LOCUS       sequence-154213-        6377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses super ecliptic A227D in mammalian cell membranes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6377)
  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 6377)
  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: Farnesylated SE pcDNA3.1
FEATURES             Location/Qualifiers
     source          1..6377
                     /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"
     CDS             970..1683
                     /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(1000..1028)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1623..1643
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1802..1819)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1808..2032
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2078..2506
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2165..2184)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2375..2396
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2515..2535)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2520..2849
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2700..2835
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2762..2781
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2898..3923
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2898..3923
                     /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    4053..4174
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4090..4109)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4144..4163
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4223..4239)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4223..4239)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4236..4258)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4247..4263
                     /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(4271..4277,4278..4295,4296..4301))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4316..4337
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4454..4471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4625..5210)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4705..4724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5381..6172,6173..6241))
                     /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     6004..6023
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6242..6346)
                     /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 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc
      961 agatatcgca tgagtaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa
     1021 ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgca
     1081 acatacggaa aacttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg
     1141 ccaacacttg tcactacttt aacttatggt gttcaatgct tttcaagata cccagatcat
     1201 atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaaagaact
     1261 atatttttca aagatgacgg gaactacaag acacgtgctg aagtcaagtt tgaaggtgat
     1321 acccttgtta atagaatcga gttaaaaggt attgatttta aagaagatgg aaacattctt
     1381 ggacacaaat tggaatacaa ctataacgat caccaggtgt acatcatggc agacaaacaa
     1441 aagaatggaa tcaaagctaa cttcaaaatt agacacaaca ttgaagatgg aggcgttcaa
     1501 ctagcagacc attatcaaca aaatactcca attggcgatg ggcccgtcct tttaccagac
     1561 aaccattacc tgtttacaac ttctactctt tcgaaagatc ccaacgaaaa gagagaccac
     1621 atggtccttc ttgagtttgt aacagctgat gggattacac atggcatgga tgaactatac
     1681 aaatccggac tcagatctaa gctgaaccct cctgatgaga gtggccccgg ctgcatgagc
     1741 tgcaagtgtg tgctctccta atagctcgag tctagagggc ccgtttaaac ccgctgatca
     1801 gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc
     1861 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg
     1921 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg
     1981 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag
     2041 gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta
     2101 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2161 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2221 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2281 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2341 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2401 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     2461 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg
     2521 tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2581 tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa
     2641 gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     2701 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     2761 ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag
     2821 gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg
     2881 gatctgatca gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc
     2941 tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc
     3001 gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg
     3061 atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc
     3121 cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg
     3181 cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg
     3241 tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc
     3301 cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg
     3361 ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg
     3421 cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg
     3481 tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca
     3541 ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct
     3601 ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg
     3661 agcttgcagg atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct
     3721 atcagagctt ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg
     3781 caatcgtccg atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg
     3841 ccgtctggac cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca
     3901 ctcgtccgag ggcaaaggaa tagcacgtgc tacgagattt cgattccacc gccgccttct
     3961 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4021 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4081 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4141 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4201 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4261 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4321 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4381 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4441 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4501 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4561 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4621 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4681 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4741 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4801 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4861 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4921 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4981 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5041 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5101 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5161 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5221 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5281 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5341 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5401 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5461 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5521 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5581 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5641 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5701 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5761 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5821 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5881 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5941 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6001 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6061 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6121 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6181 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6241 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6301 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6361 aagtgccacc tgacgtc
//
