LOCUS       sequence-292709-        7800 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses modified Danio rerio VSP without phosphatase activity 
            (inactive mutant of enhanced VSP) fused with mCherry in mammalian 
            cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7800)
  AUTHORS   Kawanabe A, Mizutani N, Polat OK, Yonezawa T, Kawai T, Mori MX, 
            Okamura Y
  TITLE     Engineering an enhanced voltage-sensing phosphatase.
  JOURNAL   J Gen Physiol. 2020 May 4;152(5). pii: 133870. doi: 
            10.1085/jgp.201912491.
  PUBMED    32167537
REFERENCE   2  (bases 1 to 7800)
  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 Gen 
            Physiol."; date: "2020-05-4"; volume: "152(5). pii: 133870. doi"; 
            pages: " 10.1085/jgp.201912491"
COMMENT     Sequence Label: CiDr-VSP L223F-mCherry pcDNA3.1 (eVSP)
FEATURES             Location/Qualifiers
     source          1..7800
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             2409..3119
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(2554..2572)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2822..2841
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3157..3174)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3163..3387
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3433..3861
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3520..3539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3730..3751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3870..3890)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3875..4204
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4055..4190
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4117..4136
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4271..5065
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4325..4344)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4935..4954
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5239..5360
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5276..5295)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5330..5349
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5409..5425)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5409..5425)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5422..5444)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5433..5449
                     /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(5457..5463,5464..5481,5482..5487))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5502..5523
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5640..5657)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5811..6399)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5891..5910)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6570..7361,7362..7430))
                     /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     7193..7212
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7431..7535)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7610..7629)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctg
      661 gctagcgttt aaacgggccc ctcgagatgg agggattcga cggttcagat tttagtcctc
      721 cagctgattt agttggcgtt gacggtgcag tcatgcggaa cgtcgttgac gtcacgataa
      781 atggtgacgt cactgctccg ccgaaagctg cgccaagaaa aagtgaatcg gtaaagaaag
      841 ttcattggaa tgatgtagac caaggaccga gtgaaaaacc agagacaaga caggaggaac
      901 gaatagatat acccgagatt tcaggtctat ggtggggcga gaatgaacat ggagtggacg
      961 atgggagaat ggagatacct actactggtg taggtaccca ggtccggaaa aagataactc
     1021 cttttgtgat gtcctttggt tttcgtgtat ttggcctggt actgatcatc ttggacatca
     1081 ttatggtcat tgtggacttg tctctttctg agaagagtcg tgatgttgga ggtgcactgg
     1141 agactgtttc tctggtcatc tctttctttt tcctcatcga tgtattgctc cgtgtttatg
     1201 tggagggatt caaagtgtat ttcagctcaa aactgaatat agtagatgct tgtattgtgg
     1261 tcattactct ggtggtcacc atgatctacg cattctctga tttctcagga gccagtctga
     1321 ttcccagggt ggtgacattc ctgaggtctc tgagaatcct aattctggta cgcatcttca
     1381 gactggcctc acagaagaga gagcttgaaa aagtcactag gagaatggtt tcagagaaca
     1441 aaagacggta tcaaaaagat ggatttgatc tggacctcac atacgtcaca gaaagagtca
     1501 tcgccatgtc ttttccatct tctgggaagc aggcgttcta taggaaccca atcagagagg
     1561 tcgttagatt cttggacacc aaacatatgg atcattacaa ggtttttaat ctctgcagtg
     1621 agaaaggtta tgatccaaag ttctttcact acagagtaga gcgtgtcatg atagatgacc
     1681 ataatgtccc ttcactggat gatatgctgc gctacaccgc ctgtgtcaga gactggatgg
     1741 cagcagactc caggaatgtt attgccatcc actgcaaagg aggaaagggt cggaccggca
     1801 ctatggtttg cacatggctc attgacagcg atcagtttga gagtgcacag gagagcctgg
     1861 attactttgg agagagacgg actgataaaa gcatgagctc caagtttcaa ggtgttgaaa
     1921 ctccgtctca gagcaggtat gttggctatt atgagatcat gaagaatcag tacaaccgtc
     1981 agcttcctcc tcggaagagc ctgaaaatca agagcatccg aatccactcc atcgcaggtg
     2041 tggggaaggg caacggcagt gacctgaaac tgaagatcat agtgaagcac gagcttgttt
     2101 tccagtgtgt ttgtgccaaa cagcacaact gtacagtgtt tcctgacact ggtagcaatg
     2161 cggtggtcat cagtttacag gacgggccta tagtgactgg agatgtgaag gtcatgtttg
     2221 agtccagtgc tggtttgccg aagggttatg aggactgtcc cttctatttc tggttcaaca
     2281 cctcatttgt ggagaactac agactttttc tgtcgaggga agaactggac aatcctcaca
     2341 aacccaagac ctgggacatt tacaaggaag actttggagt gacgctgtct ttcactgagc
     2401 ctggatccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     2461 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     2521 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     2581 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     2641 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     2701 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     2761 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     2821 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     2881 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     2941 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     3001 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     3061 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagtaaa
     3121 agctttctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     3181 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     3241 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     3301 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     3361 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     3421 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     3481 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     3541 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     3601 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     3661 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     3721 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     3781 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     3841 aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc
     3901 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag
     3961 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     4021 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4081 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4141 ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4201 caaaaagctc ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg
     4261 atcgtttcgc atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga
     4321 gaggctattc ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt
     4381 ccggctgtca gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct
     4441 gaatgaactg caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg
     4501 cgcagctgtg ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt
     4561 gccggggcag gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc
     4621 tgatgcaatg cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc
     4681 gaaacatcgc atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga
     4741 tctggacgaa gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg
     4801 catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat
     4861 ggtggaaaat ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg
     4921 ctatcaggac atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc
     4981 tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta
     5041 tcgccttctt gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg
     5101 acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     5161 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     5221 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     5281 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     5341 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     5401 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     5461 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     5521 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     5581 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     5641 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5701 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5761 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5821 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5881 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5941 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6001 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6061 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6121 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6181 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6241 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6301 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6361 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6421 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6481 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6541 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6601 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6661 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6721 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6781 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6841 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6901 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6961 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7021 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7081 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7141 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7201 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7261 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7321 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7381 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7441 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7501 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7561 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     7621 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     7681 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     7741 atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt
//
