LOCUS       sequence-200452-        6927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6927)
  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 6927)
  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-Mito-pHRed
FEATURES             Location/Qualifiers
     source          1..6927
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             189..263
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             285..359
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             381..455
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             477..551
                     /codon_start=1
                     /product="mitochondrial presequence of human cytochrome c 
                     oxidase subunit VIII (Rizutto et al., 1989)"
                     /label=COX8 presequence
                     /translation="MSVLTPLLLRGLTGSARRLPVPRAK"
     CDS             join(621..623,624..626,627..1289)
                     /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    1420..1554
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1457..1476)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1511..1530
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1577..1593)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1577..1593)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1590..1612)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1601..1617
                     /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(1625..1631,1632..1649,1650..1655))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(1807..1824)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1978..2566)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2058..2077)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2737..3528,3529..3597))
                     /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     3360..3379
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3654..4167)
                     /direction=LEFT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3849..3870)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4061..4080
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4339..4357
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4395..4417)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4517..4536
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4768..4790
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4782..4799
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4783..4799
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        5365..5744
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5745..5948
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5899..5919
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     5945..5969
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          6084..6910
                     /label=CMV intron A
                     /note="human cytomegalovirus intron A (Chapman et al., 
                     1991)"
ORIGIN
        1 aagcttcccg gccacagcaa tcgatccatg acacaaccag tgacaagcag tcccccagtg
       61 aaaccgcatc cgcatccgag tccgataccg ataaagattc tgaatcggag tgagtgccgc
      121 gtccgagagc gttccctgtc cacgtccacc atcggcggag caggtgtgcc tgaggcctct
      181 agaccaccat gagcgtgctg acaccattgt tgctcagagg cctcactggt tcagccaggc
      241 ggctgccagt cccaagagct aagatccata gtctgggaga tccaatgtca gttctcactc
      301 cccttttgct taggggactg accggaagcg ctcggcggtt gcctgtccca cgcgccaaga
      361 ttcactccct gggcgaccct atgagtgtcc ttactcctct tttgctgaga ggcttgacag
      421 ggagcgctcg gaggctccca gtgcctcgcg caaagatcca ttctctcgga gatcccatgt
      481 ccgtcctcac tcctctgctg ctgcgcgggc ttacaggttc cgctcgccgc ctgcctgttc
      541 ctcgggcaaa gatacacagt ctgggcgatc ctcaccggcg ctatctcatg ggcggtgcaa
      601 gcggcggcgc aagcggtggt atggtttccg ttatcgcaaa gcaaatgacc tataaagttt
      661 atatgtctgg caccgttaac ggccactact tcgaggttga aggtgacggc aagggcaagc
      721 cttacgaggg cgagcagacc gttaagctga ccgttaccaa aggcggccca ctgcctttcg
      781 cctgggatat cctgtcccct cagctccaat acggctccat ccctttcacc aaataccccg
      841 aggacatccc tgactacttt aagcagagct tcccagaggg ctacacctgg gagcgctcta
      901 tgaacttcga ggacggcgca gtttgtaccg tttccaacga cagctccatc cagggtaact
      961 gcttcatcta caacgttaag atttccggcg agaacttccc acctaacggc ccagttatgc
     1021 agaagaaaac ccaaggctgg gagcccagca ccgaacgcct gtttgcccgc gacggtatgc
     1081 tgattggtaa cgattatatg gccctgaaac tggaaggcgg cggtcactac ctgtgcgagt
     1141 tcaagagcac ttacaaggct aaaaagcctg ttcgcatgcc tggccgccac gaaatcgacc
     1201 gcaagctgga tgttacctct cacaaccgcg actacaccag cgttgagcag tgtgaaatct
     1261 ctatcgcccg ccactctctg ctgggttgag ctagctgagt cgacgaattc attgatcata
     1321 atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc
     1381 ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat
     1441 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     1501 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgtacc
     1561 gagctcgcgt aatcatgtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     1621 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     1681 aactacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     1741 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     1801 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     1861 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     1921 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     1981 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     2041 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     2101 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     2161 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     2221 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     2281 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     2341 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     2401 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     2461 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     2521 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     2581 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     2641 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     2701 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     2761 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     2821 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     2881 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     2941 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3001 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     3061 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     3121 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     3181 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     3241 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     3301 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     3361 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     3421 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     3481 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     3541 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     3601 cttccttttt caataagcgg ccgcggccat gccggccact agtctcgagg gccaccccgg
     3661 ttgataatca gaaaagcccc aaaaacagga agattgtata agcaaatatt taaattgtaa
     3721 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     3781 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     3841 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     3901 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     3961 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4021 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4081 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     4141 cgcttaatgc gccgctacag ggcgcgtggg gatccctcga gttattattg aagcatttat
     4201 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4261 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     4321 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt
     4381 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
     4441 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
     4501 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgaagacg
     4561 tcgcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc ctctgcataa
     4621 ataaaaaaaa ttagtcaggg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     4681 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     4741 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg
     4801 cccgtgtcaa ggacggtgac tgcagtgaat aataaaatgt gtgtttgtcc gaaatacgcg
     4861 ttttgagatt tctgtcgccg actaaattca tgtcgcgcga tagtggtgtt tatcgccgat
     4921 agagatggcg atattggaaa aatcgatatt tgaaaatatg gcatattgaa aatgtcgccg
     4981 atgtgagttt ctgtgtaact gatatcgcca tttttccaaa agtgattttt gggcatacgc
     5041 gatatctggc gatagcgctt atatcgttta cgggggatgg cgatagacga ctttggtgac
     5101 ttgggcgatt ctgtgtgtcg caaatatcgc agtttcgata taggtgacag acgatatgag
     5161 gctatatcgc cgatagaggc gacatcaagc tggcacatgg ccaatgcata tcgatctata
     5221 cattgaatca atattggcca ttagccatat tattcattgg ttatatagca taaatcaata
     5281 ttggctattg gccattgcat acgttgtatc catatcataa tatgtacatt tatattggct
     5341 catgtccaac attaccgcca tgttgacatt gattattgac tagttattaa tagtaatcaa
     5401 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     5461 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     5521 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     5581 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg
     5641 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc
     5701 ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc
     5761 agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca
     5821 ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta
     5881 acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa
     5941 gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
     6001 tccatagaag acaccgggac cgatccagcc tccgcggccg ggaacggtgc attggaacgc
     6061 ggattccccg tgccaagagt gacgtaagta ccgcctatag agtctatagg cccaccccct
     6121 tggcttctta tgcatgctat actgtttttg gcttggggtc tatacacccc cgcttcctca
     6181 tgttataggt gatggtatag cttagcctat aggtgtgggt tattgaccat tattgaccac
     6241 tcccctattg gtgacgatac tttccattac taatccataa catggctctt tgccacaact
     6301 ctctttattg gctatatgcc aatacactgt ccttcagaga ctgacacgga ctctgtattt
     6361 ttacaggatg gggtctcatt tattatttac aaattcacat atacaacacc accgtcccca
     6421 gtgcccgcag tttttattaa acataacgtg ggatctccac gcgaatctcg ggtacgtgtt
     6481 ccggacatgg gctcttctcc ggtagcggcg gagcttctac atccgagccc tgctcccatg
     6541 cctccagcga ctcatggtcg ctcggcagct ccttgctcct aacagtggag gccagactta
     6601 ggcacagcac gatgcccacc accaccagtg tgccgcacaa ggccgtggcg gtagggtatg
     6661 tgtctgaaaa tgagctcggg gagcgggctt gcaccgctga cgcatttgga agacttaagg
     6721 cagcggcaga agaagatgca ggcagctgag ttgttgtgtt ctgataagag tcagaggtaa
     6781 ctcccgttgc ggtgctgtta acggtggagg gcagtgtagt ctgagcagta ctcgttgctg
     6841 ccgcgcgcgc caccagacat aatagctgac agactaacag actgttcctt tccatgggtc
     6901 ttttctgcag tcaccgtcct tgacacg
//
