LOCUS       sequence-161276-        6801 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Danio rerio VSP (without phosphatase activity) with 
            separated EGFP in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6801)
  AUTHORS   Hossain MI, Iwasaki H, Okochi Y, Chahine M, Higashijima S, Nagayama 
            K, Okamura Y
  TITLE     Enzyme domain affects the movement of the voltage sensor in ascidian
            and zebrafish voltage-sensing phosphatases.
  JOURNAL   J Biol Chem. 2008 Jun 27;283(26):18248-59. doi: 
            10.1074/jbc.M706184200. Epub 2008 Mar 28.
  PUBMED    18375390
REFERENCE   2  (bases 1 to 6801)
  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"; doi: 
            "10.1074/jbc.M706184200"; journalName: "J Biol Chem"; date: 
            "2008-06-27- 27"; volume: "283"; issue: "26"; pages: "18248-59"
COMMENT     Sequence Label: pDrVSP(C302S)-IRES2-EGFP
FEATURES             Location/Qualifiers
     source          1..6801
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        157..460
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        461..664
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     615..635
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     misc_feature    join(2256..2830,2831..2833,2834..2842)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2423..2440)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2650..2669
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(2843..2845,2846..2848,2849..3562)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2843..2845,2846..2848,2849..3559)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2888..2909)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3149..3168)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3496..3517
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3685..3806
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3722..3741)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3776..3795
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3813..4268)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3950..3971)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4162..4181
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4295..4399
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4401..4758
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4401..4592
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4424..4444)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4609..4744
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4793..5587
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4793..5587
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4847..4866)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5457..5476
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5775..5794)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5819..5866
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6195..6783
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6684..6703
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
       61 cctgattctg tggataaccg tattaccgcc atgcattagt tattaatagt aatcaattac
      121 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
      181 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
      241 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      301 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      361 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      421 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      481 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      541 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      601 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      661 agctggttta gtgaaccgtc agatccgcta gagccgccac catgacgtct gtgcatttta
      721 accctgggtt agattccaaa gaagttaatg gtaattcggt gaaagaagag gcagaggtgc
      781 agatcggtga tggaaaggaa gaaaccaaag acccagacac aatgtaccat caggtccgga
      841 aaaagataac tccttttgtg atgtcctttg gttttcgtgt atttggcctg gtactgatca
      901 tcttggacat cattatggtc attgtggact tgtctctttc tgagaagagt cgtgatgttg
      961 gaggtgcact ggagactgtt tctctggtca tctctttctt tttcctcatc gatgtattgc
     1021 tccgtgttta tgtggaggga ttcaaagtgt atttcagctc aaaactgaat atagtagatg
     1081 cttgtattgt ggtcattact ctggtggtca ccatgatcta cgcattctct gatttctcag
     1141 gagccagtct gattcccagg gtggtgacat tcctgaggtc tctgaggatc ctaattctgg
     1201 tacgcatctt cagactggcc tcacagaaga gagagcttga aaaagtcact aggagaatgg
     1261 tttcagagaa caaaagacgg taccaaaaag atggatttga tctggacctc acatacgtca
     1321 cagaaagagt catcgccatg tcttttccat cttctgggaa gcaggcgctt tataggaacc
     1381 caatcagaga ggtcgttaga ttcttggaca ccaaacatat ggatcattac aaggttttta
     1441 atctctgcag tgagaaaggt tatgatccaa agttctttca ctacagagta gagcgtgtca
     1501 tgatagatga ccataatgtc ccttcactgg atgatatgct gcgctacacc gcctgtgtca
     1561 gagactggat ggcagcagac tccaggaatg ttattgccat ccactctaaa ggaggaaagg
     1621 gtcggaccgg cactatggtt tgcacatggc tcattgacag cgatcagttt gagagtgcac
     1681 aggagagcct ggattacttt ggagagagac ggactgataa aagcatgagc tccaagtttc
     1741 aaggtgttga aactccgtct cagagcaggt atgttggcta ttatgagatc atgaagaatc
     1801 agtacaaccg tcagcttcct cctcggaaga gcctgaaaat caagagcatc cgaatccact
     1861 ccatcgcagg tgtggggaag ggcaacggca gtgacctgaa actgaagatc atagtgaagc
     1921 acgagcttgt tttccagtgt gtttgtgcca aacagcacaa ctgtacagtg tttcctgaca
     1981 ctggtagcaa tgcggtggtc atcagtttac aggacgggcc tatagtgact ggagatgtga
     2041 aggtcatgtt tgagtccagt gctggtttgc cgaagggtta tgaggactgt cccttctatt
     2101 tctggttcaa cacctcattt gtggagaact acagactttt tctgtcgagg gaagaactgg
     2161 acaatcctca caaacccaag acctgggaca tttacaagga agactttgga gtgacgctgt
     2221 ctttcactga gccttgaccg cgggcccggg atccgcccct ctccctcccc cccccctaac
     2281 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc
     2341 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg
     2401 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg
     2461 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc
     2521 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa
     2581 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa
     2641 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta
     2701 ccccattgta tgggatctga tctggggcct cggtacacat gctttacatg tgtttagtcg
     2761 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca
     2821 cgatgataat atggccacaa ccatggtgag caagggcgag gagctgttca ccggggtggt
     2881 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2941 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     3001 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
     3061 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     3121 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     3181 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     3241 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     3301 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     3361 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     3421 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     3481 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3541 catggacgag ctgtacaagt aaagcggccg cgactctaga tcataatcag ccataccaca
     3601 tttgtagagg ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
     3661 aaaatgaatg caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa
     3721 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3781 ttgtccaaac tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa
     3841 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     3901 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     3961 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     4021 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     4081 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     4141 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     4201 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     4261 gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     4321 ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata
     4381 atattgaaaa aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
     4441 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     4501 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4561 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4621 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4681 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4741 ggcctaggct tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga
     4801 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     4861 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     4921 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga
     4981 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     5041 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     5101 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     5161 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     5221 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     5281 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga
     5341 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     5401 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     5461 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     5521 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     5581 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     5641 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     5701 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct
     5761 agggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     5821 gcaataaaaa gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt
     5881 cggtcccagg gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc
     5941 cgcgtttctt ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca
     6001 gccaacgtcg gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt
     6061 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6121 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6181 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6241 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     6301 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     6361 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     6421 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     6481 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     6541 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     6601 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     6661 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     6721 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     6781 aaaaacgcca gcaacgcggc c
//
