411
                     /label=2u ori
                     /note="yeast 2u plasmid origin of replication"
     protein_bind    401..448
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        1438..1542
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1543..1611,1612..2403)
                     /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(1761..1780)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2574..3162
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3063..3082
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3316..3333
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3450..3471
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3486..3491,3492..3509,3510..3516)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3524..3540
                     /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     3529..3551
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3548..3564
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3548..3564
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3583..3603
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3585..3603
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3654..4003
                     /gene="S. cerevisiae MET17"
                     /gene_synonym="MET15; MET25"
                     /label=MET17 promoter
                     /note="expression is repressed by methionine"
     CDS             4011..4724
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNEHLVYITADKQKNGTKA
                     IFQVHHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLHTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(4041..4069)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     4664..4684
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      4728..4975
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(4744..4762)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(4993..5012)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4994..5012)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5022..5039)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5022..5038)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5031..5053)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5179..5634
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5266..5285)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5476..5497
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(5765..6568)
                     /codon_start=1
                     /gene="S. cerevisiae URA3"
                     /product="orotidine-5'-phosphate decarboxylase, required 
                     for uracil biosynthesis"
                     /label=URA3
                     /note="yeast auxotrophic marker, counterselectable with 
                     5-fluoroorotic acid (5-FOA)"
                     /translation="MSKATYKERAATHPSPVAAKLFNIMHEKQTNLCASLDVRTTKELL
                     ELVEALGPKICLLKTHVDILTDFSMEGTVKPLKALSAKYNFLLFEDRKFADIGNTVKLQ
                     YSAGVYRIAEWADITNAHGVVGPGIVSGLKQAAEEVTKEPRGLLMLAELSCKGSLSTGE
                     YTKGTVDIAKSDKDFVIGFIAQRDMGGRDEGYDWLIMTPGVGLDDKGDALGQQYRTVDD
                     VVSTGSDIIIVGRGLFAKGRDAKVEGERYRKAGWEAYLRRCGQQN"
     promoter        complement(6569..6784)
                     /gene="S. cerevisiae URA3"
                     /label=URA3 promoter
     primer_bind     complement(6808..6827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6927..6949
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttagtatga tccaatatca aaggaaatga tagcattgaa ggatgagact aatccaattg
      121 aggagtggca gcatatagaa cagctaaagg gtagtgctga aggaagcata cgataccccg
      181 catggaatgg gataatatca caggaggtac tagactacct ttcatcctac ataaatagac
      241 gcatataagt acgcatttaa gcataaacac gcactatgcc gttcttctca tgtatatata
      301 tatacaggca acacgcagat ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc
      361 gcgttgcatt ttcggaagcg ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt
      421 cctattctct agaaagtata ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa
      481 gacgcacttt caaaaaacca aaaacgcacc ggactgtaac gagctactaa aatattgcga
      541 ataccgcttc cacaaacatt gctcaaaagt atctctttgc tatatatctc tgtgctatat
      601 ccctatataa cctacccatc cacctttcgc tccttgaact tgcatctaaa ctcgacctct
      661 acatttttta tgtttatctc tagtattact ctttagacaa aaaaattgta gtaagaacta
      721 ttcatagagt gaatcgaaaa caatacgaaa atgtaaacat ttcctatacg tagtatatag
      781 agacaaaata gaagaaaccg ttcataattt tctgaccaat gaagaatcat caacgctatc
      841 actttctgtt cacaaagtat gcgcaatcca catcggtata gaatataatc ggggatgcct
      901 ttatcttgaa aaaatgcacc cgcagcttcg ctagtaatca gtaaacgcgg gaagtggagt
      961 caggcttttt ttatggaaga gaaaatagac accaaagtag ccttcttcta accttaacgg
     1021 acctacagtg caaaaagtta tcaagagact gcattataga gcgcacaaag gagaaaaaaa
     1081 gtaatctaag atgctttgtt agaaaaatag cgctctcggg atgcattttt gtagaacaaa
     1141 aaagaagtat agattctttg ttggtaaaat agcgctctcg cgttgcattt ctgttctgta
     1201 aaaatgcagc tcagattctt tgtttgaaaa attagcgctc tcgcgttgca tttttgtttt
     1261 acaaaaatga agcacagatt cttcgttggt aaaatagcgc tttcgcgttg catttctgtt
     1321 ctgtaaaaat gcagctcaga ttctttgttt gaaaaattag cgctctcgcg ttgcattttt
     1381 gttctacaaa atgaagcaca gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc
     1441 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     1501 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     1561 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     1621 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     1681 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     1741 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa
     1801 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     1861 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     1921 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     1981 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     2041 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca
     2101 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     2161 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     2221 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     2281 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     2341 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     2401 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     2461 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     2521 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     2581 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     2641 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     2701 gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac
     2761 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     2821 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     2881 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     2941 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     3001 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     3061 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     3121 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     3181 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     3241 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     3301 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
     3361 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
     3421 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
     3481 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
     3541 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
     3601 agggaacaaa agctggagct ccggatgcaa gggttcgaat cccttagctc tcattatttt
     3661 ttgctttttc tcttgaggtc acatgatcgc aaaatggcaa atggcacgtg aagctgtcga
     3721 tattggggaa ctgtggtggt tggcaaatga ctaattaagt tagtcaaggc gccatcctca
     3781 tgaaaactgt gtaacataat aaccgaagtg tcgaaaaggt ggcaccttgt ccaattgaac
     3841 acgctcgatg aaaaaaataa gatatatata aggttaagta aagcgtctgt tagaaaggaa
     3901 gtttttcctt tttcttgctc tcttgtcttt tcatctacta tttccttcgt gtaatacagg
     3961 gtcgtcagat acatagatac aattctatta cccccatcca tactctagaa atgagcaaag
     4021 gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg
     4081 ggcacaaatt ttctgtccgt ggagagggtg aaggtgatgc tacaaacgga aaactcaccc
     4141 ttaaatttat ttgcactact ggaaaactac ctgttccgtg gccaacactt gtcactactc
     4201 tgtcttatgg tgttcaatgc ttttcccgtt atccggatca catgaaacgg catgactttt
     4261 tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc aaagatgacg
     4321 ggacctacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg
     4381 agttaaaggg tattgatttt aaagaagatg gaaacattct tggacacaaa ctcgagtaca
     4441 actttaacga gcacttggta tacatcacgg cagacaaaca aaagaatgga accaaagcta
     4501 tcttccaagt tcaccacaac gttgaagatg gttccgttca actagcagac cattatcaac
     4561 aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac ctgcacacac
     4621 aatctgtcct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt cttgagtttg
     4681 taactgctgc tgggattaca catggcatgg atgagctcta caaataatca tgtaattagt
     4741 tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa aggaaggagt
     4801 tagacaacct gaagtctagg tccctattta tttttttata gttatgttag tattaagaac
     4861 gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat
     4921 tatactgaaa accttgcttg agaaggtttt gggacgctcg aaggctttaa tttgcggccg
     4981 gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa
     5041 cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct
     5101 ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc
     5161 agcctgaatg gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5221 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     5281 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5341 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     5401 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5461 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     5521 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     5581 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat
     5641 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatagggt aataactgat
     5701 ataattaaat tgaagctcta atttgtgagt ttagtataca tgcatttact tataatacag
     5761 ttttttagtt ttgctggccg catcttctca aatatgcttc ccagcctgct tttctgtaac
     5821 gttcaccctc taccttagca tcccttccct ttgcaaatag tcctcttcca acaataataa
     5881 tgtcagatcc tgtagagacc acatcatcca cggttctata ctgttgaccc aatgcgtctc
     5941 ccttgtcatc taaacccaca ccgggtgtca taatcaacca atcgtaacct tcatctcttc
     6001 cacccatgtc tctttgagca ataaagccga taacaaaatc tttgtcgctc ttcgcaatgt
     6061 caacagtacc cttagtatat tctccagtag atagggagcc cttgcatgac aattctgcta
     6121 acatcaaaag gcctctaggt tcctttgtta cttcttctgc cgcctgcttc aaaccgctaa
     6181 caatacctgg gcccaccaca ccgtgtgcat tcgtaatgtc tgcccattct gctattctgt
     6241 atacacccgc agagtactgc aatttgactg tattaccaat gtcagcaaat tttctgtctt
     6301 cgaagagtaa aaaattgtac ttggcggata atgcctttag cggcttaact gtgccctcca
     6361 tggaaaaatc agtcaagata tccacatgtg tttttagtaa acaaattttg ggacctaatg
     6421 cttcaactaa ctccagtaat tccttggtgg tacgaacatc caatgaagca cacaagtttg
     6481 tttgcttttc gtgcatgata ttaaatagct tggcagcaac aggactagga tgagtagcag
     6541 cacgttcctt atatgtagct ttcgacatga tttatcttcg tttcctgcag gtttttgttc
     6601 tgtgcagttg ggttaagaat actgggcaat ttcatgtttc ttcaacacta catatgcgta
     6661 tatataccaa tctaagtctg tgctccttcc ttcgttcttc cttctgttcg gagattaccg
     6721 aatcaaaaaa atttcaagga aaccgaaatc aaaaaaaaga ataaaaaaaa aatgatgaat
     6781 tgaaaaggtg gtatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     6841 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     6901 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     6961 atcaccgaaa cgcgcga
//
