LOCUS       sequence-242761-        5432 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mCherry-pHluorin fusion protein in E. coli, a fluorescent 
            reporter for cytoplasmic pH.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5432)
  AUTHORS   Zarkan A, Cano-Muniz S, Zhu J, Al Nahas K, Cama J, Keyser UF, 
            Summers DK
  TITLE     Indole Pulse Signalling Regulates the Cytoplasmic pH of E. coli in a
            Memory-Like Manner.
  JOURNAL   Sci Rep. 2019 Mar 7;9(1):3868. doi: 10.1038/s41598-019-40560-3.
  PUBMED    30846797
REFERENCE   2  (bases 1 to 5432)
  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: "2019-03-7"; volume: "9(1):3868. doi"; pages: " 
            10.1038/s41598-019-40560-3"
COMMENT     Sequence Label: pSCM001
FEATURES             Location/Qualifiers
     source          1..5432
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(49..66)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      219..305
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      397..424
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        443..534
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(535..603,604..1395)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(753..772)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1566..2154
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2055..2074
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2308..2325
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2340..2480
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2406..2425)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2566..2588
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2744..3622)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPTDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3649..3933
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     3856..3875
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             3960..3982
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(4138..4156)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4406..4425
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             4719..5432
                     /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="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNDHQVYIMADKQKNGIKAN
                     FKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELYK"
     primer_bind     complement(4746..4774)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     5369..5389
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
ORIGIN
        1 aaacggtctc cagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat
       61 taaatcagaa cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt
      121 ggtcccacct gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt
      181 ggggtctccc catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt
      241 cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga
      301 caaatccgcc gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag
      361 gacgcccgcc ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc
      421 tttttgcgtt tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg
      481 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      541 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      601 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      661 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      721 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
      781 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
      841 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
      901 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
      961 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1021 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1081 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1141 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1201 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1261 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1321 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1381 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1441 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1501 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1561 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1621 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1681 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1741 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1801 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1861 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     1921 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     1981 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2041 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2101 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2161 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2221 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2281 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2341 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     2401 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     2461 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     2521 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     2581 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gcagatcaat tcgcgcgcga
     2641 aggcgaagcg gcatgcataa tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc
     2701 tatgctactc cgtcaagccg tcaattgtct gattcgttac caattatgac aacttgacgg
     2761 ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg gccccggtgc
     2821 attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg cgaccgacgg
     2881 tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct
     2941 cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac agacgcgacg
     3001 gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct gccaggtgat
     3061 cgctgatgta ctgacaagcc tcgcgtaccc gattatccgt cggtggatgg agcgactcgt
     3121 taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc agcagctccg
     3181 aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg
     3241 gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac ggccagttaa
     3301 gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac cattcgcgag
     3361 cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa atatcacccg
     3421 gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga atggtgagat
     3481 tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg
     3541 cctcaatcgg cgttaaaccc gccaccagat gggcattaaa cgagtatccc ggcagcaggg
     3601 gatcattttg cgcttcagcc atacttttca tactcccgcc attcagagaa gaaaccaatt
     3661 gtccatattg catcagacat tgccgtcact gcgtctttta ctggctcttc tcgctaacca
     3721 aaccggtaac cccgcttatt aaaagcattc tgtaacaaag cgggaccaaa gccatgacaa
     3781 aaacgcgtaa caaaagtgtc tataatcacg gcagaaaagt ccacattgat tatttgcacg
     3841 gcgtcacact ttgctatgcc atagcatttt tatccataag attagcggat cctacctgac
     3901 gctttttatc gcaactctct actgtttctc catacccgtt tttttgggct agaaataatt
     3961 ttgtttaact ttaagaagga gatatacata ccatggtttc aaaaggcgaa gaagacaaca
     4021 tggcgattat caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc
     4081 acgagttcga gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca
     4141 agctgaaggt gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt
     4201 tcatgtacgg ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc
     4261 tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg
     4321 tgaccgtgac ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc
     4381 gcggcaccaa cttcccctcc gacggccccg taatgcagaa gaagaccatg ggctgggagg
     4441 cctcctccga gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc
     4501 tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga
     4561 agcccgtgca gctgcccggc gcctacaacg tcaacatcaa gttggacatc acctcccaca
     4621 acgaggacta caccatcgtg gaacagtacg aacgcgccga gggccgccac tctaccggcg
     4681 gtatggatga actgtacggc ggctctggcg gctctataag taaaggagaa gaacttttca
     4741 ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac aaattttctg
     4801 tcagtggaga gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca
     4861 ctactggaaa actacctgtt ccttggccaa cacttgtcac tactttaact tatggtgttc
     4921 aatgcttttc aagataccca gatcatatga aacggcatga ctttttcaag agtgccatgc
     4981 ccgaaggtta tgtacaggaa agaactatat ttttcaaaga tgacgggaac tacaagacac
     5041 gtgctgaagt caagtttgaa ggtgataccc ttgttaatag aatcgagtta aaaggtattg
     5101 attttaaaga agatggaaac attcttggac acaaattgga atacaactat aacgatcacc
     5161 aggtgtacat catggcagac aaacaaaaga atggaatcaa agctaacttc aaaattagac
     5221 acaacattga agatggaggc gttcaactag cagaccatta tcaacaaaat actccaattg
     5281 gcgatgggcc cgtcctttta ccagacaacc attacctgtt tacaacttct actctttcga
     5341 aagatcccaa cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga
     5401 ttacacatgg catggatgaa ctatacaaat ga
//
