LOCUS       sequence-194621-        6969 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A variant of Bongwoori with H237R mutation in its linker region. (* 
            super ecliptic pHluorin A227D).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6969)
  AUTHORS   Lee S, Geiller T, Jung A, Nakajima R, Song YK, Baker BJ
  TITLE     Improving a genetically encoded voltage indicator by modifying the 
            cytoplasmic charge composition.
  JOURNAL   Sci Rep. 2017 Aug 15;7(1):8286. doi: 10.1038/s41598-017-08731-2.
  PUBMED    28811673
REFERENCE   2  (bases 1 to 6969)
  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: "2017-08-15"; volume: "7(1):8286. doi"; pages: " 
            10.1038/s41598-017-08731-2"
COMMENT     Sequence Label: Bongwoori-R3
FEATURES             Location/Qualifiers
     source          1..6969
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     112..131
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     365..382
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    499..520
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(535..540,541..558,559..565)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    573..589
                     /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     578..600
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     597..613
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     597..613
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    662..783
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(673..692)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     727..746
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(913..1938)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             complement(913..1938)
                     /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"
     promoter        complement(1987..2316)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2001..2136
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2055..2074)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2301..2321
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2330..2758)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2440..2461)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2652..2671
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2804..3028
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     3017..3034
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(3079..3795)
                     /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
                     NFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLFTTSTLSKDPNEKRDHMVLLE
                     FVTADGITHGMDELYK"
     primer_bind     complement(3122..3142)
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     3737..3765
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     complement(4543..4562)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4544..4562)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4607..4810)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4636..4656)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4811..5190
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     5362..5381
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        5456..5560
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5561..5629,5630..6421)
                     /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(5779..5798)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6592..6969,1..211)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
       61 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
      121 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
      181 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
      241 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
      301 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
      361 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
      421 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
      481 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
      541 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
      601 aaacagctat gaccatgatt acgccaagct ctagctagag gtcgacggta tacagacatg
      661 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
      721 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
      781 gttggggtgg gcgaagaact ccagcatgag atccccgcgc tggaggatca tccagccggc
      841 gtcccggaaa acgattccga agcccaacct ttcatagaag gcggcggtgg aatcgaaatc
      901 tcgtagcacg tgctattcct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc
      961 ggcgagtact tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt
     1021 acgcccgaca gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc
     1081 tgcatcatcg aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag
     1141 catatacgcc cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg
     1201 cgtctgctgc tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta
     1261 ttgggaatcc ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc
     1321 cgtcaggaca ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc
     1381 ctcggcccaa agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc
     1441 catcacagtt tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca
     1501 tgtagtgtat tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag
     1561 atcggccgca gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc
     1621 ggtttcaggc aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag
     1681 gctctcgctg aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa
     1741 gtgccgataa acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac
     1801 atatccacgc cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg
     1861 catcaggtcg gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt
     1921 gagttcaggc tttttcatca cgtgctgatc agatccgaaa atggatatac aagctcccgg
     1981 gagctttttg caaaagccta ggcctccaaa aaagcctcct cactacttct ggaatagctc
     2041 agaggcagag gcggcctcgg cctctgcata aataaaaaaa attagtcagc catggggcgg
     2101 agaatgggcg gaactgggcg gagttagggg cgggatgggc ggagttaggg gcgggactat
     2161 ggttgctgac taattgagat gcatgctttg catacttctg cctgctgggg agcctgggga
     2221 ctttccacac ctggttgctg actaattgag atgcatgctt tgcatacttc tgcctgctgg
     2281 ggagcctggg gactttccac accctaactg acacacattc cacagaatta attcgcgtta
     2341 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat
     2401 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca
     2461 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
     2521 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta
     2581 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg
     2641 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg
     2701 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg
     2761 ggataccccc tagagcccca gctggttctt tccgcctcag aagccataga gcccaccgca
     2821 tccccagcat gcctgctatt gtcttcccaa tcctccccct tgctgtcctg ccccacccca
     2881 ccccccagaa tagaatgaca cctactcaga caatgcgatg caatttcctc attttattag
     2941 gaaaggacag tgggagtggc accttccagg gtcaaggaag gcacggggga ggggcaaaca
     3001 acagatggct ggcaactaga aggcacagtc gaggctgatc agcgggttta aacgggccct
     3061 ctagactcga gttctagatc atttgtatag ttcatccatg ccatgtgtaa tcccatcagc
     3121 tgttacaaac tcaagaagga ccatgtggtc tctcttttcg ttgggatctt tcgaaagagt
     3181 agaagttgta aacaggtaat ggttgtctgg taaaaggacg ggcccatcgc caattggagt
     3241 attttgttga taatggtctg ctagttgaac gcctccatct tcaatgttgt gtctaatttt
     3301 gaagttagct ttgattccat tcttttgttt gtctgccatg atgtacacct ggtgatcgtt
     3361 atagttgtat tccaatttgt gtccaagaat gtttccatct tctttaaaat caataccttt
     3421 taactcgatt ctattaacaa gggtatcacc ttcaaacttg acttcagcac gtgtcttgta
     3481 gttcccgtca tctttgaaaa atatagttct ttcctgtaca taaccttcgg gcatggcact
     3541 cttgaaaaag tcatgccgtt tcatatgatc tgggtatctt gaaaagcatt gaacaccata
     3601 agttaaagta gtgacaagtg ttggccatgg aacaggtagt tttccagtag tgcaaataaa
     3661 tttaagggta agttttccgt atgttgcatc accttcaccc tctccactga cagaaaattt
     3721 gtgcccatta acatcaccat ctaattcaac aagaattggg acaactccag tgaaaagttc
     3781 ttctccttta ctcatgggat ccccttgttg cctggaataa aatattcttg ctaagataac
     3841 cacacgcagc aatctggcca acacaaccaa ctgccccaaa ccatcggctc ctgtttcggt
     3901 accgtattca tctaacacag tgtaaaatat cgtgacgacg aatgtgacaa cgataatcaa
     3961 accatcagcc acctcccaag ggttggtgaa gaaaagcttg ggcccgtagg caaatatcct
     4021 taatcctaaa tccagcatga aataacaaga aagagccaag tccatcccgt cataaaagga
     4081 ttgtgaagat tcacttttat cgataagact gagatcaatg atcatgagga tgatgtccaa
     4141 gaaaattagg aagactccaa agactcgcat ccctagatga tcaatcactg ctcgaatgcg
     4201 aaactggacg cgacctacac cagtagtagg tatctccatt ctcccatcgt ccactccatg
     4261 ttcattctcg ccccaccata gacctgaaat ctcgggtata tctattcgtt cctcctgtct
     4321 tgtctctggt ttttcactcg gtccttggtc tacatcattc caatgaactt tctttaccga
     4381 ttcacttttt cttggcgcag ctttcggcgg agcagtgacg tcaccattta tcgtgacgtc
     4441 aacgacgttc cgcatgactg caccgtcaac gccaactaaa tcagctggag gactaaaatc
     4501 tgaaccgtcg aatccctcca tggtgctagc cagcttgggt ctccctatag tgagtcgtat
     4561 taatttcgat aagccagtaa gcagtgggtt ctctagttag ccagagagct ctgcttatat
     4621 agacctccca ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag ttgttacgac
     4681 attttggaaa gtcccgttga ttttggtgcc aaaacaaact cccattgacg tcaatggggt
     4741 ggagacttgg aaatccccgt gagtcaaacc gctatccacg cccattgatg tactgccaaa
     4801 accgcatcac catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt
     4861 cccataaggt catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa
     4921 tagggggcgt acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta
     4981 aatactccac ccattgacgt caatggaaag tccctattgg cgttactatg ggaacatacg
     5041 tcattattga cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg
     5101 taagttatgt aacgcggaac tccatatatg ggctatgaac taatgacccc gtaattgatt
     5161 actattaata actagtcaat aatcaatgtc aacgcgtata tctggcccgt acatcgcgaa
     5221 gcagcgcaaa acgcctaacc ctaagcagat tcttcatgca attgtcggtc aagccttgcc
     5281 ttgttgtagc ttaaattttg ctcgcgcact actcagcgac ctccaacaca caagcaggga
     5341 gcagatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccataggg
     5401 gatcgggaga tctcccgatc cgtcgacgtc aggtggcact tttcggggaa atgtgcgcgg
     5461 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata
     5521 accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg
     5581 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     5641 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     5701 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     5761 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     5821 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     5881 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     5941 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     6001 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     6061 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     6121 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     6181 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     6241 gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact
     6301 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac
     6361 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     6421 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     6481 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     6541 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     6601 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     6661 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     6721 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     6781 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     6841 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     6901 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     6961 actgagata
//
