LOCUS       sequence-148888-        7007 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  More dimeric SE attached to Triple Mutant GEVI from Piao et al 2015.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7007)
  AUTHORS   Kang BE, Baker BJ
  TITLE     Pado, a fluorescent protein with proton channel activity can 
            optically monitor membrane potential, intracellular pH, and map gap 
            junctions.
  JOURNAL   Sci Rep. 2016 Apr 4;6:23865. doi: 10.1038/srep23865.
  PUBMED    27040905
REFERENCE   2  (bases 1 to 7007)
  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: "2016-04-4"; volume: "6:23865. doi"; pages: " 
            10.1038/srep23865"
COMMENT     Sequence Label: TM SE T206A
FEATURES             Location/Qualifiers
     source          1..7007
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      901..910
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1675..2391
                     /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
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSALSKDPNEKRDHMVLLE
                     FVTADGITHGMDELYK"
     primer_bind     complement(1705..1733)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2328..2348
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(2432..2449)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2438..2662
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2708..3136
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2795..2814)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3005..3026
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3145..3165)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3150..3479
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3330..3465
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3392..3411
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3528..4553
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             3528..4553
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    4683..4804
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4720..4739)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4774..4793
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4853..4869)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4853..4869)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4866..4888)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4877..4893
                     /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(4901..4907,4908..4925,4926..4931))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4946..4967
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5084..5101)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5255..5840)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5335..5354)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6011..6802,6803..6871))
                     /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     6634..6653
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6872..6976)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gccaccatgg agggattcga cggttcagat tttagtcctc cagctgattt agttggcgtt
      961 gacggtgcag tcatgcggaa cgtcgttgac gtcacgataa atggtgacgt cactgctccg
     1021 ccgaaagctg cgccaagaaa aagtgaatcg gtaaagaaag ttcattggaa tgatgtagac
     1081 caaggaccga gtgaaaaacc agagacaaga caggaggaac gaatagatat acccgagatt
     1141 tcaggtctat ggtggggcga gaatgaacat ggagtggacg atgggagaat ggagatacct
     1201 actactggtg taggtcgcgt ccagtttcgg atccgagcag tgattgatca tctagggatg
     1261 cgagtctttg gagtcttcct aattttcttg gacatcatcc tcatgatcat tgatctcagt
     1321 cttatcgata aaagtgaatc ttcacaatcc ttttatgacg ggatggactt ggctctttct
     1381 tgttatttca tgctggattt aggattaagg atatttgcct acgggcccaa gcttttcttc
     1441 accaaccctt gggaggtggc tgatggtttg attatcgttg tcacattcgt cgtcacgata
     1501 ttttacactg tgttagatga atacggtacc gaaacaggag ccgatggttt ggggcagttg
     1561 gttgtgttgg ccagattgct gcgtgtggtt atcttagcaa gaatatttta ttcccaccaa
     1621 caaatgaagg cttcaagcag aagaacaata tcacagaaca aaagaagata tcgcatgagt
     1681 aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga tggtgatgtt
     1741 aatgggcaca aattttctgt cagtggagag ggtgaaggtg atgcaacata cggaaaactt
     1801 acccttaaat ttatttgcac tactggaaaa ctacctgttc catggccaac acttgtcact
     1861 actttaactt atggtgttca atgcttttca agatacccag atcatatgaa acggcatgac
     1921 tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa gaactatatt tttcaaagat
     1981 gacgggaact acaagacacg tgctgaagtc aagtttgaag gtgataccct tgttaataga
     2041 atcgagttaa aaggtattga ttttaaagaa gatggaaaca ttcttggaca caaattggaa
     2101 tacaactata acgatcacca ggtgtacatc atggcagaca aacaaaagaa tggaatcaaa
     2161 gctaacttca aaattagaca caacattgaa gatggaggcg ttcaactagc agaccattat
     2221 caacaaaata ctccaattgg cgatgggccc gtccttttac cagacaacca ttacctgttt
     2281 acaacttctg ccctttcgaa agatcccaac gaaaagagag accacatggt ccttcttgag
     2341 tttgtaacag ctgatgggat tacacatggc atggatgaac tatacaaatg atgactcgag
     2401 tctagagggc ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     2461 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     2521 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     2581 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     2641 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     2701 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     2761 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2821 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2881 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2941 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     3001 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     3061 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     3121 aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3181 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3241 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3301 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3361 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg
     3421 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     3481 agctcccggg agcttgtata tccattttcg gatctgatca gcacgtgatg aaaaagcctg
     3541 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
     3601 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
     3661 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
     3721 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
     3781 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     3841 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
     3901 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     3961 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     4021 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     4081 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     4141 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     4201 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     4261 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
     4321 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     4381 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     4441 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     4501 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagcacgtgc
     4561 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     4621 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     4681 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4741 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4801 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4861 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4921 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4981 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     5041 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     5101 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     5161 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     5221 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     5281 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     5341 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     5401 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     5461 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     5521 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     5581 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     5641 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5701 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5761 gatccggcaa acaaaccacc gctggtagcg gtttttttgt ttgcaagcag cagattacgc
     5821 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5881 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5941 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6001 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6061 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6121 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6181 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6241 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6301 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6361 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6421 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6481 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6541 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6601 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6661 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6721 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6781 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6841 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6901 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6961 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
